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The U7 snRNP and the hairpin binding protein: Key players in histone mRNA metabolism
seminars in C E L L & D E V E L OP M E N T A L B I OL OG Y , Vol 8, 1997: pp 567]576
The U7 snRNP and the hairpin binding protein: Key players in histone mRNA metabolism Berndt Muller ¨ and Daniel Schumperli ¨ Animal replication-dependent histone mRNAs are subject to several post-transcriptional regulatory processes. Their nonpolyadenylated 39 ends are formed preferentially during S phase by a unique nuclear cleavage event. This requires the base pairing between U7 snRNA and a histone spacer element 39 of the cleavage site. Cleavage occurs preferentially after adenosine, at a fixed distance from the hybrid region. A conserved RNA hairpin just upstream of the cleavage site is recognised by the hairpin binding protein (HBP) that acts as an auxiliary processing factor, stabilising the interaction of the histone pre-mRNA with the U7 snRNP. The interaction between HBP and the RNA hairpin is very stable and HBP is also found associated with histone mRNAs on polysomes. The hairpin and presumably, HBP are also required for nuclear export and translation of histone mRNA. Furthermore, histone mRNAs are selectively destabilised in the G2 phase or upon inhibition of DNA synthesis and this regulation is also associated with the hairpin. Recently, HBP-encoding cDNAs were isolated from various organisms. Human, mouse and Xenopus laevis HBPs are similar, while the Caenorhabditis elegans protein has significant homology to the others only in a central RNA binding domain.
five to 20 copies of these genes for each histone subtype, concentrated in two clusters on chromosomes 1 and 6 in humans1 and chromosomes 3 and 13 in mice.2,3 The main distinctive features of these genes are their lack of introns and the fact that their mRNAs are not polyadenylated but produced by a unique RNA 39 processing reaction Žreviewed in ref 4; see below.. Histone genes with these characteristics are common to all metazoans, whereas other organisms such as fungi and plants have histone mRNAs with polyŽA. tails and frequently, introns.5,6 However, there are notable exceptions. The flagellate algae Volvox 7 and Chlamydomonas 8,9 have histone genes similar to the metazoan type. In contrast, a histone H4 gene from the nematode Ascaris lumbricoides Žpossibly the only H4 gene of this organism. has introns and encodes a polyadenylated mRNA.10 Despite their name, the replication-dependence of these genes is far from absolute. First, the relationship between DNA synthesis and histone gene expression has not been analysed in many organisms. Then there are cases where histone mRNAs are maternally expressed and stored in oocytes Že.g. Xenopus, Drosophila, sea urchins..11 ] 13 In Xenopus these translationally inactive maternal histone mRNAs have short polyŽA. tails added to the normal 39 ends,11 whereas for all other maternally stored mRNAs the inactive forms lack polyŽA. and the actively translated forms are polyadenylated.14 In mammals and birds some of the genes of the replication-dependent type are also expressed during spermatogenesis and this expression is not tightly coupled to DNA synthesis.15,16 Finally, sea urchins have several sets of histone genes of the replication-dependent type which are expressed in a partly overlapping fashion at different developmental stages or in different tissues.13 In addition, most metazoans and certainly all vertebrates have histone genes with the standard organization for protein coding genes Žintrons, polyŽA. tails..17 These are either constitutively expressed such as the H1.0, H2A.Z and H3.3 genes or strictly cell typespecific as the H5 genes of animals with nucleated erythrocytes. The products of the former class are called basal or replacement histones since they grad-
Key words: histone r RNA 39 processing r U7 small nuclear ribonucleoprotein r hairpin binding protein r stem-loop binding protein r RNA]protein interaction Q1997 Academic Press Ltd
Introduction: The replication-dependent histone genes IN ANIMAL CELLS, the packaging of DNA into chromatin during DNA replication is maintained by S phase- specific expression of the so- called replication-dependent histone genes. Mammals have U
From the Abteilung fur ¨ Entwicklungsbiologie, Zoologisches Institut der Universitat ´ Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Q1997 Academic Press Ltd 1084-9521r 97r 060567q 10 $25.00r 0r sr970182
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ually replace the products of the replication-dependent genes in the chromatin of non-proliferating and terminally differentiated cells. In recent years, several additional histone-like proteins Žand genes. have been described which share with the ‘real’ histones a common structural motif called the histone fold.18 These proteins are thought to play important roles in regional chromosome and chromatin structure and in the modulation of gene expression. The cell cycle-dependent production of replication-dependent histone proteins, at least in mammalian cells, is entirely caused by a modulation of mRNA levels Žreviewed in refs 5, 19 and 20.. This, in turn is achieved by a combination of three regulatory events. Transcription5,21,22 and histone RNA 39 processing19 are both activated late in G1 and turned off at the end of the S phase. Together, these two events can cause a 30 to 50-fold modulation of histone mRNA production.23 Furthermore, at the end of the S phase, the half-life of histone mRNAs drops from 40]60 to 5]12 min, resulting in a rapid elimination of all remaining histone mRNAs from the cell.23 Partly identical 39-terminal sequences are involved in controlling and regulating the 39 end processing and degradationrstability of histone mRNAs as well as other steps of histone mRNA metabolism. In this review, we will concentrate on the events leading to the formation of the mature mRNA 39 ends and the factors influencing mRNA stability.
terised by a four base uridine-rich loop and a six base pair stem. The uppermost base pair is always U]A, followed by three consecutive pyrimidine]purine pairs and two obligatory G]C pairs at the base.20 Flanking nucleotides on both sides of the hairpin are also conserved and together with the hairpin itself, constitute the binding site for a hairpin binding processing factor ŽHBF.. The presence, in nuclear extracts, of a protein binding to the hairpin was first indicated by nuclease protection studies.29 Mutational analyses and competition experiments then showed that the hairpin interacts with a titratable processing factor, called hairpin-binding factor, or HBF.30,31 This factor is not absolutely required but contributes to efficient processing.30 ] 32 However, this auxiliary effect is variable, depending on the gene and type of nuclear extract used.33 Further mutational and competition analyses33,34 indicated that HBF acts in the processing reaction by stabilising the interaction of the pre-mRNA with the U7 snRNP Žsee below.. A human cDNA for a protein displaying HBF function and homologues from several other organisms have recently been cloned.35,36 To reflect this more precise characterization, we have proposed to change the name from HBF to hairpin binding protein, HBP. After processing, this protein remains associated with the mature histone mRNA and controls further steps of its metabolism Žsee below.. Immediately juxtaposed to the hairpin are conserved nucleotides surrounding and specifying the processing site. In vertebrates, the consensus sequence for the nucleotides following the hairpin is ACCCA’C Žthe apostrophe indicates the preferred processing site;84 whereas in sea urchins the corresponding sequence is ACCA’AA.25 Mutational analyses indicate that cleavage preferably occurs after A residues37,84 . The preference for the nucleotide 59 to the processing site appears to be A ) C ) U ) G and a G immediately after the processing site is inhibitory.84 Several nucleotides downstream of the processing site, there is a purine-rich spacer element 24 which is complementary to and binds to the 59 end of U7 snRNA present in the U7 snRNP.38 ] 40 This interaction is essential for processing to occur. In vertebrates the purine-richness only applies to the 59 portion of the spacer element and the complementary region is more extensive than in sea urchins, reflecting the greater length of the U7 RNA 59 end ŽFigure 1..34 The U7 snRNP is a minor nucleoplasmic snRNP, present in mammalian cells in 2000]15,000 copiesrnucleus.41 The 59 end of U7 RNA is modified
Histone mRNA 39 end formation and the minor U7 snRNP The 39 ends of replication-dependent histone mRNAs are formed by an endonucleolytic cleavage reaction that is distinct from the normal 39 processing Žpolyadenylation. event involved in forming all other mRNAs Žreviewed in refs 4 and 24.. Histone mRNAs have short 39 untranslated regions of 50]70 nucleotides and the signalŽs. controlling the processing reaction and its regulation are contained within a region of ; 60 nucleotides surrounding the processing site.25 ] 27 The reaction has been studied mainly in two systems, Xenopus laevis oocytes25,26 and nuclear extracts from mammalian cells.28 The 60-nucleotide region can be subdivided into three functional parts ŽFigure 1.. Completely contained within the mature mRNA portion is a highly conserved stretch of ; 26 nucleotides. Within these lies a dyad symmetry element that forms a stable hairpin structure in the RNA. This hairpin is charac568
The U7 snRNP and the hairpin binding protein: key players in histone mRNA metabolism
Figure 1. Aligned sequences of mammalian Žtop. and sea urchin Žbottom. histone pre-mRNAs and U7 snRNAs. For the histone pre-mRNAs Župper sequences., the figure shows the consensus sequences of the hairpin element, the nucleotides surrounding the processing site and the spacer element. The dashed line represents unconserved sequences and reproduces the correct spacing. Relevant distances discussed in the text are indicated and the processing site is represented by a thick downward arrow. For the U7 snRNAs Žlower sequences., only the 59 part is shown, whereas the 39-terminal hairpin has been omitted. The regions complementary to the histone pre-mRNA spacer element and the Sm binding sites are shown in upper case. The positions of the 59-terminal trimethyl guanosine cap structure Ž3mG. and of a cytosine that can be crosslinked to a U7-specific protein46 Župward arrow. are indicated.
