‘Snurpogenesis’: The transcription and assembly of U snRNP components

TIBS 14-January 1989

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Reviews, 'Snurpogenesis': The transcription and assembly of U snRNP components Huw D. Parry, Daniel Scherly and lain W. Mattaj The major class of small r,uclear ribonucleoproteins (snRNPs) are part of the nuclear machinery for processing pre-messenger RNAs. The genes encoding the RNA components of these particles (snRNAs) have several unique features which distinguish them from other pol H- and pol Ill-transcribed genes. Assembly of the snRNAs with proteins to form snRNP particles has been studied in vivo, and new in vitro systems promise to advance our understanding of the assembly process stillfurther. The U snRNA (Uracil-rich small nuclear RNA) family in vertebrates is subdivided into major and minor members on the basis of abundance ~. U1 to U6, the major U snRNAs, form a group of highly expressed multicopy genes. At least 200 000 transcripts of each of these RNAs are fotmd in each vertebrate cell nucleus. The minor U snRNAs are one or twc, orders of magnitude less abundant. The number of different minor U snRNAs in any organism and the degree ot their conservation in evolution is currently unknown; however, there is probably a larger number of minor slzecies than major species. U1, U2, U4/U6 and U5 snRNPs (small nuclear ribonucleoproteins) are localized in the nucleoplasm and are involved in the processing of pre-messenger R N A 2. In this review we will look at how the major vertebrate U snRNAs are transcribed and subsequently assembled with proteins into snRNPs ('Snurps'). U snRNA gene promoter structure is unusual

U snRNAs are ubiquitously expressed and in vertebrates their genes are often arranged in tandemly repeated units. All of the major U snRNA genes, with the ex,zeption of U6 which will be considered separately, are transcribed solely by R N A polymerase II (pol II) 3. U snRNA transcripts initially have a 7-methyl H. D. Parry, D. Scherly and 1. W. Mattaj are at the European Molecular Biology Laboratory, Meyerhofstrasse 1, 6900 Heidelberg, FRG.

guanosine cap, like other pol II transcripts, which is modified posttranscriptionally in the cytoplasm to the 2,2,7-trimethyl guanosine cap characteristic of and unique to this group of RNAs ~'4. The structure of the U snRNA genes is, however, different from those of other genes transcribed by pol II 3. Most noticeably, their promoters lack elements found in other classes of pol II-transcribed genes 5"6. Unlike most other pol II transcripts U snRNAs are not polyadenylated. Figure 1 shows diagrammatically the structure of the Xenopus U2 and U6 promoters. These are among the best characterized U snRNA gene promoters and are shown as typical examples. There are two major promoter elements in the 5' flanking region of U snRNA genes: a Distal Sequence Element (DSE) located upstream of position - 2 0 0 relative to the start site of transcription and a Proximal Sequence Element (PSE) which is located between 40 and 70 bp upstream of the start site 3. The DSE is responsible for the enhancement of U s n R N A transcription: its action is independent of orientation and to some extent of position within the 5' non-coding region. Some DSEs, for example Human U27 and Xenopus U58, also function when positioned downstream of the RNA coding sequence. The PSE functions like a T A T A box, in that it is essential for transcription and specifies the site of transcription initiation 3. However, it does not resemble a T A T A box either in primary sequence or in its position relative to the transcription start site.

As we will now discuss, the DSE and PSE are specialized promoter elements which in combination give rise to a transcription complex with unique properties. The DSEs of U snRNA genes have some common features. They are all in roughly the same position relative to the RNA coding sequence and all contain an octamer motif ( A T G C A A A T ) which may be present in either orientation. In U1 DSEs this motif is not always exactly conserved but appears nevertheless to be functionally important. The Octamer sequence is found in combination with binding sites for other transcription factors. For example, Spl binding sites are often, but not always, present. The auxiliary sites vary among different DSEs; most of the DSEs functionally analysed to date (human U1 and U2, Xenopus U1, U2 and U5 and chicken U1 and U4) 3"7 12 have been shown to have unique structures. The DSEs appear to fall into two different functional classes when assayed in vivo in homologous or heterologous systems. In one class, including Xenopus U1 and U5 and human U2, mutation in individual transcription factor binding sites abolishes enhancer activity. This suggests that the individual functional units of these DSEs not only act synergistically but are totally interdependent. In the second class, mutation of individual elements leaves residual activity, which may be considerable. Human U1, chicken U1 and Xenopus U2 are examples of this type. The Xenopus U2 DSE is composed of an Octamer motif and an Spl binding site which are positioned adjacent to each other, and two, less well defined, elements nearby (D2 in Fig. 1). The D2 activity was not revealed until both the octamer motif and the Spl binding site had been mutated. Thus, single units of this second class of DSE may be partially or entirely functionally redundant. Although DSEs appear to bind transcription factors like Spl which are involved in the transcription of many pol II genes they do not, where tested, efficiently activate transcription from 'TATA'-containing or other types of pol II promoter (Ref. 13 and unpublished data). This characteristic distinguishes the DSE from conventional eukaryotic enhancer elements and suggests that there is something unique

