RNA processing

RNA processing Donald University

of California,

C. Rio Berkeley,

California,

USA

Significant progress has been made over the last year in our understanding of the roles that RNA-binding proteins play in pre-mRNA splicing, the components of the spliceosome and how these components relate to the mechanism of splicing. Of particular importance has been the sequence analysis of the first mammalian splicing factors and structural determination of an RNA-binding domain.

Current

Opinion

in Cell

Biology

Introduction

of eukaryotic

4444-452

action intermediates and products. This work led to the understanding that splicing of pre-mRNAs proceeds via a two-step transesterification reaction with the generation of 5’ exon and intron-3’ exon intermediates. Both the intron product and intron-3’ exon intermediate exist as branched RNA lariat species in which the 5’ guanosine residue of the intron is joined to an adenosine residue in the intron via a 2’-5’ phosphodiester bond [ 1**,2*.4°].

During the biogenesis of functional mRNAs. rRNA and tRNAs in eukaryotic cells, the primary transcription products undergo an extensive series of RNA-processing reactions in the nucleus before export to the cytoplasm. In this review, the primary focus will be on recent insights into the process of RNA splicing, which leads to the generation of eukaryotic mRNAs. Splicing results in the removal of introns from pre-mRNA and it joins exons to produce a functional mRNA Splicing occurs in all eukaryotic organisms from yeast to Drosophila to humans. These three organisms each have their own advantages for studying the splicing process. Yeast provides an opportunity to combine genetics with biochemistr) to gain an understanding of the genes and gene products involved in pre-mRNA splicing. Drosophila has also yielded important insights into the regulation of altemative pre-mRNA splicing using genetics and biochemistry. The mammalian system has formed the basis for detailed biochemical identification and study of the structure and function of splicing factors. This review will focus on pre-mRNA processing and on the determination of the structure of an RNA-binding domain found in a number of heterogeneous and small nuclear ribonucleoproteins (hnRNP and snRNPs) and pre-mRNA splicing factors. A number of reviews of this field [ 1**,2*-4*] and of the 3’ end processing of mRNAs (5-71 have appeared recently and the reader is referred to these for background or additional reading. This article will not deal with the interesting topics of RNA transport, self-splicing (catalytic) RNAs, or RNA editing, which have also been recently reviewed [%ll]. The mechanism splicing

1992,

These cleavage-ligation reactions in pre-mRNA splicing take place in a large ribonucleoprotein complex that has been dubbed the ‘spliceosome’ [ 1**,2*,4*]. The abundant snRNPs LJI, Ll2, LJw’LJGand LJ5 are integral cmlponents of the spliceosome. The snRNPs are themselves made up of snRNAs and a set of common, as well as snRNP-specific, proteins. A variety of studies have shown that the snRNPs assemble on the pre-mRNA substrate in an ordered fashion to form the mature catalytically ac tive spliceosome [ 1l *.2-,4*] Ul snKNP recognizes and binds to the intron 5’ splice site, a signal in the RNA that marks the 5’ end of the intron. U2 snRNP then binds to the intron branchpoint sequence near the 3’ splice site. Unlike the Lll snRNP-5’ splice site interaction, the U2 snRNP-branchpoint interaction requires ATP and accessory protein factors [ 1**,2*,4*). This Ul-U2 snRNP-pre-mRNA complex then binds the U4Ub/U5 trisnRNP complex in further ATP-dependent steps to generate the mature spliceosome in order to begin catalysis of intron removal. It has been estimated that 5ClOO pro teins are required for spliceosome assembly and splicing [ 1**,2*,4*,12*].

In addition to the sheer complexity of the basic or general pre-mRNA splicing reaction, a variety of alternative intron splicing patterns can occur [ 13,14**]. This cornplexity is best illustrated by the human dystrophin gene that spans 2 megabases of DNA and contains at least 65 introns. Thus, the complexity of accurately and reproducibly recognizing splice sites and removing introns within large pre-mRNAs is an interesting problem in bi-

pre-mRNA

The pathway of eukatyotic pre-mRNA splicing has been determined from the analysis of in l&l-o and in lu’llo reAbbreviations dsx-double-sex;

hnRNP-heterogeneous

pPTB-polypyrimidine snRNP-small

nuclear

tract-binding; nuclear

RNP; suwa-

@

Current

IVS&P-transposable

ribonucleoprotein;