with a trimethyl guanosine cap structure and the murine U7 snRNP contains a complete set of Sm proteins as well as U7-specific proteins of 50 and 14 kDa.42 In mammals, U7 snRNA is encoded by a single copy gene 43,44 which is located at chromosomal position 6F3 in mice 45 and 12p13 in humans ŽGenEMBL acc.no. U47924.. The U7 RNAs of sea urchins and vertebrates are 57]62 nucleotides long, not including the cap nucleotide. Between the 59 end complementary to the histone spacer element and an extensive 39-terminal hairpin Žnot shown in Figure 1., all U7 R N A s have a version of the sequence AAr GUUU Gr C UCUAG, which is reminiscent of the conserved Sm binding sites found in other nucleoplasmic snRNAs. In assembly and UV-crosslinking studies in Xenopus oocytes, it could be shown that this sequence indeed interacts with Sm proteins but also with at least one U7-specific protein of 40 kDa, presumably the Xenopus homologue of the mouse 50 kDa U7-specific protein.46 Conversion of the atypical Sm binding site to the consensus AAUUUUUGGAG led to more efficient assembly and nuclear accumulation, indicating that the U7 Sm binding site is suboptimal.41,46 However, the U7 RNA with the consensus
Sm binding site did not form the UV-crosslink with the U7-specific protein and was inactive in histone RNA 39 processing.46 Thus it seems that the special Sm binding site of U7 RNA and perhaps the 40 kDa U7-specific protein are critical for the processing reaction. A similar overlap between the binding sites for Sm proteins and a snRNP-specific protein has recently been described for the spliced leader RNA of nematodes47 but has not been observed in any of the spliceosomal snRNPs. In both mammalian cells 48 and Xenopus oocytes49 the U7 RNA is primarily localised in coiled bodies. Since these nuclear structures are often associated with histone loci, this raises the interesting possibility that the localization to coiled bodies may help to direct the U7 snRNPs to their target histone premRNAs. Intriguingly, the U7 RNA with the consensus Sm binding site is not localized to coiled bodies in Xenopus oocytes and the special Sm binding site of U7 RNA is both necessary and sufficient for correct targeting.50 Depending on the definition of the spacer element, it seems that its position, length and sequence are quite variable among vertebrate histone genes, in 569
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contrast to its virtual invariability in sea urchins. Scharl and Steitz 37 analysed the constraints on the distance between the spacer element and the hairpin in a mouse H3 pre-mRNA. They found that increasing the distance shifted the processing site in the 39 direction by a corresponding number of nucleotides. Processing at the novel sites was always less efficient than in the wild-type configuration. Part of this reduction could be traced back to the lack of an appropriate nucleotide for cleavage Žan adenosine. while the rest was probably due to problems in achieving the proper conformation between the hairpinrHBP and the U7 snRNP. These results led to the proposal of a ‘molecular ruler’ model for the U7 snRNP. In this model, the cleavage site is chosen at a more or less fixed distance upstream from the U7rpre-mRNA hybrid. Interestingly, if one selects a fixed position within this hybrid, rather than either end of it, as the reference point Že.g the centre of the highly conserved hexamer GAGCUG in mammalianrvertebrate histone genes, Figure 1., then the distance to the processing site becomes almost invariable Ž17 or 18 nucleotides.. At this position, cleavage activity then occurs at the most appropriate phosphodiester bond Žalmost always 39 of an adenosine, see above. within a window of maximally four nucleotides.37,84 By introducing corresponding length insertions between the Sm binding site and the base-pairing region of U7 snRNA and testing these in a complementation assay in X. laevis oocytes, Scharl and Steitz 51 could show that length suppression does occur as predicted by the molecular ruler model, but only if the inserted sequences are complementary to those present in the histone pre-mRNA. In other words, besides the length correspondence, the position of the beginning of the U7rpre-mRNA hybrid also appeared to be critical. This was interpreted as a requirement for rigidity in this region of the processing complex. Inspection of vertebrate histone gene sequences in the databases shows that the distance between the beginning of the U7rpre-mRNA hybrid and the cleavage site varies between nine and 11 nucleotides, but is actually 11 nucleotides for most genes ŽFigure 1.. It is interesting to speculate which parts of the processing complex become juxtaposed to the processing site by the molecular ruler effect. If one simply counts the nucleotides in the two RNAs, then the dinucleotide CU within the conserved Sm binding site of U7 RNA comes to lie at this position ŽFigure 1.. On the one hand, the uridine of U7 could
form a base-pair with the adenosine upstream of the cleavage site, whereas the cytidine of U7 could not; note that a guanosine following the cleavage site is inhibitory 84 . On the other hand, the UV crosslink to the 40-kDa U7-specific protein was mapped precisely to this C residue,46 making this protein an excellent candidate for the cleavage activity. In this context it is interesting that a chimeric RNA consisting of an upstream histone pre-mRNA and a downstream U7 RNA moiety, when injected into Xenopus oocytes, assembles with proteins similarly to a single U7 RNA. However, the chimeric RNP remains cytoplasmic and yet the RNA is cleaved at the right position in a cis-catalytic fashion.52 Thus it is possible that all enzymatic components for the cleavage reaction are contained within the U7 snRNP. Two other activities involved in histone RNA 39 processing have been identified. The first is a heatlabile processing factor ŽHLF..53 This factor is inactivated by mild heat-treatment Ž15 min at 45]508C. whereas U7 snRNPs capable of complementing a U7-depleted extract withstand temperatures up to 658C. However it has to be noted that HBP 54 and a 59]39 exonuclease ŽWalther et al, unpublished; see below. are also inactivated at 508C. At least in certain cellular systems, changes in the HLF appear to be responsible for the cell cycle-dependent regulation of histone RNA 39 processing.55,56 Attempts at purifying HLF revealed an extensive co-fractionation with U7 snRNPs and the total cleavage activity.53 On gel filtration columns, the HLF could be separated into two peaks of apparent molecular mass 300 and 40 kDa. The larger peak might represent a U7 snRNPassociated form whereas the 40-kDa peak could be the free protein. If this is the case, then the 50r40 kDa U7-specific polypeptide is an obvious candidate for the HLF. The free form could either reflect an excess of the protein or a tendency of it to dissociate from U7 snRNPs. Another activity has recently been identified in attempts to study the generation of 39 fragments during histone RNA 39 processing ŽWalther et al, unpublished.. These fragments are only observed in processing reactions carried out in the presence of EDTA and they are usually shorter than expected, i.e. their Žheterogeneous. 59 ends lie several nucleotides downstream of the end of the 59 fragment.28,33 The new experiments suggest that these shortened fragments are generated by a 59]39 exonuclease after the initial endonucleolytic cleavage has occurred ŽWalther et al, unpublished.. This activity could be important in recycling U7 snRNPs for additional rounds of cleavage. 570
The U7 snRNP and the hairpin binding protein: key players in histone mRNA metabolism
Structural and functional features of hairpinbinding proteins
recombinant human protein is able to participate in histone RNA 39 end processing.36 To signify that this protein is, or is part of the HBF as originally defined by Birnstiel et al,30 we are calling it hairpin-binding protein ŽHBP. whereas Marzluff et al use the name SLBP. Despite this difference, both laboratories agree that this protein is the common RNA binding component of HBF and SLBP. The human hbp gene is located on chromosome 4p16.3 and composed of eight exons covering 19.5 kb.36,63 An alternative splicing event causes exon 3 to be skipped in a small fraction of HBP mRNAs in both human and mouse cells. The resulting in-frame fusion deletes 35 amino acids but this does not interfere with the binding to hairpin RNA ŽEglite et al, unpublished.. The function of this alternative splicing event is not clear. The C. elegans gene contains only five exons and covers ; 1.8 kb on chromosome 2.36 Figure 2 summarizes an alignment of the amino acid sequences of the human, mouse, X. laevis and C. elegans HBPs. The proteins contain 270, 275, 254 and 367 amino acids, respectively. However, all the proteins migrate with abnormal mobility during denaturing gel electrophoresis; for example the human protein, with a predicted molecular mass of ; 31 kDa, migrates like a ; 43 kDa protein. The human and mouse proteins are nearly identical with 89% amino acid identity. Human and X. laevis proteins are to 62% identical, and human and C. elegans proteins are 38% identical. The human, mouse and X. laevis proteins contain several conserved blocks of seven amino acids or more Žshown as grey boxes in Figure 2. distributed throughout the proteins. The only region where all four proteins have significant homology is a 62-amino acid stretch Žshown in black.. In this sequence Žalso shown in Figure 2, bottom., the human, mouse and X. laevis proteins are to 82% identical and human and C. elegans proteins are 55% identical. This region is both necessary and sufficient for RNA binding 35 and both human and C. elegans polypeptides with the sequences shown in Figure 2 are able to bind specifically to wild-type hairpin RNA ŽMichel et al, unpublished.. The sequence of this RNA binding domain ŽRBD. is not related to any of the known RNA-binding motifs and computer structure predictions based on all four protein sequences propose that the RBD contains two a-helices Žindicated in Figure 2.. In the C. elegans hairpin element, a cytosine replaces the first uracil in the loop Žsee Figure 1.. Interestingly, the human and C. elegans RBDs show
As described above, the 39-terminal hairpin and a hairpin-binding processing factor play an important role in histone RNA 39 processing. Here we will discuss the characterization and cDNA cloning of hairpin-binding proteins. The evidence indicating that the hairpin structure is involved in additional and, notably, cytoplasmic steps of histone mRNA metabolism will be reviewed further below. Activities binding to the histone RNA hairpin have been detected in different extracts from mammalian cell lines and calf thymus by a variety of biochemical and functional assays.29,30,54,57,58 The initially described nuclear activity implicated in histone RNA 39 processing was termed hairpin binding factor ŽHBF..29,30,54 Marzluff et al then described nuclear and polysomal activities from a mouse cell line that bound hairpin RNA in a structure- and sequencedependent manner 57,59 and designated them as stem]loop binding proteins ŽSLBP.. A ; 45 kDa protein was enriched from polysomes and identified by Northwestern blotting as the RNA-binding component.60 Since the SLBP prepared from polysomes could substitute for the nuclear HBF in in vitro processing reactions,61 it appeared that these activities are related, if not identical. We have described a similar RNA-binding activity from calf thymus and by biochemical fractionation of whole cell extract, have obtained highly enriched fractions capable of both binding to hairpin RNA and substituting HBF in histone RNA 39 processing.58 The most enriched fraction, although not active in processing for technical reasons, contained two proteins of 40 and 43 kDa. Together with the finding that complexes of hairpin RNA formed in vitro with either SLBP 59 or HBF ŽSchaller, A. and B.M., unpublished. have half-lives in excess of 4]6 h, these results suggest that one and the same protein participates in histone RNA processing and then remains associated with the mature mRNA 39 end until it reaches its cytoplasmic Žpolysomal. destination. Using the yeast three hybrid system designed for the characterization of RNA-binding proteins,62 human and Xenopus laevis cDNAs encoding proteins that bind to the histone RNA hairpin were isolated.35,36 In addition, a homologous mouse cDNA was isolated by hybridization screening 35 and a Caenorhabditis elegans homologue was identified by data base search and subsequent functional characterisation.36 Complementation assays showed that the 571
B. Muller ¨ and D. Schumperli ¨
Figure 2. Alignment of human, mouse, Xenopus laevis and Caenorhabditis elegans HBP sequences. Top: the proteins are shown as bars and are not drawn to scale. Shaded areas and the sequences above them represent blocks of seven or more amino acids that are virtually identical between the three vertebrate HBPs. At divergent positions within these blocks the residue present in the two mammalian HBPs and the one present in the Xenopus HBP are separated by a slash Žr.. A dot Ž.. indicates a missing amino acid. Black areas are conserved between all four proteins and encompass the RNA-binding domain. Bottom: sequences of human and C. elegans polypeptides binding specifically to hairpin RNA. The consensus sequence shows only those amino acids that are identical in three of the four proteins. In the human and C. elegans sequences, the amino acids diverging from the consensus are shown in lower case. Two potential a-helical regions were predicted by a computer algorithm. Highlighted T and S represent potential phosphorylation sites.
preferential binding to their conspecific hairpin RNAs, indicating that the differences in amino acid sequence of the RBDs confer different binding preferences ŽMichel et al, unpublished.. However, it is possible that other parts of the protein outside of the RBD also contribute to the specificity of the protein]RNA interaction. Interestingly, some of the most conserved sequence elements of the known HBPs are potential phosphorylation sites. In Figure 2, the sequence element TPNK at the N-terminal border of the second proposed a-helix is a potential site for threonine-phosphorylation by Cdc2 kinase, the first serine in the sequence SRRS in the same second a-helix is a potential target for phosphorylation by cGMP-dependent kinase or protein kinase C and the second serine in the same sequence could be phosphorylated
by either cGMP-dependent or cAMP-dependent kinase.64 The fact that all these sequences are found in the RBD raises the interesting possibility that HBP function might be regulated by phosphorylation of residues in this domain.