© 1989, Elsevier Science Publishers Ltd, (UK)

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TIBS 14 - January 1989 DSE D2S 0 D2

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Fig. 1. Diagram of the structure of the Xenopus U2 and U6 promoters'. DEE, distal sequence element; PSE, proximal sequence element; O, octamer motif; S, Spl binding site; D2 stands for DSE, U2.

about the DSE-PSE interaction (see below). The PSEs are less well characterized and thus far have only been found in genes encoding short RNAs, including some which are not of the U snRNA type. The Xenopus U2 PSE was initially shown to be located upstream of position - 4 8 and downstream of position - 8 2 (Ref. 4). Many point mutations made in this region have further defined the U2 PSE (unpublished data). Changes within the region from -61 to - 5 6 tend to severely reduce, or abolish, transcription of the U2 gene whereas changes between - 5 5 and - 4 9 generally have a less severe effect. Both sequence comparison of various mammalian U snRNA promoters 3 and mutagenic analysis of the human U2 gene 14 suggest that mammalian PSEs are considerably longer than the Xenopus U2 PSE thus defined. It is therefore possible that in Xenopus this element binds a single transcription factor while mammalian PSEs may consist of two adjacent factor binding sites. However, human and chicken U snRNA genes are transcribed after microinjection into Xenopus oocytes, indicating that differences between vertebrate U snRNA gene promoters are not in essential regions. Nuclear proteins which bind to the Xenopus U2 ~° and human UI ~'~PSEs have been identified but they have not yet been well characterized. Downstream of U snRNA genes is a conserved sequence called the 3' box which directs the formation of the 3' ends of the primary snRNA transcripts 3. However, efficient recognition of the Y-end signal only occurs if transcription is initiated from a U snRNA

gene promoter. In constructs where a U snRNA coding region was attached to an m R N A promoter transcription continued through the Y-end signal and polyadenylation of the transcript occurred at cryptic or engineered cleavage and polyadenylation sites downstream of the 3' box 16"17. Conversely, transcripts initiated at a U snRNA gene promoter can ignore polyadenylation signals and terminate instead at cryptic Y box signals 17. Some upstream element of the U snRNA gene promoter must therefore be involved in Y-end signal recognition. Since it is possible to delete the DSE and to replace much of the U snRNA coding region without affecting Y-end formation, the proximal region of the promoter has seemed the most likely candidate. A study in which the proximal part of the human U2 gene promoter was altered by means of a series of linker-scanning mutations failed to locate any element involved solely in Y-end formation 14. Mutations outside the PSE had little or no effect either on transcription or on Y-end formation, while mutation of the PSE reduced transcription without affecting the efficiency of Y-end formation. Together with the results discussed above this suggests that the combination of a DSE and a PSE produces a unique type of transcription complex which is capable of either altering the termination properties of 'standard' pol II in some way or of selecting an RNA polymerase of unusual subunit composition which recognizes U snRNA gene-specific signals on the DNA. The complicated nature of the signals encoded in the DNA of eukaryotic