PRP-pre-RNA

processing;

suppressor-of-while-apricof; Biology

Ltd

ISSN

RNP-CS-RNP

Sxl-Sex-lethal; 0955-0674

element consensus

third type;

tra--lrans/ormer;

intron;

RNA

ological recognition and one that will occupy the minds of people in the splicing field for years to come. Pre-mRNA

splicing

in yeast

In the budding yeast Saccharomyces cereuisiae, it has been possible to dissect the process of pre-mRNA splicing using a combination of genetics and biochemistry [2=,4=]. A number of mutations, known as pre-RNA processing (PRP) mutants, can block pre-mRNA splicing at different steps in the reaction pathway. Furthermore, in many instances, these temperature-sensitive mutations also show temperature-dependent activity in rlitro, allowing a biochemical assay for the wild type or mutant gene product in splicing. A number of genetic approaches have been used in yeast to isolate specific mutations that effect particular steps in the splicing pathway or to detect interactions among different components of the spliceosome. For instance, analysis of the PRP 16 mutant [ 151, which was isolated as a suppressor of a pre-mRNA branchpoint mutation, indicated that PRP 16 showed sequence homology to a family of proteins (DEAD family) known to have ATPdependent double-stranded nucleic acid helicase or unwinding activity [ 161. A number of other PRP mutants encode products that are also members of this family. More recently, it has been demonstrated that PRP 16 has an intrinsic RNA-dependent nucleotide hydrolysis activity [ 17**] and that the purified protein can complement immunodepleted spliceosomes that are blocked after the first step in the splicing reaction [ 17**]. Another member of the DEAD family, PRP 22, is required for the release of mRNA from spliceosomes. The PRP 22 protein also shares homologous regions with bacterial ribosomal protein Sl and Escherichia co/i polynucleotide phosphorylase [ 18.1. These similarities with translation proteins may not be fortuitous since both processes involve large ribonucleoprotein complex assembly, d&assembly and recycling. Furthermore, the requirement for multiple ATP-dependent assembly and conformational steps in splicing suggests an analogy between translation and splicing at the level of kinetic proofreading, a process by which each nucleoside triphosphate hydrolytic event is used as a checkpoint for fidelity of complex multistep assembly processes [ 151. Other PRP genes are also members of the RNA helicase family of proteins such as PRP 28 [ 191 and PRP 2 [20], suggesting that multiple RNA duplex unwinding events are required for the proper assembly and conformational changes of the spliceosome, as well as perhaps for the actual execution of the splicing transesterilication reactions [2*,4*,16]. Genetic experiments have also examined the interaction between Ud and ~6 snRNP. Identification of suppressors of a U4 snRNA mutation identified a ~6 snRNP protein (PRP 24) with an RNP consensus-rype (RNPCS) RNA-binding motif and led to identification of the PRP 24 binding site in ~6 snRNA [21]. This study illustrates the power of genetic analysis to identify molecular interactions among spliceosomal components. The role of Ul snRNP in 5’ splice site recognition has been known for many years, yet the detailed mecha-

processing

Rio

nisms by which Ul snRNP discriminates among 5’ splice sites are not understood. Biochemical studies in both yeast and mammalian cell extracts have shown that Ul snRNP plays an early and hierarchical role in spliceosome assembly [ 1==,2*--4*]. In yeast, an additional role of Ul snRNP in recognition or promoting the stability of complexes formed at the branchpoint sequence has been suggested by in zlitro experiments using wild type or mutant pre-mRNA substrates with lesions in the branchpoint sequence or in biochemical competition experiments, as well as from experiments using extracts derived from Ul or U2 snRNP-depleted cells [ 221. Supporting this ida, it has been possible to demonstrate an effect of Ul snRNA mutations on 3’ splice site selection [ 231. A different set of experiments in mammalian cell extracts supports the idea that Ul snRNP can influence the stability of U2 snRNP-branchpoint interactions, in addition to the well documented role of Ul snRNP in 5’ splice site recognition [24*-l. A recent study clearly demonstrates that in mammalian extracts, as well as in yeast, Ul snRNP can form stable ATP-independent complexes with the 5’ splice site [25]. An understanding of the mechanism by which Ul snRNP selects and stably interacts with 5’ splice sites will be essential to understanding mechanisms of splice site selection in complex pre-mRNAs. Recently, mutational analysis of Ul snRNA has yielded suppressor mutations (second site-unlinked mutations) in a consenfed loop of U5 snRNA, raising the interesting possibility that in the catalytically active spliceosome U5 snRNA may be closely juxtaposed to the 5’ splice site or Ul snRNA itself [ 26.1.