Involvement of the 39-terminal hairpin in steps subsequent to histone RNA 39 processing The conserved hairpin at the 39 ends of animal replication-dependent histone mRNAs is involved in virtually all processes and regulatory events that occur after the mature mRNA has been generated by RNA 39 processing Žreviewed in ref 20.. The fact that at least the RNA-binding component of the nuclear
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The U7 snRNP and the hairpin binding protein: key players in histone mRNA metabolism
HBFrHBP and the polysomal SLBP are identical suggests that all these functions of the histone mRNA 39 end are mediated by this protein. Here we will briefly review the evidence linking the hairpin to these post-transcriptional events and thereby concentrate on newer findings. Initial evidence for the involvement of the histone hairpin in nucleo-cytoplasmic transport of mRNAs was obtained by animal cell transfections of DNA constructs with a cis-acting hammerhead ribozyme that allowed the production of virtually normal histone mRNA 39 ends independently of the 39 processing signal Žof which the hairpin is an important part..65 Mutations in the hairpin led to complete nuclear retention of the cleaved RNAs, whereas an RNA with the wild-type hairpin was exported. However, in some cases, the ribozyme-generated RNAs with wild-type hairpin were also partly or completely export-deficient, depending on the sequences present upstream in the RNA or the cell line used. These latter results suggest that histone mRNA export might be directly coupled to 39 processing. Similarly, a histone RNA with the first and third uridines in the loop changed to adenosines was processed, albeit inefficiently, but was severely impaired in nucleocytoplasmic transport, both in animal cells and in Xenopus oocytes.66 Since the same mutation causes a 20]50-fold reduction in affinity to HBP, this provided circumstantial evidence for an involvement of HBP in nucleo-cytoplasmic transport. Similarly to the polyŽA. tail on other mRNAs, the hairpin is also essential for recruiting histone mRNA to polysomes.66,67 In a recent study using electroporation of luciferase reporter transcripts into tissue culture cells, the hairpin was directly shown to be required and co-dependent on a 59 cap structure for translation in mammalian cells.68 However, only a polyŽA. tail but not the histone hairpin was able to stimulate translation in electroporated plant protoplasts. During S phase in mammalian cells, histone mRNAs have a half-life of 40]60 min19,23 and histone mRNA degradation starts at the 39 end.69 Inhibition of DNA synthesis leads to a rapid destabilisation of histone mRNA, probably by a process similar to the destabilisation of histone mRNA upon exit from S phase.23 Inversely, histone mRNA is stabilised by the addition of cycloheximide to the cells. These stability changes are dependent on the presence of a wild-type hairpin element at the RNA 39 end and can be conferred onto non-histone RNAs by adding the 39terminal processing site with the hairpin.66,70 Inter-
estingly, the destabilisation requires that the mRNA be translated and it is crucial that the distance between the stop codon and the hairpin is shorter than ; 300 nucleotides.71 Two non-exclusive models have been proposed to explain how this degradation of histone mRNAs may be triggered: Ž1. a regulatory signal, possibly an unusual nucleotide, might coordinately regulate DNA replication, deoxynucleotide metabolism and histone mRNA levels;72 and Ž2. excess histone proteins might regulate their own biosynthesis.73 The addition of histone proteins, either mixed or individually, to an in vitro histone RNA decay system consisting of polysomes, as the source of mRNA and the RNA-degrading enzymeŽs., and a post-polysomal supernatant ŽS130. destabilises histone mRNA,74 ] 76 lending support to the autoregulation model. This in vitro decay reaction starts at the 39 end and proceeds from 39 to 59,74 like the decay reaction occurring in vivo.69 A 39-59 exoribonuclease of 33 kDa with the required specificity has been purified.77 The formation of a characteristic intermediate ending within the loop suggests that one of the early steps in the decay reaction is the removal of HBP from the mRNA, and it will be interesting to know what the role of HBP in this decay reaction is. Fractionation of the S130 fraction demonstrated the presence of an as yet undefined histone-activatable destabiliser, and a stabiliser identified as the La protein,78 a RNA-binding protein found associated with different RNAs. Addition of La protein leads to the accumulation of the intermediate ending in the loop. This intermediate has a string of uridines at the 39 end. Although the molecular mechanism of the stabilisation is not clear, it is interesting that La protein is also found associated with U-rich termini of polymerase III transcripts79 and it is tempting to speculate that a similar association of La protein with the decay intermediate might protect histone mRNA from further exonucleolytic degradation. In summary, the histone hairpin element is important for correct histone gene expression and is involved in all the post-transcriptional regulatory events described so far. Mutations in the hairpin element reducing histone gene expression, RNA export and mRNA stability also reduce the affinity for HBP,59,66,80 indirectly implicating HBP in all the steps affected by these mutations, i.e. nuclear export, translation and mRNA stability. However, direct evidence exists only for the participation of the human HBP in mRNA 39 end formation. 573
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Table 1. Comparison of factors controlling the metabolism of histone versus polyadenylated mRNAs
Upstream sequence element ŽUSE. Downstream sequence element ŽDSE. Specificity factor Žfor cleavage. Auxiliary factor Žfor cleavage.U Auxiliary factor Žfor polyadenylation.U Export factor† Translation factor† Stabilisation factor‡
Histone mRNAs
Polyadenylated mRNAs
Hairpin Spacer element U7 snRNP Žbinds DSE. HBP Žbinds USE. } HBP Ž? . HBP Ž? . HBP Ž? .
AAUAAA GU-rich sequence CPSF Žbinds USE. CStF Žbinds DSE. PABP2 Žbinds growing polyŽA. tail. PABP2 or PABP1 Ž? . PABP1 PABP1
U
Cleavage of polyadenylated mRNAs additionally requires cleavage factors I and II. PolyŽA. polymerase is required for both the cleavage and polyadenylation steps.81 † Export and translation of both types of mRNAs Žpresumably. requires a 59 cap structure, the nuclear cap binding complex and the cytoplasmic cap binding factor eIF-4FrG.82,83 ‡ For both types of mRNAs a connection between mRNA degradation and translation has been demonstrated.71,82
Factors controlling the metabolism of histone and polyadenylated mRNAs
downstream fragment. In histone RNAs, the auxiliary processing factor HBP later becomes the molecular escort of the mature mRNA and therefore has to be associated with the 59 fragment whereas the U7 snRNP is presumably not needed for further modifications of this particular RNA and hence is best attached to the 39 fragment destined for degradation. After processing Žand polyadenylation. have occurred, it appears that HBP fulfills all the functions proven or ascribed to the nuclear and cytoplasmic polyŽA. binding proteins, PABP1 and PABP2.81,82
Similarities and differences between the metabolic processes controlling the replication-dependent animal histone mRNAs as opposed to all other, polyadenylated mRNAs have been discussed previously.6,24,37 Among the similaritites between the cleavage reactions are the lack of a requirement for cleavable ATP or divalent cations, the preference for cleaving downstream of an adenosine and the types of ends generated Ž39-OH and 59-phosphate.. In contrast, no shared trans-acting factors have been identified so far. Both types of mRNA 39 processing reactions require upstream and downstream sequence elements ŽTable 1; for a review on polyadenylation see ref 81.. A main difference is that the specificity factor binds upstream of the cleavage site in polyadenylation ŽCPSF binding to the AAUAAA sequence. and downstream in histone RNA 39 processing Žthe U7 snRNP binding to the spacer element.; the respective auxiliary factors ŽCstF and HBP. stabilise the processing complex by binding on the other side of the processing site. This difference in the arrangement of specificity and auxiliary factors is most likely related to the different metabolic pathways the cleaved RNAs engage in. In polyadenylation, the specificity factor CPSF, together with polyŽA. polymerase and PABP2, is required for the coupled step of polyŽA. addition; thus CPSF has to be associated with the upstream cleavage fragment, whereas CStF which is not required in the polyŽA. addition step is attached to the
Acknowledgements Work in our laboratory was supported by the Kanton Bern and by the Swiss National Science Foundation.
References 1. Allen BS, Stein JL, Stein GS, Osterer H Ž1991. Single-copy flanking sequences in human histone gene clusters map to chromosomes 1 and 6. Genomics 10:486]488 2. Wang ZF, Krasikov T, Frey MR, Wang J, Matera AG, Marzluff WF Ž1996. Characterization of the mouse histone gene cluster on chromosome 13: 45 histone genes in three patches spread over 1 Mb. Genome Res 6:688]701 3. Wang ZF, Tisovec R, DeBry RW, Frey MR, Matera AG, Marzluff WF Ž1996. Characterization of the 55-kb mouse histone gene cluster on chromosome 3. Genome Res 6:702]714 4. Birnstiel ML, Schaufele FJ Ž1988. Structure and function of minor snRNPs, in Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles ŽBirnstiel ML, ed. pp 155]182. Springer Verlag, New York 5. Osley MA Ž1991. The regulation of histone synthesis in the cell cycle. Annu Rev Biochem 60:827]861 6. Wittop Koning TH, Schumperli D Ž1994. RNAs and ribonu¨
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7. 8. 9.
10.
11. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22.
23.
24. 25.
26.
27.
28. Gick O, Kramer A, Keller W, Birnstiel ML Ž1986. Generation ¨ of histone mRNA 39 ends by nucleolytic cleavage of the pre-mRNA in a snRNP-dependent in vitro reaction. EMBO J 5:1319]1326 29. Mowry KL, Steitz JA Ž1987. Both conserved signals on mammalian histone pre-mRNAs associate with small nuclear ribonucleoproteins during 39 end formation in vitro. Mol Cell Biol 7:1663]1672 30. Vasserot AP, Schaufele FJ, Birnstiel ML Ž1989. Conserved terminal hairpin sequences of histone mRNA precursors are not involved in duplex formation with the U7 RNA but act as a target site for a distinct processing factor. Proc Natl Acad Sci USA 86:4345]4349 31. Mowry KL, OH R, Steitz JA Ž1989. Each of the conserved sequence elements flanking the cleavage site of mammalian histone pre-mRNAs has a distinct role in the 39 end processing reaction. Mol Cell Biol 9:3105]3108 32. Cotten M, Gick O, Vasserot A, Schaffner G, Birnstiel ML Ž1988. Specific contacts between mammalian U7 snRNA and histone precursor RNA are indispensable for the in vitro 39 RNA processing reaction. EMBO J 7:801]808 33. Streit A, Wittop Koning T, Soldati D, Melin L, Schumperli D ¨ Ž1993. Variable effect of the conserved RNA hairpin element upon 39 end processing of histone pre-mRNA in vitro. Nucleic Acids Res 21:1569]1575 34. Spycher C, Streit A, Stefanovic B, Albrecht D, Wittop Koning TH, Schumperli D Ž1994. 39 end processing of mouse histone ¨ pre-mRNA: Evidence for additional base-pairing between U7 snRNA and pre-mRNA. Nucleic Acids Res 22:4023]4030 35. Wang ZF, Whitfield ML, Ingledue TC, III, Dominski Z, Marzluff WF Ž1996. The protein that binds the 39 end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing. Genes Dev 10:3028]3040 36. Martin F, Schaller A, Eglite S, Schumperli D, Muller B Ž1997. ¨ ¨ The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein. EMBO J 16:769]778 37. Scharl EC, Steitz JA Ž1994. The site of 39 end formation of histone messenger RNA is a fixed distance from the downstream element recognized by the U7 snRNP. EMBO J 13:2432]2440 38. Strub K, Galli G, Busslinger M, Birnstiel ML Ž1984. The cDNA sequences of the sea urchin U7 small nuclear RNA suggest specific contacts between histone mRNA precursors and U7 RNA during RNA processing. EMBO J 3:2801]2807 39. Schaufele F, Gilmartin GM, Bannwarth W, Birnstiel ML Ž1986. Compensatory mutations suggest that base-pairing with a small nuclear RNA is required to form the 39 end of H3 messenger RNA. Nature 323:777]781 40. Bond U, Yario TA, Steitz JA Ž1991. Multiple processing defective mutations in a mammalian histone messenger RNA are suppressed by compensatory changes in U7 RNA both in vivo and in vitro. Genes Dev 5:1709]1722 41. Grimm C, Stefanovic B, Schumperli D Ž1993. The low abun¨ dance of U7 snRNA is partly determined by its Sm binding site. EMBO J 12:1229]1238 42. Smith HO, Tabiti K, Schaffner G, Soldati D, Albrecht U, Birnstiel ML Ž1991. Two-step purification of U7 small nuclear robonucleoprotein particles using complementary biotinylated 29-O-methyl oligoribonucleotides. Proc Natl Acad Sci USA 88:9784]9788 43. Gruber A, Soldati D, Burri M, Schumperli D Ž1991. Isolation ¨ of an active gene and of two pseudogenes for mouse U7 small nuclear RNA. Biochim Biophys Acta 1088:151]154 44. Philips SC, Turner PC Ž1991. Nucleotide sequence of the mouse U7 snRNA gene. Nucleic Acids Res 19:1344 45. Turner PC, Whalen A, Schumperli D, Matera AG Ž1996. The ¨ bona fide mouse U7 snRNA gene maps to a different chromosome than two U7 pseudogenes. Genomics 31:250]252