promoters is reflected in the large number of sequence-specific DNA-binding proteins that are involved in the activation of transcription. Promoter complexity does not, however, end with these DNA-protein interactions. The individual, DNA-bound, transcription factors interact with one another, and with other, non-DNA bound, components of the transcription apparatus through protein-protein interactions. These interactions strengthen the resulting 'stable transcription complex' such that it remains in place during many rounds of transcription initiation. Stable complex formation has been studied in vitro using many eukaryotic genes transcribed by either pol I, II or III (see Ref. 18 for references). The high efficiency with which U snRNA genes are transcribed in vivo after microinjection in Xenopus oocytes has made them an ideal subject for the study of stable transcription complex formation in vivo. While the PSE and DSE are the only promoter elements required for U snRNA transcription to initiate, they are not sufficient for the formation of a stable transcription complex on the gene is. Two additional requirements for stable complex formation have been identified, in the case of the Xenopus U2 gene, by means of in vivo assays: a short gene internal sequence is required and the nucleotide at the normal point of transcription initiation must be a purine. These results have been interpreted by proposing that pol II must make exact, stereospecifically defined, contact with DNA-bound transcription factors in vivo in order to stablilize the transcription complex on the U2 promoteP s.

TIBS 14 - January 1989

U6, the exception which incorporates the rule Unlike other U snRNA gene promoters, the U6 promoter is utilized by both pol II and pol III, although the transcripts defined as U6 RNA appear to be solely due to pol III transcription 19"2°. The pol II transcripts of the gene are longer, probably heterogenous, and of uncertain significance. U6 RNA accumulation is resistant to high levels of c~-amanitin, requires a run of 4 thymidine residues for termination and can be completed by cotranscription of 5S RNA or tRNA genes, which characterize U6 as an RNA pol III transcript 2°. Despite this the Xenopus U6 promoter is, at le~.st in part, very similar to that of other U snRNA genes (Fig. 1). It has a functional DSE, which contains an octamer rnotif 2~ and an Spl binding site (unpublished data). Deletion of the U6 DSE results in a reduction in the level of transcription analogous to that seen on removal of the DSE from a pol ll-transcribed U snRNA gene. U6 also has a PSE which is functionally interchangeable with the U2 PSE and whose deletion or mutation abolishes U6 transcription 21'22. In addition, U6 contains the sequence T T A T A A G which resembles a pol II T A T A box 5, centred at position -27. Some mutations in this sequence result in the loss of pol III, but not pol II, transcription of U 6 1 9 . Transcription of Xenopus U6 by pol III is also dependent on the sequence around the initiation site. For example, mutation of the purine (G) ',at position +1 to a pyrimidine reduces the level of transcription to less than 5% of wild type. Analysis of various promoter mutants reveals a strong preference for particular initiation sites. The choice of the transcription start site has been proposed to be due to direct interaction of pol III itself with the initiation region 19. Unlike almost all other pol IIItranscribed genes, U6 does not have promoter elements which are located downstream of + 1 (Ref. 23). From the data accumulated on U6 transcription it has been possible to conslruct a U6 promoter which retains sol,ely pol I1 activity. This is accomplished by mutating the 'TATA' box but leaving the PSE and DSE intact. Additionally, the specificity of the Xenopus U2 promoter can be changed from pol II te pol III by the addition of the 'TATA' and initiation elements from the U6 gene (Fig. 2). Although the U6 'TATA' element shows strong sequence similarity to pol II T A T A boxes it is not clear whether

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the transcription factor which recognizes this site in U6 is also involved in the pol II transcription of other genes. The U6 promoter is thus both unusual and complex, incorporating in its pol 1II moiety promoter sequences and types of element which were previously thought to be specific for pol II promoters. This raises the question of whether the pol III responsible for U6 transcription is identical to that which is utilized by classical types of pol III promoter. Following transcription, U snRNAs are immediately transported to the cytoplasm where they undergo maturation and assembly into snRNPs before re-entering the nucleus. In the sections which follow we will describe this assembly process and the R N A protein interactions that are the basis of U snRNP structure.