A number of examples of ‘regulated’ splicing of yeast pre-mRNA.s have appeared during this past year. Perhaps most interesting is the demonstration of meiosisspecific splicing in yeast [27**]. The yeast MEh2 gene is transcribed during both meiosis and mitosis, but is only spliced properly to give a functional product during meiosis. This regulated splicing event is dependent upon the product of a second gene, MEi71, which is also implicated in control of the splicing of other premRNAs [ 27**]. A role of RNA structure and RNA-protein interactions in splicing control was suggested by experiments on the yeast L32 ribosomal protein gene (RPI32) intron where mutations that abolished an RNA stem-loop structure near the 5’ splice site prevented regulation of splicing by the L32 protein [ 28**]. Several other examples of alternative pre-mRNA splicing in yeast have been studied with artificial genes carrying duplicated 5’ or 3’ splice sites. The results show that a U-rich tract adjacent to the 3’ splice site can activate splice site usage when it is in competition with a non-U-rich tract containing 3’ splice site [29]. Similar 3’ splice site competition results based on pyrimidine tract sequences were observed in mammalian cells (see below) [30]. Duplication of 5’ splice sites demonstrated cis-competition and the fact that an adjacent 5’ splice site can influence 5’ splice site usage [23]. This idea is important in the context of understanding splice site selection and is related to similar experiments in mammalian cells [ 31-341. Yeast genetics should allow identification of genes whose products can influence 5’ or 3’ splice site selection in these assays.

445

446

Nucleus

andsgene

expression

. Mammalian

splicing

factors

splicing pathway and immunodepletion-complementation experiments have indicated that SC-35 is an essential splicing factor [ 491. Recent gene cloning analysis indicated that SC-35 is similar in size and sequence to SFZ/ASF, and like SF2/ASF, contains an RNP-CS RNAbinding domain and an R/S domain [50*]. However, there is enough divergence in sequence to be sure that, although similar in molecular weight, SC-35 is not identical to SF2jASF. Another factor, SF-88, was similarly identified, using monoclonal antibodies, but its analysis has only just begun [ 511.

Biochemical studies have indicated that in addition to the spliceosomal snRNPs, a myriad of additional protein factors are required for pre-mRNA splicing [ 1**,2*,4*]. The use of biochemical complementation assays and RNAbinding assays have provided a means to begin to identify factors essential for the early steps of spliceosome assembly and splicing [I**]. These assays have led to the identification of many such factors [ 1**I, the purification of four of them to homogeneity, and the isolation of cDNA clones for four of them. One factor, SF2/ASF, was identified independently by its ability to complement a cytoplasmic snRNP-containing S-100 extract for splicing [35*] and by its ability to shift splicing from the SV40 T (distal) to the t (proximal) 5’ splice site [36**]. These assays led to the purification of SF2/ASF to homogeneit) as a 33-kD polypeptide [ 35*,3ti*]. Gene cloning analysis over the past year has shown that SF2IASF is related in sequence to the Drosophila splicing regulators IEW.$O~-~?WI (&a), suppressor-of-‘whiteapricot (SN”‘C) and ttu.?, that have either an arginine-serine (R/S) dipeptide repeat domain and/or an RNP-CS RNA-binding domain [37**,38**]. SF2/ASF is required for spliceosome assembly and can shift 5’ splice site usage from distal to proximal sites itI zjitro [ 39=*], Another identified acthity, SF5 or DSF, counteracts the action of SF2iASF and directs splicing to distal 5’ splice sites [40*,41*]. The SF5 activity was purified and shown to correspond to hnRNP Al [ 400]. The recombinant SF2/ASF protein expressed and purified from E. co/i possesses all the activities of the native protein isolated from mammalian cells [ 37**,38**]. The availability of this cDNA clone should allow a structure-function analysis of the SF2/ASF molecule and lead to elucidation of the role of the R/S domain in splicing. There is some suggestion that the R/S domain may function as a nuclear, and possibly a splicing organelle, targeting signal [ 421. Inconsistent with this simple idea, however, in zgitro splicing assays suggest that the R/S domain is essential for splicing activity, indicating that it must serve a more active role in splicing than simply that of a nuclear targeting signal [43**1.