cleoproteins in recognition and catalysis. Eur J Biochem 219:25]42 Muller K, Schmitt R Ž1988. Histone genes of Volvox carteri: ¨ DNA sequence and organization of two H3-H4 loci. Nucleic Acids Res 16:4121]4136 Walther Z, Hall JL Ž1995. The uni chromosome of Chlamydomonas: histone genes and nucleosome structure. Nucleic Acids Res 23:3756]3763 Fabry S, Muller K, Lindauer A, Park PB, Cornelius T, Schmitt ¨ R Ž1995. The organization, structure and regulatory elements of Chlamydomonas histone genes reveal features linking plant and animal genes. Curr Genet 28:333]345 Wyler-Duda P, Bernard V, Stadler M, Suter D, Schumperli D ¨ Ž1997. Histone H4 mRNA from the nematode Ascaris lumbricoides is cis-spliced and polyadenylated. Biochim Biophys Acta 1350:259]261 Woodland HR Ž1980. Histone synthesis during the development of Xenopus. FEBS Lett 121:1]7 Rudell A, Jacobs-Lorena M Ž1985. Biphasic pattern of histone gene expression during Drosophila oogenesis. Proc Natl Acad Sci USA 82:3316]3319 Busslinger M, Schumperli D, Birnstiel ML Ž1985. Regulation ¨ of histone gene expression. Cold Spring Harbor Symp Quant Biol 50:665]670 Hake LE, Richter JD Ž1997. Translational regulation of maternal mRNA. Biochim Biophys Acta 1332:M31]M38 Challoner P, B., Moss SB, Groudine M Ž1989. Expression of replication-dependent histone genes in avian spermatids involves an alternate pathway of mRNA 39-end formation. Mol Cell Biol 9:902]913 Moss SB, Challoner PB, Groudine M Ž1989. Expression of a novel histone 2B during mouse spermiogenesis. Dev Biol 133:83]92 Wells D, Brown D Ž1991. Histone and histone gene compilation and alignment update. Nucleic Acids Res 19:2173]2188 Wolffe AP, Pruss D Ž1996. Deviant nucleosomes: the functional specialization of chromatin. Trends Genet 12:58]62 Schumperli D Ž1988. Multilevel regulation of replication¨ dependent histone genes. Trends Genet 4:187]191 Marzluff WF Ž1992. Histone 39ends: essential and regulatory functions. Gene Expr 2:93]97 Heintz N Ž1991. The regulation of histone gene expression during the cell cycle. Biochim Biophys Acta 1088:327]339 Stein GS, Stein JL, Van Wijnen AJ, Lian JB Ž1994. Histone gene transcription: a model for responsiveness to an integrated series of regulatory signals mediating cell cycle control and proliferationrdifferentiation interrelationships. J Cell Biochem 54:393]404 Harris ME, Bohni R, Schneiderman MH, Ramamurthy L, ¨ Schumperli D, Marzluff WF Ž1991. Regulation of histone ¨ mRNA in the unperturbed cell cycle: evidence suggesting control at two posttranscriptional steps. Mol Cell Biol 11:2416]2424 Birnstiel ML, Busslinger M, Strub K Ž1985. Transcription termination and 39 processing: the end is in site! Cell 41:349]359 Birchmeier C, Grosschedl R, Birnstiel ML Ž1982. Generation of authentic 39 termini of an H2A mRNA in vivo is dependent on a short inverted DNA repeat and on a spacer sequence. Cell 26:739]745 Birchmeier C, Schumperli D, Sconzo D, Birnstiel ML Ž1984. ¨ 39 editing of mRNAs: sequence requirements and involvement of a 60 nucleotide RNA in maturation of histone mRNA precursors. Proc Natl Acad Sci USA 81:1057]1061 Stauber C, Luscher B, Eckner R, Lotscher E, Schumperli D ¨ ¨ ¨ Ž1986. A signal regulating mouse histone H4 mRNA levels in a mammalian cell cycle mutant and sequences controlling RNA 39 processing are both contained within the same 80]bp fragment. EMBO J 5:3297]3303
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46. Stefanovic B, Hackl W, Luhrmann R, Schumperli D Ž1995. ¨ ¨ Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes. Nucleic Acids Res 23:3141]3151 47. Denker JA, Maroney PA, Yu Y, Kanost A, Nilsen TW Ž1996. Multiple requirements for nematode spliced leader RNP function in trans-splicing. RNA 2:746]755 48. Frey MR, Matera AG Ž1995. Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells. Proc Natl Acad Sci USA 92:5915]5919 49. Wu CH, Gall JG Ž1993. U7 small nuclear RNA in C snurpososmes of the Xenopus germinal vesicle. Proc Natl Acad Sci USA 90:6257]6259 50. Wu CH, Murphy C, Gall JG Ž1996. The Sm binding site targets U7 snRNA to coiled bodies Žspheres. of amphibian oocytes. RNA 2:811]823 51. Scharl EC, Steitz JA Ž1996. Length suppression in histone messenger RNA 39-end maturation: processing defects of insertion mutant premessenger RNAs can be compensated by insertions into the U7 small nuclear RNA. Proc Natl Acad Sci USA 93:14659]14664 52. Stefanovic B, Wittop Koning TH, Schumperli D Ž1995. A ¨ synthetic histone pre-mRNA-U7 small nuclear RNA chimera undergoing cis cleavage in the cytoplasm of Xenopus oocytes. Nucleic Acids Res 23:3152]3160 53. Gick O, Kraemer A, Vasserot A, Birnstiel ML Ž1987. Heat-labile regulatory factor is required for 39 processing of histone precursor mRNAs. Proc Natl Acad Sci USA 84:8937]8940 54. Melin L, Soldati D, Mital R, Streit A, Schumperli D Ž1992. ¨ Biochemical demonstration of complex formation of histone pre-mRNA with U7 small nuclear ribonucleoprotein and hairpin binding factors. EMBO J 11:691]697 55. Luscher B, Schumperli D Ž1987. RNA 39 processing regulates ¨ ¨ histone mRNA levels in a mammalian cell cycle mutant. A processing factor becomes limiting in G1]arrested cells. EMBO J 6:1721]1726 56. Stauber C, Schumperli D Ž1988. 39 processing of pre-mRNAs ¨ plays a major role in proliferation-dependent regulation of histone gene expression. Nucleic Acids Res 16:9366]9414 57. Pandey NB, Sun J, Marzluff WF Ž1991. Different complexes are formed on the 39 end of histone mRNA with nuclear and polyribosomal proteins. Nucleic Acids Res 19:5653]5659 58. Schaller A, Martin F, Muller B Ž1997. Characterization of the ¨ calf thymus hairpin-binding factor involved in histone premRNA 39 end processing. J Biol Chem 272:10435]10441 59. Williams AS, Marzluff WF Ž1995. The sequence of the stem and flanking sequences at the 39 end of histone mRNA are critical determinants for the binding of the stem-loop binding protein. Nucleic Acids Res 23:654]662 60. Hanson RJ, Sun JH, Willis DG, Marzluff WF Ž1996. Efficient extraction and partial purification of the polyribosome-associated stem-loop binding protein bound to the 39 end of histone mRNA. Biochemistry 35:2146]2156 61. Dominski Z, Sumerel J, Hanson R, Marzluff W Ž1996. The polyribosomal protein bound to the 39 end of histone mRNA can function in histone pre- mRNA processing. RNA 1:915]923 62. Sengupta DJ, Zhang BL, Kraemer B, Pochart P, Fields S, Wickens M Ž1996. A three-hybrid system to detect RNA-protein interactions in vivo. Proc Natl Acad Sci USA 93:8496]8501 63. McCombie WR, Martin-Gallardo A, Gocayne JD, Fitzgerald M, Dubnick M, Kelley JM, Castilla L, Wallace S Ž1992. Expressed genes, Alu repeats and polymorphisms in cosmids
64. 65. 66.
67. 68. 69. 70.
71. 72. 73. 74. 75. 76. 77. 78. 79. 80.
81. 82. 83. 84.
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sequenced from chromosome 4p16.3. Nature Genet 1:348]353 Pearson RB, Kemp BE Ž1991. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Meth Enzymol 200:62]81 Eckner R, Ellmeier W, Birnstiel ML Ž1991. Mature mRNA 39 end formation stimulates RNA export from the nucleus. EMBO J 10:3513]3522 Williams AS, Ingledue TC, III, Kay BK, Marzluff WF Ž1994. Changes in the stem-loop at the 39 terminus of histone mRNA affects its nucleocytoplasmic transport and cytoplasmic regulation. Nucleic Acids Res 22:4660]4666 Sun J, Pilch DR, Marzluff WF Ž1992. The histone mRNA 39 end is required for localisation of histone mRNA to polyribosomes. Nucleic Acids Res 20:6057]6066 Gallie DR, Lewis NJ, Marzluff WF Ž1996. The histone 39terminal stem-loop is necessary for translation in Chinese hamster ovary cells. Nucleic Acids Res 24:1954]1962 Ross J, Peltz SW, Kobs G, Brewer G Ž1986. Histone mRNA degradation in vivo: the first detectable step occurs at or near the 39 terminus. Mol Cell Biol 6:4362]4371 Pandey NB, Marzluff WF Ž1987. The stem-loop structure at the 39 end of histone pre-mRNA is necessary and sufficient for regulation of histone mRNA stability. Mol Cell Biol 7:4557]4559 Graves RA, Pandey NB, Chodchoy N, Marzluff W Ž1987. Translation is required for regulation of histone mRNA degradation. Cell 48:615]626 Graves RA, Marzluff WF Ž1984. Rapid reversible changes in the rate of histone gene transcription and histone mRNA levels in mouse myeloma cells. Mol Cell Biol 4:351]357 Sariban E, Wu RS, Erickson LC, Bonner WM Ž1985. Interrelationships of protein and DNA syntheses during replication of mammalian cells. Mol Cell Biol 5:1279]1286 Ross J, Kobs G Ž1986. H4 histone messenger RNA decay in cell-free extracts initiates at or near the 39 terminus and proceeds 39 to 59. J Mol Biol 188:579]593 Peltz SW, Ross J Ž1987. Autogenous regulation of histone mRNA decay by histone proteins in a cell free system. Mol Cell Biol 7:4345]4356 McLaren RS, Ross J Ž1993. Individual purified core and linker histones induce histone H4 mRNA destabilisation in vitro. J Biol Chem 268:14637]14644 Caruccio N, Ross J Ž1994. Purification of a human polyribosome-associated 39 to 59 exoribonuclease. J Biol Chem 269:31814]31821 McLaren RS, Caruccio N, Ross J Ž1997. Human La protein: a stabilizer of histone mRNA. Mol Cell Biol 17:3028]3036 vanVenrooji WJ, Slobbe RL, Prujin GJM Ž1993. Structure and function of La and Ro RNPs. Mol Biol Rep 18:113]119 Pandey NB, Williams AS, Sun J-H, Brown VD, Bond U, Marzluff WF Ž1994. Point mutations in the stem-loop at the 39 end of mouse histone mRNA reduce expression by reducing the efficiency of 39 end formation. Mol Cell Biol 14:1709]1720 Wahle E, Keller W Ž1996. The biochemistry of polyadenylation. Trends Biochem Sci 21:247]250 Sachs A, Wahle E Ž1993. PolyŽA. tail metabolism and function in eucaryotes. J Biol Chem 268:22955]22958 Izaurralde E, Mattaj IW Ž1995. RNA export. Cell 81:153]159 Furger A, Schaller A, Schumperli D Ž1998. Functional impor¨ tance of conserved nucleotides at the histone RNA 39 processing site. RNA 4: in press
The U7 snRNP and the hairpin binding protein: Key players in histone mRNA metabolism Berndt Muller ¨ and Daniel Schumperli ¨ Animal replication-dependent histone mRNAs are subject to several post-transcriptional regulatory processes. Their nonpolyadenylated 39 ends are formed preferentially during S phase by a unique nuclear cleavage event. This requires the base pairing between U7 snRNA and a histone spacer element 39 of the cleavage site. Cleavage occurs preferentially after adenosine, at a fixed distance from the hybrid region. A conserved RNA hairpin just upstream of the cleavage site is recognised by the hairpin binding protein (HBP) that acts as an auxiliary processing factor, stabilising the interaction of the histone pre-mRNA with the U7 snRNP. The interaction between HBP and the RNA hairpin is very stable and HBP is also found associated with histone mRNAs on polysomes. The hairpin and presumably, HBP are also required for nuclear export and translation of histone mRNA. Furthermore, histone mRNAs are selectively destabilised in the G2 phase or upon inhibition of DNA synthesis and this regulation is also associated with the hairpin. Recently, HBP-encoding cDNAs were isolated from various organisms. Human, mouse and Xenopus laevis HBPs are similar, while the Caenorhabditis elegans protein has significant homology to the others only in a central RNA binding domain.