U snRNP assembly studied in vivo U snRNPs are composed of one (U1, U2, U5) or two (U4/U6) molecules of U snRNA associated with at least seven polypeptides 24. The proteins named B' (29 kDa), B (28 kDa), D (16 kDa), D' (15.5 kDa), E (12 kDa), F (11 kDa) and G (9 kDa) are common to all U snRNPs. In addition, some U snRNPs possess specific proteins: there are three for U1 snRNP, 70K (56 kDa), A (34 kDa) and C (22 kDa) and two for U2 snRNP, A' (33 kDa) and B" (28.5 kDa). Several proteins have been suggested to be associated with U5 snRNP, IBP (100 kDa or 70 kDa) and 25 K (25 kDa). Different subsets of the proteins are recognized by antibodies present in the sera of patients with certain autoimmune disorders 2"24. The antibodies have proved a major tool in the analysis of the struc-

ture of U snRNPs. Some snRNP proteins appear to have been strongly conserved in evolution. For example epitopes recognized by several of the autoimmune antisera have been conserved on snRNP proteins from humans to fungi 25"26. Fungal snRNAs are recognized and bound specifically by Xenopus snRNP proteins 26'27, providing additional evidence of the conservation of functional regions of snRNP proteins. While cDNAs corresponding to several of the snRNP proteins have been isolated 24, thus far the transcription of their mRNAs has not been studied. It will be of interest to determine whether the expression of U snRNP components is coregulated and, if so, how this is accomplished. Analysis of the promoter of a Xenopus U1 70K protein gene has revealed no similarity to either the DSE or PSE elements present in U snRNA promoters 2~. In any case, coordinate accumulation of snRNP components might be more easily accomplished by post-transcriptional regulatory mechanisms. An early step in the structural analysis of snRNPs was the determination of protein binding sites on U snRNAs. They were first defined experimentally by micrococcal nuclease digestion of U snRNPs and the analysis of U snRNA fragments protected against digestion 29. Those studies identified a protected region containing the sequence, PuA(U)~6GPu, which is conserved in U1, U2, U4 and U5 snRNAs. More recently in vitro mutagenesis has been used to dissect the protein binding sites of U1 and U2 snRNAs 3C~32. When mutant Xenopus U2 or U1 snRNAs lacking the A(U)4~6G motif were injected into

18

T I B S 1 4 - J a n u a r y 1989

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Fig. 3. A model of U1 snRNP structure. The protein components (clouds) are drawn on UI RNA. Continuous lines represent regions in volved in RNA-protein or protein-protein interactions essentialfor RNP assembly. Broken lines and crosses represent regions involved in non-essential RNA-protein or protein-protein interactions.

X e n o p u s oocytes, the RNAs were no

longer immunoprecipitable by antibodies recognizing the common sn RNP proteins (anti-Sm antibodies). Conversely, insertion of this sequence into heterologous RNAs resulted in their association with the common snRNP proteins. The binding motif is thus often referred to as the Sm binding site. Binding of the common proteins to this site in U2 and U1 is required for the conversion of the transcriptionally added monomethyl guanosine cap to the trimethyl cap characteristic of the U snRNAs and for the transport of assembled U snRNPs from the cytoplasm to their site of action, the nucleus 3°. The importance of this region for U snRNP morphogenesis probably explains its evolutionary conservation 2~''27. By similar techniques it has been shown that the region essential for binding of the two U2-specific proteins A' and B" is located within the last two (3') hairpin loop structures of U2 snRNA and that binding of the UIspecific proteins 70K, A and C requires the first (5') hairpin of U1 snRNA. The common and unique U snRNP proteins can bind independently but, once bound, protein-protein interactions

between the two groups of proteins stabilize the particle 31-33. The order of assembly of snRNP components has been analysed by sucrose gradient analysis and immunoprecipitation of snRNPs pulse/chase labelled in vivo 34. Evidence has been obtained that an intermediate in snRNP particle assembly is a cytoplasmic '6S core' particle containing the four common proteins D , E , F and G. The next assembly step is probably the addition of U snRNA to the '6S core' particle and finally the addition of B, B' and specific proteins 3°. In vitro assembly