U2AF, another essential splicing factor, is required to rem cruit U2 snRNP to the intron branchpoint [l**]. This factor was shon;n to interact with the intron polypyrimidine tract [l**] and apparently acts in conjunction with two other factors, SF1 and SF3, to mediate U2 snRNP-branchpoint binding and early splicing complex assembl!, [52]. U2AF w;ts purified to homogeneity and consists of two subunits of 65 and 35 kD [I**]. It has been shown that the 65.kD subunit is sufficient for all the activities ascribed to LJiXF [ 53**], Furthermore, U2AF activity was detected in Drosophila splicing extracts and two proteins that crossreact with U2AF subunit-specific antibodies may correspond to the two Drosophila U2AF subunits [ 53**] Thus, it appears that many splicing conlponents, in addition to the U snRNPs, will be conserved between Drosophilu and mammals. Gene cloning analysis indicates that the 65.kD U&F subunit contains an R/S domain and three RNP-CS RNA-binding motifs [43**]. The R/S domain is required for splicing activity in rlitro and all three RNP-CS motifs are required for RNA-binding activit) [43**].

Interestingly, there is apparently a functional homologue of SFZ/ASF in Drosophila. A monoclonal antibody raised against Xenopus oocyte nuclei cross-reacts with a farnil) of nuclear phosphoproteins in mammals and Drosophilrr [44,45*]. A DrosopMu protein, SRp55, was isolated that cross-reacts with this antibody [45*]. SRp55 will functionally substitute for mammalian SF2/ASF in in t+tro splicing assays, and gene cloning analysis indicates that this protein is highly homologous with mammali*an SF~IASF [46*]. The Drosophila homologue of SF2/ASF was independently identified by an antibody to a chromatinassociated protein, B52, which is present in transcribed chromosomal regions [ 471. Therefore, these proteins seem to be members of a larger family of proteins that contain both RNP-CS RNA-binding domains and R/S domains (see below).

Thus, the theme emerging from these biochemical and gene cloning studies is that many splicing factors will contain RNP-CS type RNA-binding domains (see below) and R/S domains. The RNP-CS motif is required for interaction with pre-mRNA and the R/S domain is required for splicing activity. It will be critical to understand the role and interactions of the R/S domain in splicing to gain further mechanistic insights into the regulation of pre-mRNA splicing.

Splicing factors have also been identified by other means. For instance, the SC-35 antigen was identified with a series of monoclonal antibodies raised against purified spliceosomes [48]. This factor appears to act early in the

Another protein that interacts with intron polypyrimidine tracts is found in spliceosomes and its binding correlates with splicing activity. This polypyrimidine tract binding protein (pPTB), which has been purified to homogeneig and cDNA clones for which have been isolated and sequenced, shows homology to the RNA-binding domains of hnRNP L and the Drosophila elaztgene, both of which contain RNP-CS binding domains [54*,55-l, The binding of this protein correlates with the use of 3’ splice sites that contain extended polypyrimidine tracts in situations where 3’ splice site competition exists, such as in the ptropomyosin gene (301.

Alternative

pre-mRNA

splicing

in mammals

Numerous examples of alternative pre-mRNA splicing have been described using mammalian cells [ I-*,13,1 ?**I. It is conceivable that alternative pre-mRNA splicing could play a central role in the diversification of mammalian Cell types, particularly in those of the nervous system. In the

RNA

past year, several studies have analyzed the splicing control of tissue-specific mammalian exons. In a study of the preprotachykinin gene, which contains an exon that is normally excluded in HeLa cells and extracts but is included in other cell types (56,571, it was shown that Ul snRNP binding to a downstream 5’ splice site can influence splicing of an upstream intron [56]. This result is interesting because it implicates an interaction across an exon in the process of exon exclusion. Splicing control of the cellular SWproto-oncogene, which contains a small neural-specific exon that is included in the mature neural SK mRNA but not in other cell types, was investigated by Black [ 581. Using transfection assays, he showed that inclusion of this exon could be activated in neurons by increasing the size of the neural-specific exon, suggesting that inhibition of inclusion occurred by interference between splice sites at the exon terminii. Again, the unit of regulation is the exon; however, inclusion is achieved by a different mechanism, namely steric interference. The NWIgene provides another example of a neuralspecific splicing event [ 591. This gene encodes a cell adhesion molecule that is thought to play an important role in cellLcel1 interactions in the nervous ,system and is related to immunoglobin molecules. In this case, two isoforms of N-C44 are generated by exclusion or inclusion of a particular exon; inclusion of the exon increases with neuronal differentiation and seems to be regulated in part through the 5’ splice site of the optional exon (downstream intron). Replacement of this 5’ splice site with the a-globin 5’ splice site resulted in constitutive use of the optional exon in undifferentiated cells. Moving the N-GM 5’ splice site into a globin exon caused the exon to be partially excluded in undifferentiated neuroblas toma cells. The level of exon exclusion decreased when the cells underwent differentiation, indicating that part of the tissue-specificity of this exon splicing is derived from this 5’ splice site region. The P-tropomyosin gene, in both rat and chicken, has provided another system with which to study alternative splicing because there are different patterns of mutually exclusive splicing in two different cell types: skeletal muscle and fibroblasts. Recent studies have pointed to a role for RNA secondary structure [GO], as well as RNA-protein interactions [61] 1 in the control of this splicing event. Thus, mammalian cells provide numerous models to study alternative pre-mRNA splicing mechanisms and to understand the diversification of cellular phenotypes through the process of alternative splicing. Alternative