five to 20 copies of these genes for each histone subtype, concentrated in two clusters on chromosomes 1 and 6 in humans1 and chromosomes 3 and 13 in mice.2,3 The main distinctive features of these genes are their lack of introns and the fact that their mRNAs are not polyadenylated but produced by a unique RNA 39 processing reaction Žreviewed in ref 4; see below.. Histone genes with these characteristics are common to all metazoans, whereas other organisms such as fungi and plants have histone mRNAs with polyŽA. tails and frequently, introns.5,6 However, there are notable exceptions. The flagellate algae Volvox 7 and Chlamydomonas 8,9 have histone genes similar to the metazoan type. In contrast, a histone H4 gene from the nematode Ascaris lumbricoides Žpossibly the only H4 gene of this organism. has introns and encodes a polyadenylated mRNA.10 Despite their name, the replication-dependence of these genes is far from absolute. First, the relationship between DNA synthesis and histone gene expression has not been analysed in many organisms. Then there are cases where histone mRNAs are maternally expressed and stored in oocytes Že.g. Xenopus, Drosophila, sea urchins..11 ] 13 In Xenopus these translationally inactive maternal histone mRNAs have short polyŽA. tails added to the normal 39 ends,11 whereas for all other maternally stored mRNAs the inactive forms lack polyŽA. and the actively translated forms are polyadenylated.14 In mammals and birds some of the genes of the replication-dependent type are also expressed during spermatogenesis and this expression is not tightly coupled to DNA synthesis.15,16 Finally, sea urchins have several sets of histone genes of the replication-dependent type which are expressed in a partly overlapping fashion at different developmental stages or in different tissues.13 In addition, most metazoans and certainly all vertebrates have histone genes with the standard organization for protein coding genes Žintrons, polyŽA. tails..17 These are either constitutively expressed such as the H1.0, H2A.Z and H3.3 genes or strictly cell typespecific as the H5 genes of animals with nucleated erythrocytes. The products of the former class are called basal or replacement histones since they grad-
Key words: histone r RNA 39 processing r U7 small nuclear ribonucleoprotein r hairpin binding protein r stem-loop binding protein r RNA]protein interaction Q1997 Academic Press Ltd
Introduction: The replication-dependent histone genes IN ANIMAL CELLS, the packaging of DNA into chromatin during DNA replication is maintained by S phase- specific expression of the so- called replication-dependent histone genes. Mammals have U
From the Abteilung fur ¨ Entwicklungsbiologie, Zoologisches Institut der Universitat ´ Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Q1997 Academic Press Ltd 1084-9521r 97r 060567q 10 $25.00r 0r sr970182
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ually replace the products of the replication-dependent genes in the chromatin of non-proliferating and terminally differentiated cells. In recent years, several additional histone-like proteins Žand genes. have been described which share with the ‘real’ histones a common structural motif called the histone fold.18 These proteins are thought to play important roles in regional chromosome and chromatin structure and in the modulation of gene expression. The cell cycle-dependent production of replication-dependent histone proteins, at least in mammalian cells, is entirely caused by a modulation of mRNA levels Žreviewed in refs 5, 19 and 20.. This, in turn is achieved by a combination of three regulatory events. Transcription5,21,22 and histone RNA 39 processing19 are both activated late in G1 and turned off at the end of the S phase. Together, these two events can cause a 30 to 50-fold modulation of histone mRNA production.23 Furthermore, at the end of the S phase, the half-life of histone mRNAs drops from 40]60 to 5]12 min, resulting in a rapid elimination of all remaining histone mRNAs from the cell.23 Partly identical 39-terminal sequences are involved in controlling and regulating the 39 end processing and degradationrstability of histone mRNAs as well as other steps of histone mRNA metabolism. In this review, we will concentrate on the events leading to the formation of the mature mRNA 39 ends and the factors influencing mRNA stability.
terised by a four base uridine-rich loop and a six base pair stem. The uppermost base pair is always U]A, followed by three consecutive pyrimidine]purine pairs and two obligatory G]C pairs at the base.20 Flanking nucleotides on both sides of the hairpin are also conserved and together with the hairpin itself, constitute the binding site for a hairpin binding processing factor ŽHBF.. The presence, in nuclear extracts, of a protein binding to the hairpin was first indicated by nuclease protection studies.29 Mutational analyses and competition experiments then showed that the hairpin interacts with a titratable processing factor, called hairpin-binding factor, or HBF.30,31 This factor is not absolutely required but contributes to efficient processing.30 ] 32 However, this auxiliary effect is variable, depending on the gene and type of nuclear extract used.33 Further mutational and competition analyses33,34 indicated that HBF acts in the processing reaction by stabilising the interaction of the pre-mRNA with the U7 snRNP Žsee below.. A human cDNA for a protein displaying HBF function and homologues from several other organisms have recently been cloned.35,36 To reflect this more precise characterization, we have proposed to change the name from HBF to hairpin binding protein, HBP. After processing, this protein remains associated with the mature histone mRNA and controls further steps of its metabolism Žsee below.. Immediately juxtaposed to the hairpin are conserved nucleotides surrounding and specifying the processing site. In vertebrates, the consensus sequence for the nucleotides following the hairpin is ACCCA’C Žthe apostrophe indicates the preferred processing site;84 whereas in sea urchins the corresponding sequence is ACCA’AA.25 Mutational analyses indicate that cleavage preferably occurs after A residues37,84 . The preference for the nucleotide 59 to the processing site appears to be A ) C ) U ) G and a G immediately after the processing site is inhibitory.84 Several nucleotides downstream of the processing site, there is a purine-rich spacer element 24 which is complementary to and binds to the 59 end of U7 snRNA present in the U7 snRNP.38 ] 40 This interaction is essential for processing to occur. In vertebrates the purine-richness only applies to the 59 portion of the spacer element and the complementary region is more extensive than in sea urchins, reflecting the greater length of the U7 RNA 59 end ŽFigure 1..34 The U7 snRNP is a minor nucleoplasmic snRNP, present in mammalian cells in 2000]15,000 copiesrnucleus.41 The 59 end of U7 RNA is modified
Histone mRNA 39 end formation and the minor U7 snRNP The 39 ends of replication-dependent histone mRNAs are formed by an endonucleolytic cleavage reaction that is distinct from the normal 39 processing Žpolyadenylation. event involved in forming all other mRNAs Žreviewed in refs 4 and 24.. Histone mRNAs have short 39 untranslated regions of 50]70 nucleotides and the signalŽs. controlling the processing reaction and its regulation are contained within a region of ; 60 nucleotides surrounding the processing site.25 ] 27 The reaction has been studied mainly in two systems, Xenopus laevis oocytes25,26 and nuclear extracts from mammalian cells.28 The 60-nucleotide region can be subdivided into three functional parts ŽFigure 1.. Completely contained within the mature mRNA portion is a highly conserved stretch of ; 26 nucleotides. Within these lies a dyad symmetry element that forms a stable hairpin structure in the RNA. This hairpin is charac568
The U7 snRNP and the hairpin binding protein: key players in histone mRNA metabolism
Figure 1. Aligned sequences of mammalian Žtop. and sea urchin Žbottom. histone pre-mRNAs and U7 snRNAs. For the histone pre-mRNAs Župper sequences., the figure shows the consensus sequences of the hairpin element, the nucleotides surrounding the processing site and the spacer element. The dashed line represents unconserved sequences and reproduces the correct spacing. Relevant distances discussed in the text are indicated and the processing site is represented by a thick downward arrow. For the U7 snRNAs Žlower sequences., only the 59 part is shown, whereas the 39-terminal hairpin has been omitted. The regions complementary to the histone pre-mRNA spacer element and the Sm binding sites are shown in upper case. The positions of the 59-terminal trimethyl guanosine cap structure Ž3mG. and of a cytosine that can be crosslinked to a U7-specific protein46 Župward arrow. are indicated.