New possibilities for studying snRNP assembly have been created by the development of in vitro systems which offer the advantage of flexibility and ease of manipulation. In the first studies, total human poly(A) + RNAs were translated in wheat germ 35 or rabbit reticulocyte 36 extracts to generate all of the snRNP proteins. Association of the translated proteins with endogenous or exogenously added U snRNAs demonstrated the feasibility of the in vitro approach. Mature X e n o p u s oocytes and eggs

accumulate a large amount of U snRNP protein in their cytoplasm 3°. Taking advantage of this, an in vitro assembly system was developed using X e n o p u s eggs as a source of U snRNP proteins and in vitro synthesized wild type (wt) or mutant X e n o p u s U1 snRNAs. By immunoprecipitation of in vitro assembled particles with antibodies against U1 snRNP components it was possible to define the region of U1 snRNA essential for assembly with Ul-specific proteins to the first hairpin loop structure of U1 snRNA. Other, more sensitive, assays revealed the role of protein-protein interactions and nonessential R N A - p r o t e i n interactions in the stabilization of U1 snRNP 31. In vitro assembly experiments using human U1 snRNA and H e L a cell S-100 extracts have also suggested that the primary binding site for the 70K protein is on the first hairpin and that this protein interacts with the common snRNP proteins 37"3~. Detailed analysis of the protein binding site(s) within the first U 1 hairpin (which are essential for R N A assembly) was performed 32 making use of a set of point mutations in X e n o p u s U1 snRNA and of two plant U1 snRNAs. The plant RNAs differ significantly from X e n o p u s U1 in sequence, but have identical predicted secondary structures. It was shown that the only RNA sequence essential for assembly with Ul-specific proteins is localized in the loop of the first U1 hairpin. Two different double point mutations introduced into this sequence prevented the binding of proteins 70K, A and C. Since it is unlikely that all three proteins interact individually with two pairs of nucleotides this implied that the Ul-specific proteins did not assemble independently on the RNA. Alterations of the stem structure of the hairpin weakened the binding of A and 70K differentially but did not affect the binding of C. Similarly, in high salt conditions, where electrostatic interactions are weakened, it was shown that the common snRNP proteins and C could still bind wild-type U1 R N A while A and 70K could not. These results showed that C could bind U1 RNA in the absence of 70K and A, and implied that electrostatic interactions with the RNA helix were more important for A and 70K binding than for C. A possible model for U1 assembly is suggested by these data and kinetic experiments 3~. In this model U1 snRNP assembly is driven by specific binding of protein C and the common proteins to the RNA. Interestingly, the

TIBS 14-January 1989 RNA sequences essential for binding are in both cases regions of U1 R N A predicted to be single-stranded. The proteins apparently bind te the RNA in a sequence-specific manner. Subsequently proteins A and 70K associate with the RNP probably mainly by protein-protein interactions, although their binding is likely to depend partly on contacts with the RNA helices or bases which are not essential for assembly. The elements of this assembly model are incorporated in the U1 snRNP structure shown in Fig. 3. Sequence comparison A very different way to predict protein binding sites on RNAs is the comparative analysis of RNA sequences. Protein binding sites are predicted to be among the elements conserved in primary sequence ant secondary structure when phylogenetically distant RNAs are compared. U snRNA sequences from many species are now available and their comparison reveals that the regions essential for protein binding (for example, the loop sequence of the first U1 hairpin, the last U2 hairpin loop, the Sm binding site) have been highly conserved 39. The significance of other conserved regions whose function is currently unknown will have to be tested experimentally. Prospects U snRNA genes have several features which distinguish them from other pol II- or pol III-.transcribed genes. The fact that the U6 gene is transcribed in vitro in standard pol III transcription extracts should allow a biochemical approach to understanding the basis of these differences. U snRNA gene-specific transcription factors can be purified and their interactions with other comportents of the basic transcription machinery analysed. Many snRNP protein cDNAs have now been isolated. Analysis of the predicted amino acid sequences has already revealed features which are common to different snRNP proteins and the basis of the imraunological relatedness between some ~nRNP proteins is under study. The availability of genomic and e D N A clones ~or both the protein and RNA snRNP components, in combination with antibody probes, will enable analysis of the synthesis of snRNP components and reveal whether mechanisms to coordinate their production exist. The structure of RNA binding domains in the proteins can also be analysed by utilizing the

19

cDNAs. The way is almost open for a complete in vitro assembly system allowing the production of snRNPs of defined composition whose interactions with other snRNPs and components of the splicing machinery can be tested. Acknowledgements We thank Graham Tebb and J6rg Hamm for many discussions, improvements to the manuscript, and unpublished data. We also thank Angus Lamond and David Tollervey for criticism of the manuscript and Heide Seifert for secretarial help. We apologize to the colleagues whose original references we were unable to quote for reasons of space.

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