with a trimethyl guanosine cap structure and the murine U7 snRNP contains a complete set of Sm proteins as well as U7-specific proteins of 50 and 14 kDa.42 In mammals, U7 snRNA is encoded by a single copy gene 43,44 which is located at chromosomal position 6F3 in mice 45 and 12p13 in humans ŽGenEMBL acc.no. U47924.. The U7 RNAs of sea urchins and vertebrates are 57]62 nucleotides long, not including the cap nucleotide. Between the 59 end complementary to the histone spacer element and an extensive 39-terminal hairpin Žnot shown in Figure 1., all U7 R N A s have a version of the sequence AAr GUUU Gr C UCUAG, which is reminiscent of the conserved Sm binding sites found in other nucleoplasmic snRNAs. In assembly and UV-crosslinking studies in Xenopus oocytes, it could be shown that this sequence indeed interacts with Sm proteins but also with at least one U7-specific protein of 40 kDa, presumably the Xenopus homologue of the mouse 50 kDa U7-specific protein.46 Conversion of the atypical Sm binding site to the consensus AAUUUUUGGAG led to more efficient assembly and nuclear accumulation, indicating that the U7 Sm binding site is suboptimal.41,46 However, the U7 RNA with the consensus
Sm binding site did not form the UV-crosslink with the U7-specific protein and was inactive in histone RNA 39 processing.46 Thus it seems that the special Sm binding site of U7 RNA and perhaps the 40 kDa U7-specific protein are critical for the processing reaction. A similar overlap between the binding sites for Sm proteins and a snRNP-specific protein has recently been described for the spliced leader RNA of nematodes47 but has not been observed in any of the spliceosomal snRNPs. In both mammalian cells 48 and Xenopus oocytes49 the U7 RNA is primarily localised in coiled bodies. Since these nuclear structures are often associated with histone loci, this raises the interesting possibility that the localization to coiled bodies may help to direct the U7 snRNPs to their target histone premRNAs. Intriguingly, the U7 RNA with the consensus Sm binding site is not localized to coiled bodies in Xenopus oocytes and the special Sm binding site of U7 RNA is both necessary and sufficient for correct targeting.50 Depending on the definition of the spacer element, it seems that its position, length and sequence are quite variable among vertebrate histone genes, in 569
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contrast to its virtual invariability in sea urchins. Scharl and Steitz 37 analysed the constraints on the distance between the spacer element and the hairpin in a mouse H3 pre-mRNA. They found that increasing the distance shifted the processing site in the 39 direction by a corresponding number of nucleotides. Processing at the novel sites was always less efficient than in the wild-type configuration. Part of this reduction could be traced back to the lack of an appropriate nucleotide for cleavage Žan adenosine. while the rest was probably due to problems in achieving the proper conformation between the hairpinrHBP and the U7 snRNP. These results led to the proposal of a ‘molecular ruler’ model for the U7 snRNP. In this model, the cleavage site is chosen at a more or less fixed distance upstream from the U7rpre-mRNA hybrid. Interestingly, if one selects a fixed position within this hybrid, rather than either end of it, as the reference point Že.g the centre of the highly conserved hexamer GAGCUG in mammalianrvertebrate histone genes, Figure 1., then the distance to the processing site becomes almost invariable Ž17 or 18 nucleotides.. At this position, cleavage activity then occurs at the most appropriate phosphodiester bond Žalmost always 39 of an adenosine, see above. within a window of maximally four nucleotides.37,84 By introducing corresponding length insertions between the Sm binding site and the base-pairing region of U7 snRNA and testing these in a complementation assay in X. laevis oocytes, Scharl and Steitz 51 could show that length suppression does occur as predicted by the molecular ruler model, but only if the inserted sequences are complementary to those present in the histone pre-mRNA. In other words, besides the length correspondence, the position of the beginning of the U7rpre-mRNA hybrid also appeared to be critical. This was interpreted as a requirement for rigidity in this region of the processing complex. Inspection of vertebrate histone gene sequences in the databases shows that the distance between the beginning of the U7rpre-mRNA hybrid and the cleavage site varies between nine and 11 nucleotides, but is actually 11 nucleotides for most genes ŽFigure 1.. It is interesting to speculate which parts of the processing complex become juxtaposed to the processing site by the molecular ruler effect. If one simply counts the nucleotides in the two RNAs, then the dinucleotide CU within the conserved Sm binding site of U7 RNA comes to lie at this position ŽFigure 1.. On the one hand, the uridine of U7 could
form a base-pair with the adenosine upstream of the cleavage site, whereas the cytidine of U7 could not; note that a guanosine following the cleavage site is inhibitory 84 . On the other hand, the UV crosslink to the 40-kDa U7-specific protein was mapped precisely to this C residue,46 making this protein an excellent candidate for the cleavage activity. In this context it is interesting that a chimeric RNA consisting of an upstream histone pre-mRNA and a downstream U7 RNA moiety, when injected into Xenopus oocytes, assembles with proteins similarly to a single U7 RNA. However, the chimeric RNP remains cytoplasmic and yet the RNA is cleaved at the right position in a cis-catalytic fashion.52 Thus it is possible that all enzymatic components for the cleavage reaction are contained within the U7 snRNP. Two other activities involved in histone RNA 39 processing have been identified. The first is a heatlabile processing factor ŽHLF..53 This factor is inactivated by mild heat-treatment Ž15 min at 45]508C. whereas U7 snRNPs capable of complementing a U7-depleted extract withstand temperatures up to 658C. However it has to be noted that HBP 54 and a 59]39 exonuclease ŽWalther et al, unpublished; see below. are also inactivated at 508C. At least in certain cellular systems, changes in the HLF appear to be responsible for the cell cycle-dependent regulation of histone RNA 39 processing.55,56 Attempts at purifying HLF revealed an extensive co-fractionation with U7 snRNPs and the total cleavage activity.53 On gel filtration columns, the HLF could be separated into two peaks of apparent molecular mass 300 and 40 kDa. The larger peak might represent a U7 snRNPassociated form whereas the 40-kDa peak could be the free protein. If this is the case, then the 50r40 kDa U7-specific polypeptide is an obvious candidate for the HLF. The free form could either reflect an excess of the protein or a tendency of it to dissociate from U7 snRNPs. Another activity has recently been identified in attempts to study the generation of 39 fragments during histone RNA 39 processing ŽWalther et al, unpublished.. These fragments are only observed in processing reactions carried out in the presence of EDTA and they are usually shorter than expected, i.e. their Žheterogeneous. 59 ends lie several nucleotides downstream of the end of the 59 fragment.28,33 The new experiments suggest that these shortened fragments are generated by a 59]39 exonuclease after the initial endonucleolytic cleavage has occurred ŽWalther et al, unpublished.. This activity could be important in recycling U7 snRNPs for additional rounds of cleavage. 570
The U7 snRNP and the hairpin binding protein: key players in histone mRNA metabolism
Structural and functional features of hairpinbinding proteins
recombinant human protein is able to participate in histone RNA 39 end processing.36 To signify that this protein is, or is part of the HBF as originally defined by Birnstiel et al,30 we are calling it hairpin-binding protein ŽHBP. whereas Marzluff et al use the name SLBP. Despite this difference, both laboratories agree that this protein is the common RNA binding component of HBF and SLBP. The human hbp gene is located on chromosome 4p16.3 and composed of eight exons covering 19.5 kb.36,63 An alternative splicing event causes exon 3 to be skipped in a small fraction of HBP mRNAs in both human and mouse cells. The resulting in-frame fusion deletes 35 amino acids but this does not interfere with the binding to hairpin RNA ŽEglite et al, unpublished.. The function of this alternative splicing event is not clear. The C. elegans gene contains only five exons and covers ; 1.8 kb on chromosome 2.36 Figure 2 summarizes an alignment of the amino acid sequences of the human, mouse, X. laevis and C. elegans HBPs. The proteins contain 270, 275, 254 and 367 amino acids, respectively. However, all the proteins migrate with abnormal mobility during denaturing gel electrophoresis; for example the human protein, with a predicted molecular mass of ; 31 kDa, migrates like a ; 43 kDa protein. The human and mouse proteins are nearly identical with 89% amino acid identity. Human and X. laevis proteins are to 62% identical, and human and C. elegans proteins are 38% identical. The human, mouse and X. laevis proteins contain several conserved blocks of seven amino acids or more Žshown as grey boxes in Figure 2. distributed throughout the proteins. The only region where all four proteins have significant homology is a 62-amino acid stretch Žshown in black.. In this sequence Žalso shown in Figure 2, bottom., the human, mouse and X. laevis proteins are to 82% identical and human and C. elegans proteins are 55% identical. This region is both necessary and sufficient for RNA binding 35 and both human and C. elegans polypeptides with the sequences shown in Figure 2 are able to bind specifically to wild-type hairpin RNA ŽMichel et al, unpublished.. The sequence of this RNA binding domain ŽRBD. is not related to any of the known RNA-binding motifs and computer structure predictions based on all four protein sequences propose that the RBD contains two a-helices Žindicated in Figure 2.. In the C. elegans hairpin element, a cytosine replaces the first uracil in the loop Žsee Figure 1.. Interestingly, the human and C. elegans RBDs show
As described above, the 39-terminal hairpin and a hairpin-binding processing factor play an important role in histone RNA 39 processing. Here we will discuss the characterization and cDNA cloning of hairpin-binding proteins. The evidence indicating that the hairpin structure is involved in additional and, notably, cytoplasmic steps of histone mRNA metabolism will be reviewed further below. Activities binding to the histone RNA hairpin have been detected in different extracts from mammalian cell lines and calf thymus by a variety of biochemical and functional assays.29,30,54,57,58 The initially described nuclear activity implicated in histone RNA 39 processing was termed hairpin binding factor ŽHBF..29,30,54 Marzluff et al then described nuclear and polysomal activities from a mouse cell line that bound hairpin RNA in a structure- and sequencedependent manner 57,59 and designated them as stem]loop binding proteins ŽSLBP.. A ; 45 kDa protein was enriched from polysomes and identified by Northwestern blotting as the RNA-binding component.60 Since the SLBP prepared from polysomes could substitute for the nuclear HBF in in vitro processing reactions,61 it appeared that these activities are related, if not identical. We have described a similar RNA-binding activity from calf thymus and by biochemical fractionation of whole cell extract, have obtained highly enriched fractions capable of both binding to hairpin RNA and substituting HBF in histone RNA 39 processing.58 The most enriched fraction, although not active in processing for technical reasons, contained two proteins of 40 and 43 kDa. Together with the finding that complexes of hairpin RNA formed in vitro with either SLBP 59 or HBF ŽSchaller, A. and B.M., unpublished. have half-lives in excess of 4]6 h, these results suggest that one and the same protein participates in histone RNA processing and then remains associated with the mature mRNA 39 end until it reaches its cytoplasmic Žpolysomal. destination. Using the yeast three hybrid system designed for the characterization of RNA-binding proteins,62 human and Xenopus laevis cDNAs encoding proteins that bind to the histone RNA hairpin were isolated.35,36 In addition, a homologous mouse cDNA was isolated by hybridization screening 35 and a Caenorhabditis elegans homologue was identified by data base search and subsequent functional characterisation.36 Complementation assays showed that the 571
B. Muller ¨ and D. Schumperli ¨
Figure 2. Alignment of human, mouse, Xenopus laevis and Caenorhabditis elegans HBP sequences. Top: the proteins are shown as bars and are not drawn to scale. Shaded areas and the sequences above them represent blocks of seven or more amino acids that are virtually identical between the three vertebrate HBPs. At divergent positions within these blocks the residue present in the two mammalian HBPs and the one present in the Xenopus HBP are separated by a slash Žr.. A dot Ž.. indicates a missing amino acid. Black areas are conserved between all four proteins and encompass the RNA-binding domain. Bottom: sequences of human and C. elegans polypeptides binding specifically to hairpin RNA. The consensus sequence shows only those amino acids that are identical in three of the four proteins. In the human and C. elegans sequences, the amino acids diverging from the consensus are shown in lower case. Two potential a-helical regions were predicted by a computer algorithm. Highlighted T and S represent potential phosphorylation sites.
preferential binding to their conspecific hairpin RNAs, indicating that the differences in amino acid sequence of the RBDs confer different binding preferences ŽMichel et al, unpublished.. However, it is possible that other parts of the protein outside of the RBD also contribute to the specificity of the protein]RNA interaction. Interestingly, some of the most conserved sequence elements of the known HBPs are potential phosphorylation sites. In Figure 2, the sequence element TPNK at the N-terminal border of the second proposed a-helix is a potential site for threonine-phosphorylation by Cdc2 kinase, the first serine in the sequence SRRS in the same second a-helix is a potential target for phosphorylation by cGMP-dependent kinase or protein kinase C and the second serine in the same sequence could be phosphorylated
by either cGMP-dependent or cAMP-dependent kinase.64 The fact that all these sequences are found in the RBD raises the interesting possibility that HBP function might be regulated by phosphorylation of residues in this domain.
Involvement of the 39-terminal hairpin in steps subsequent to histone RNA 39 processing The conserved hairpin at the 39 ends of animal replication-dependent histone mRNAs is involved in virtually all processes and regulatory events that occur after the mature mRNA has been generated by RNA 39 processing Žreviewed in ref 20.. The fact that at least the RNA-binding component of the nuclear
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The U7 snRNP and the hairpin binding protein: key players in histone mRNA metabolism
HBFrHBP and the polysomal SLBP are identical suggests that all these functions of the histone mRNA 39 end are mediated by this protein. Here we will briefly review the evidence linking the hairpin to these post-transcriptional events and thereby concentrate on newer findings. Initial evidence for the involvement of the histone hairpin in nucleo-cytoplasmic transport of mRNAs was obtained by animal cell transfections of DNA constructs with a cis-acting hammerhead ribozyme that allowed the production of virtually normal histone mRNA 39 ends independently of the 39 processing signal Žof which the hairpin is an important part..65 Mutations in the hairpin led to complete nuclear retention of the cleaved RNAs, whereas an RNA with the wild-type hairpin was exported. However, in some cases, the ribozyme-generated RNAs with wild-type hairpin were also partly or completely export-deficient, depending on the sequences present upstream in the RNA or the cell line used. These latter results suggest that histone mRNA export might be directly coupled to 39 processing. Similarly, a histone RNA with the first and third uridines in the loop changed to adenosines was processed, albeit inefficiently, but was severely impaired in nucleocytoplasmic transport, both in animal cells and in Xenopus oocytes.66 Since the same mutation causes a 20]50-fold reduction in affinity to HBP, this provided circumstantial evidence for an involvement of HBP in nucleo-cytoplasmic transport. Similarly to the polyŽA. tail on other mRNAs, the hairpin is also essential for recruiting histone mRNA to polysomes.66,67 In a recent study using electroporation of luciferase reporter transcripts into tissue culture cells, the hairpin was directly shown to be required and co-dependent on a 59 cap structure for translation in mammalian cells.68 However, only a polyŽA. tail but not the histone hairpin was able to stimulate translation in electroporated plant protoplasts. During S phase in mammalian cells, histone mRNAs have a half-life of 40]60 min19,23 and histone mRNA degradation starts at the 39 end.69 Inhibition of DNA synthesis leads to a rapid destabilisation of histone mRNA, probably by a process similar to the destabilisation of histone mRNA upon exit from S phase.23 Inversely, histone mRNA is stabilised by the addition of cycloheximide to the cells. These stability changes are dependent on the presence of a wild-type hairpin element at the RNA 39 end and can be conferred onto non-histone RNAs by adding the 39terminal processing site with the hairpin.66,70 Inter-
estingly, the destabilisation requires that the mRNA be translated and it is crucial that the distance between the stop codon and the hairpin is shorter than ; 300 nucleotides.71 Two non-exclusive models have been proposed to explain how this degradation of histone mRNAs may be triggered: Ž1. a regulatory signal, possibly an unusual nucleotide, might coordinately regulate DNA replication, deoxynucleotide metabolism and histone mRNA levels;72 and Ž2. excess histone proteins might regulate their own biosynthesis.73 The addition of histone proteins, either mixed or individually, to an in vitro histone RNA decay system consisting of polysomes, as the source of mRNA and the RNA-degrading enzymeŽs., and a post-polysomal supernatant ŽS130. destabilises histone mRNA,74 ] 76 lending support to the autoregulation model. This in vitro decay reaction starts at the 39 end and proceeds from 39 to 59,74 like the decay reaction occurring in vivo.69 A 39-59 exoribonuclease of 33 kDa with the required specificity has been purified.77 The formation of a characteristic intermediate ending within the loop suggests that one of the early steps in the decay reaction is the removal of HBP from the mRNA, and it will be interesting to know what the role of HBP in this decay reaction is. Fractionation of the S130 fraction demonstrated the presence of an as yet undefined histone-activatable destabiliser, and a stabiliser identified as the La protein,78 a RNA-binding protein found associated with different RNAs. Addition of La protein leads to the accumulation of the intermediate ending in the loop. This intermediate has a string of uridines at the 39 end. Although the molecular mechanism of the stabilisation is not clear, it is interesting that La protein is also found associated with U-rich termini of polymerase III transcripts79 and it is tempting to speculate that a similar association of La protein with the decay intermediate might protect histone mRNA from further exonucleolytic degradation. In summary, the histone hairpin element is important for correct histone gene expression and is involved in all the post-transcriptional regulatory events described so far. Mutations in the hairpin element reducing histone gene expression, RNA export and mRNA stability also reduce the affinity for HBP,59,66,80 indirectly implicating HBP in all the steps affected by these mutations, i.e. nuclear export, translation and mRNA stability. However, direct evidence exists only for the participation of the human HBP in mRNA 39 end formation. 573
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Table 1. Comparison of factors controlling the metabolism of histone versus polyadenylated mRNAs
Upstream sequence element ŽUSE. Downstream sequence element ŽDSE. Specificity factor Žfor cleavage. Auxiliary factor Žfor cleavage.U Auxiliary factor Žfor polyadenylation.U Export factor† Translation factor† Stabilisation factor‡
Histone mRNAs
Polyadenylated mRNAs
Hairpin Spacer element U7 snRNP Žbinds DSE. HBP Žbinds USE. } HBP Ž? . HBP Ž? . HBP Ž? .
AAUAAA GU-rich sequence CPSF Žbinds USE. CStF Žbinds DSE. PABP2 Žbinds growing polyŽA. tail. PABP2 or PABP1 Ž? . PABP1 PABP1
U
Cleavage of polyadenylated mRNAs additionally requires cleavage factors I and II. PolyŽA. polymerase is required for both the cleavage and polyadenylation steps.81 † Export and translation of both types of mRNAs Žpresumably. requires a 59 cap structure, the nuclear cap binding complex and the cytoplasmic cap binding factor eIF-4FrG.82,83 ‡ For both types of mRNAs a connection between mRNA degradation and translation has been demonstrated.71,82
Factors controlling the metabolism of histone and polyadenylated mRNAs
downstream fragment. In histone RNAs, the auxiliary processing factor HBP later becomes the molecular escort of the mature mRNA and therefore has to be associated with the 59 fragment whereas the U7 snRNP is presumably not needed for further modifications of this particular RNA and hence is best attached to the 39 fragment destined for degradation. After processing Žand polyadenylation. have occurred, it appears that HBP fulfills all the functions proven or ascribed to the nuclear and cytoplasmic polyŽA. binding proteins, PABP1 and PABP2.81,82
Similarities and differences between the metabolic processes controlling the replication-dependent animal histone mRNAs as opposed to all other, polyadenylated mRNAs have been discussed previously.6,24,37 Among the similaritites between the cleavage reactions are the lack of a requirement for cleavable ATP or divalent cations, the preference for cleaving downstream of an adenosine and the types of ends generated Ž39-OH and 59-phosphate.. In contrast, no shared trans-acting factors have been identified so far. Both types of mRNA 39 processing reactions require upstream and downstream sequence elements ŽTable 1; for a review on polyadenylation see ref 81.. A main difference is that the specificity factor binds upstream of the cleavage site in polyadenylation ŽCPSF binding to the AAUAAA sequence. and downstream in histone RNA 39 processing Žthe U7 snRNP binding to the spacer element.; the respective auxiliary factors ŽCstF and HBP. stabilise the processing complex by binding on the other side of the processing site. This difference in the arrangement of specificity and auxiliary factors is most likely related to the different metabolic pathways the cleaved RNAs engage in. In polyadenylation, the specificity factor CPSF, together with polyŽA. polymerase and PABP2, is required for the coupled step of polyŽA. addition; thus CPSF has to be associated with the upstream cleavage fragment, whereas CStF which is not required in the polyŽA. addition step is attached to the
Acknowledgements Work in our laboratory was supported by the Kanton Bern and by the Swiss National Science Foundation.
References 1. Allen BS, Stein JL, Stein GS, Osterer H Ž1991. Single-copy flanking sequences in human histone gene clusters map to chromosomes 1 and 6. Genomics 10:486]488 2. Wang ZF, Krasikov T, Frey MR, Wang J, Matera AG, Marzluff WF Ž1996. Characterization of the mouse histone gene cluster on chromosome 13: 45 histone genes in three patches spread over 1 Mb. Genome Res 6:688]701 3. Wang ZF, Tisovec R, DeBry RW, Frey MR, Matera AG, Marzluff WF Ž1996. Characterization of the 55-kb mouse histone gene cluster on chromosome 3. Genome Res 6:702]714 4. Birnstiel ML, Schaufele FJ Ž1988. Structure and function of minor snRNPs, in Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles ŽBirnstiel ML, ed. pp 155]182. Springer Verlag, New York 5. Osley MA Ž1991. The regulation of histone synthesis in the cell cycle. Annu Rev Biochem 60:827]861 6. Wittop Koning TH, Schumperli D Ž1994. RNAs and ribonu¨
574
The U7 snRNP and the hairpin binding protein: key players in histone mRNA metabolism
7. 8. 9.
10.
11. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22.
23.
24. 25.
26.
27.
28. Gick O, Kramer A, Keller W, Birnstiel ML Ž1986. Generation ¨ of histone mRNA 39 ends by nucleolytic cleavage of the pre-mRNA in a snRNP-dependent in vitro reaction. EMBO J 5:1319]1326 29. Mowry KL, Steitz JA Ž1987. Both conserved signals on mammalian histone pre-mRNAs associate with small nuclear ribonucleoproteins during 39 end formation in vitro. Mol Cell Biol 7:1663]1672 30. Vasserot AP, Schaufele FJ, Birnstiel ML Ž1989. Conserved terminal hairpin sequences of histone mRNA precursors are not involved in duplex formation with the U7 RNA but act as a target site for a distinct processing factor. Proc Natl Acad Sci USA 86:4345]4349 31. Mowry KL, OH R, Steitz JA Ž1989. Each of the conserved sequence elements flanking the cleavage site of mammalian histone pre-mRNAs has a distinct role in the 39 end processing reaction. Mol Cell Biol 9:3105]3108 32. Cotten M, Gick O, Vasserot A, Schaffner G, Birnstiel ML Ž1988. Specific contacts between mammalian U7 snRNA and histone precursor RNA are indispensable for the in vitro 39 RNA processing reaction. EMBO J 7:801]808 33. Streit A, Wittop Koning T, Soldati D, Melin L, Schumperli D ¨ Ž1993. Variable effect of the conserved RNA hairpin element upon 39 end processing of histone pre-mRNA in vitro. Nucleic Acids Res 21:1569]1575 34. Spycher C, Streit A, Stefanovic B, Albrecht D, Wittop Koning TH, Schumperli D Ž1994. 39 end processing of mouse histone ¨ pre-mRNA: Evidence for additional base-pairing between U7 snRNA and pre-mRNA. Nucleic Acids Res 22:4023]4030 35. Wang ZF, Whitfield ML, Ingledue TC, III, Dominski Z, Marzluff WF Ž1996. The protein that binds the 39 end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing. Genes Dev 10:3028]3040 36. Martin F, Schaller A, Eglite S, Schumperli D, Muller B Ž1997. ¨ ¨ The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein. EMBO J 16:769]778 37. Scharl EC, Steitz JA Ž1994. The site of 39 end formation of histone messenger RNA is a fixed distance from the downstream element recognized by the U7 snRNP. EMBO J 13:2432]2440 38. Strub K, Galli G, Busslinger M, Birnstiel ML Ž1984. The cDNA sequences of the sea urchin U7 small nuclear RNA suggest specific contacts between histone mRNA precursors and U7 RNA during RNA processing. EMBO J 3:2801]2807 39. Schaufele F, Gilmartin GM, Bannwarth W, Birnstiel ML Ž1986. Compensatory mutations suggest that base-pairing with a small nuclear RNA is required to form the 39 end of H3 messenger RNA. Nature 323:777]781 40. Bond U, Yario TA, Steitz JA Ž1991. Multiple processing defective mutations in a mammalian histone messenger RNA are suppressed by compensatory changes in U7 RNA both in vivo and in vitro. Genes Dev 5:1709]1722 41. Grimm C, Stefanovic B, Schumperli D Ž1993. The low abun¨ dance of U7 snRNA is partly determined by its Sm binding site. EMBO J 12:1229]1238 42. Smith HO, Tabiti K, Schaffner G, Soldati D, Albrecht U, Birnstiel ML Ž1991. Two-step purification of U7 small nuclear robonucleoprotein particles using complementary biotinylated 29-O-methyl oligoribonucleotides. Proc Natl Acad Sci USA 88:9784]9788 43. Gruber A, Soldati D, Burri M, Schumperli D Ž1991. Isolation ¨ of an active gene and of two pseudogenes for mouse U7 small nuclear RNA. Biochim Biophys Acta 1088:151]154 44. Philips SC, Turner PC Ž1991. Nucleotide sequence of the mouse U7 snRNA gene. Nucleic Acids Res 19:1344 45. Turner PC, Whalen A, Schumperli D, Matera AG Ž1996. The ¨ bona fide mouse U7 snRNA gene maps to a different chromosome than two U7 pseudogenes. Genomics 31:250]252
cleoproteins in recognition and catalysis. Eur J Biochem 219:25]42 Muller K, Schmitt R Ž1988. Histone genes of Volvox carteri: ¨ DNA sequence and organization of two H3-H4 loci. Nucleic Acids Res 16:4121]4136 Walther Z, Hall JL Ž1995. The uni chromosome of Chlamydomonas: histone genes and nucleosome structure. Nucleic Acids Res 23:3756]3763 Fabry S, Muller K, Lindauer A, Park PB, Cornelius T, Schmitt ¨ R Ž1995. The organization, structure and regulatory elements of Chlamydomonas histone genes reveal features linking plant and animal genes. Curr Genet 28:333]345 Wyler-Duda P, Bernard V, Stadler M, Suter D, Schumperli D ¨ Ž1997. Histone H4 mRNA from the nematode Ascaris lumbricoides is cis-spliced and polyadenylated. Biochim Biophys Acta 1350:259]261 Woodland HR Ž1980. Histone synthesis during the development of Xenopus. FEBS Lett 121:1]7 Rudell A, Jacobs-Lorena M Ž1985. Biphasic pattern of histone gene expression during Drosophila oogenesis. Proc Natl Acad Sci USA 82:3316]3319 Busslinger M, Schumperli D, Birnstiel ML Ž1985. Regulation ¨ of histone gene expression. Cold Spring Harbor Symp Quant Biol 50:665]670 Hake LE, Richter JD Ž1997. Translational regulation of maternal mRNA. Biochim Biophys Acta 1332:M31]M38 Challoner P, B., Moss SB, Groudine M Ž1989. Expression of replication-dependent histone genes in avian spermatids involves an alternate pathway of mRNA 39-end formation. Mol Cell Biol 9:902]913 Moss SB, Challoner PB, Groudine M Ž1989. Expression of a novel histone 2B during mouse spermiogenesis. Dev Biol 133:83]92 Wells D, Brown D Ž1991. Histone and histone gene compilation and alignment update. Nucleic Acids Res 19:2173]2188 Wolffe AP, Pruss D Ž1996. Deviant nucleosomes: the functional specialization of chromatin. Trends Genet 12:58]62 Schumperli D Ž1988. Multilevel regulation of replication¨ dependent histone genes. Trends Genet 4:187]191 Marzluff WF Ž1992. Histone 39ends: essential and regulatory functions. Gene Expr 2:93]97 Heintz N Ž1991. The regulation of histone gene expression during the cell cycle. Biochim Biophys Acta 1088:327]339 Stein GS, Stein JL, Van Wijnen AJ, Lian JB Ž1994. Histone gene transcription: a model for responsiveness to an integrated series of regulatory signals mediating cell cycle control and proliferationrdifferentiation interrelationships. J Cell Biochem 54:393]404 Harris ME, Bohni R, Schneiderman MH, Ramamurthy L, ¨ Schumperli D, Marzluff WF Ž1991. Regulation of histone ¨ mRNA in the unperturbed cell cycle: evidence suggesting control at two posttranscriptional steps. Mol Cell Biol 11:2416]2424 Birnstiel ML, Busslinger M, Strub K Ž1985. Transcription termination and 39 processing: the end is in site! Cell 41:349]359 Birchmeier C, Grosschedl R, Birnstiel ML Ž1982. Generation of authentic 39 termini of an H2A mRNA in vivo is dependent on a short inverted DNA repeat and on a spacer sequence. Cell 26:739]745 Birchmeier C, Schumperli D, Sconzo D, Birnstiel ML Ž1984. ¨ 39 editing of mRNAs: sequence requirements and involvement of a 60 nucleotide RNA in maturation of histone mRNA precursors. Proc Natl Acad Sci USA 81:1057]1061 Stauber C, Luscher B, Eckner R, Lotscher E, Schumperli D ¨ ¨ ¨ Ž1986. A signal regulating mouse histone H4 mRNA levels in a mammalian cell cycle mutant and sequences controlling RNA 39 processing are both contained within the same 80]bp fragment. EMBO J 5:3297]3303
575
B. Muller ¨ and D. Schumperli ¨
46. Stefanovic B, Hackl W, Luhrmann R, Schumperli D Ž1995. ¨ ¨ Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes. Nucleic Acids Res 23:3141]3151 47. Denker JA, Maroney PA, Yu Y, Kanost A, Nilsen TW Ž1996. Multiple requirements for nematode spliced leader RNP function in trans-splicing. RNA 2:746]755 48. Frey MR, Matera AG Ž1995. Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells. Proc Natl Acad Sci USA 92:5915]5919 49. Wu CH, Gall JG Ž1993. U7 small nuclear RNA in C snurpososmes of the Xenopus germinal vesicle. Proc Natl Acad Sci USA 90:6257]6259 50. Wu CH, Murphy C, Gall JG Ž1996. The Sm binding site targets U7 snRNA to coiled bodies Žspheres. of amphibian oocytes. RNA 2:811]823 51. Scharl EC, Steitz JA Ž1996. Length suppression in histone messenger RNA 39-end maturation: processing defects of insertion mutant premessenger RNAs can be compensated by insertions into the U7 small nuclear RNA. Proc Natl Acad Sci USA 93:14659]14664 52. Stefanovic B, Wittop Koning TH, Schumperli D Ž1995. A ¨ synthetic histone pre-mRNA-U7 small nuclear RNA chimera undergoing cis cleavage in the cytoplasm of Xenopus oocytes. Nucleic Acids Res 23:3152]3160 53. Gick O, Kraemer A, Vasserot A, Birnstiel ML Ž1987. Heat-labile regulatory factor is required for 39 processing of histone precursor mRNAs. Proc Natl Acad Sci USA 84:8937]8940 54. Melin L, Soldati D, Mital R, Streit A, Schumperli D Ž1992. ¨ Biochemical demonstration of complex formation of histone pre-mRNA with U7 small nuclear ribonucleoprotein and hairpin binding factors. EMBO J 11:691]697 55. Luscher B, Schumperli D Ž1987. RNA 39 processing regulates ¨ ¨ histone mRNA levels in a mammalian cell cycle mutant. A processing factor becomes limiting in G1]arrested cells. EMBO J 6:1721]1726 56. Stauber C, Schumperli D Ž1988. 39 processing of pre-mRNAs ¨ plays a major role in proliferation-dependent regulation of histone gene expression. Nucleic Acids Res 16:9366]9414 57. Pandey NB, Sun J, Marzluff WF Ž1991. Different complexes are formed on the 39 end of histone mRNA with nuclear and polyribosomal proteins. Nucleic Acids Res 19:5653]5659 58. Schaller A, Martin F, Muller B Ž1997. Characterization of the ¨ calf thymus hairpin-binding factor involved in histone premRNA 39 end processing. J Biol Chem 272:10435]10441 59. Williams AS, Marzluff WF Ž1995. The sequence of the stem and flanking sequences at the 39 end of histone mRNA are critical determinants for the binding of the stem-loop binding protein. Nucleic Acids Res 23:654]662 60. Hanson RJ, Sun JH, Willis DG, Marzluff WF Ž1996. Efficient extraction and partial purification of the polyribosome-associated stem-loop binding protein bound to the 39 end of histone mRNA. Biochemistry 35:2146]2156 61. Dominski Z, Sumerel J, Hanson R, Marzluff W Ž1996. The polyribosomal protein bound to the 39 end of histone mRNA can function in histone pre- mRNA processing. RNA 1:915]923 62. Sengupta DJ, Zhang BL, Kraemer B, Pochart P, Fields S, Wickens M Ž1996. A three-hybrid system to detect RNA-protein interactions in vivo. Proc Natl Acad Sci USA 93:8496]8501 63. McCombie WR, Martin-Gallardo A, Gocayne JD, Fitzgerald M, Dubnick M, Kelley JM, Castilla L, Wallace S Ž1992. Expressed genes, Alu repeats and polymorphisms in cosmids
64. 65. 66.
67. 68. 69. 70.
71. 72. 73. 74. 75. 76. 77. 78. 79. 80.
81. 82. 83. 84.
576
sequenced from chromosome 4p16.3. Nature Genet 1:348]353 Pearson RB, Kemp BE Ž1991. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Meth Enzymol 200:62]81 Eckner R, Ellmeier W, Birnstiel ML Ž1991. Mature mRNA 39 end formation stimulates RNA export from the nucleus. EMBO J 10:3513]3522 Williams AS, Ingledue TC, III, Kay BK, Marzluff WF Ž1994. Changes in the stem-loop at the 39 terminus of histone mRNA affects its nucleocytoplasmic transport and cytoplasmic regulation. Nucleic Acids Res 22:4660]4666 Sun J, Pilch DR, Marzluff WF Ž1992. The histone mRNA 39 end is required for localisation of histone mRNA to polyribosomes. Nucleic Acids Res 20:6057]6066 Gallie DR, Lewis NJ, Marzluff WF Ž1996. The histone 39terminal stem-loop is necessary for translation in Chinese hamster ovary cells. Nucleic Acids Res 24:1954]1962 Ross J, Peltz SW, Kobs G, Brewer G Ž1986. Histone mRNA degradation in vivo: the first detectable step occurs at or near the 39 terminus. Mol Cell Biol 6:4362]4371 Pandey NB, Marzluff WF Ž1987. The stem-loop structure at the 39 end of histone pre-mRNA is necessary and sufficient for regulation of histone mRNA stability. Mol Cell Biol 7:4557]4559 Graves RA, Pandey NB, Chodchoy N, Marzluff W Ž1987. Translation is required for regulation of histone mRNA degradation. Cell 48:615]626 Graves RA, Marzluff WF Ž1984. Rapid reversible changes in the rate of histone gene transcription and histone mRNA levels in mouse myeloma cells. Mol Cell Biol 4:351]357 Sariban E, Wu RS, Erickson LC, Bonner WM Ž1985. Interrelationships of protein and DNA syntheses during replication of mammalian cells. Mol Cell Biol 5:1279]1286 Ross J, Kobs G Ž1986. H4 histone messenger RNA decay in cell-free extracts initiates at or near the 39 terminus and proceeds 39 to 59. J Mol Biol 188:579]593 Peltz SW, Ross J Ž1987. Autogenous regulation of histone mRNA decay by histone proteins in a cell free system. Mol Cell Biol 7:4345]4356 McLaren RS, Ross J Ž1993. Individual purified core and linker histones induce histone H4 mRNA destabilisation in vitro. J Biol Chem 268:14637]14644 Caruccio N, Ross J Ž1994. Purification of a human polyribosome-associated 39 to 59 exoribonuclease. J Biol Chem 269:31814]31821 McLaren RS, Caruccio N, Ross J Ž1997. Human La protein: a stabilizer of histone mRNA. Mol Cell Biol 17:3028]3036 vanVenrooji WJ, Slobbe RL, Prujin GJM Ž1993. Structure and function of La and Ro RNPs. Mol Biol Rep 18:113]119 Pandey NB, Williams AS, Sun J-H, Brown VD, Bond U, Marzluff WF Ž1994. Point mutations in the stem-loop at the 39 end of mouse histone mRNA reduce expression by reducing the efficiency of 39 end formation. Mol Cell Biol 14:1709]1720 Wahle E, Keller W Ž1996. The biochemistry of polyadenylation. Trends Biochem Sci 21:247]250 Sachs A, Wahle E Ž1993. PolyŽA. tail metabolism and function in eucaryotes. J Biol Chem 268:22955]22958 Izaurralde E, Mattaj IW Ž1995. RNA export. Cell 81:153]159 Furger A, Schaller A, Schumperli D Ž1998. Functional impor¨ tance of conserved nucleotides at the histone RNA 39 processing site. RNA 4: in press