pre-mRNA

splicing

in Drosophila

In Drosqpbika, genetic analysis has been used to identie a number of loci that can alter splicing patterns in a variet) of genes. For example, control of somatic sexual differentiation depends on a cascade of alternative splicing events [ 621. A number of gene products involved in these splicing decisions have been identified and shown to contain RNP-CS domains and/or R/S domains similar to the mmmalian splicing regulators described above [ 621. Clearly, a common theme in the control of both splice site selec-

processing

Rio

tion and alternative splicing events is the involvement of RNA-binding proteins carrying the RNP-CS motif and/or the R/S domain. Three examples of negative control of splice site choice have been described in Drosophila. The Sex-lethal (Sxl) protein, which contains two RNP-CS motifs, appears to repress the use of a male-specific 3’ splice site in the Sxl gene itself in an autoregulatory mode [63] and also acts to repress the use of a non-sex specific 3’ splice site in the tra gene, situated downstream in the pathway of sexual differentiation [62]. Both molecular genetic and biochemical experiments indicate that Sxl acts by binding to the polypyrimidine tracts of the introns it controls [ 641. Another example of negative splicing control is the germline-specific splicing of the P transposable element third intron (NS3) [65,66]. Genetic and biochemical experiments have indicated that at least one aspect of this control lies in the repression of this splicing event in somatic cells, resulting in the retention of the third intron in the mature somatic mRNA [67*,&,69]. Competition between the accurate 5’ splice site and pseudo-5’ splice sites in the adjacent 5’ exon seems to play a role in somatic inhibition of NS3 splicing [ 67*]. Mutations in the 5’ exon that disrupt these 5’ splice site-like sequences activate third intron splicing both in zklo in somatic cells of transgenic Drosophkr (681 and in LYtro in somatic cell extracts [67*,69,70**]. An excess of this inhibitory exon RNA sequence relieves the inhibitory effect observed in Drosopbilu somatic cell and mammalian nuclear extracts iu zjitro [69,70**], suggesting that this exon RNA fragment titrates inhibitory factors away from the pre-mRNk Furthermore, the inhibitory activity correlates with the binding of several somatic Drosophila RNA-binding proteins to the inhibitory RNA target site in the 5’ exon [ 67*,70**]. These RNA-binding proteins appear to act by influencing U1 snRNP binding in zjitro [ 70**]. The further characterization and identification of these RNA-binding proteins should help to determine their tissue distribution and clarify which of them might be responsible for the different splicing patterns of lVS3 in the germline and soma. A third example of negative splicing control in Drosophila involves the SZI”‘~ gene [71] The suw protein, which contains an R/S domain but no RNP-CS motif, autoregulates the splicing of the SZ~~‘~ pre-mRNA to prevent synthesis of the protein by blocking removal of one of the SZI~~‘~ mtrons. Thus, several examples of splicing control in Drosophikz involve the repression of splice site use in alternative splicing decisions. One example of positive control of pre-mRNA splicing in Drosophila has been described. Genetic experiments have shown that the female-specific 3’ splice site of the double-sex (C&Y) gene, another in the sex determination pathway, is activated by the products of the tra and tra3 genes [62], It is the female product of the dsh- gene that is then responsible for directing further female development [ 621. Sequencing of the tra and tra-2 genes has revealed that the tra protein possesses an R/S domain [62] and that the tra-2 protein has RNP-CS and R/S domains [62]. A tra-2 protein can also act to autoregulate splicing of the trn-2 gene itself and it is clear that the tra-2 primary transcript is subject to alternative RNA splicing in a tissue-specific manner 172,731. Genetic ex-

447

448

Nucleus

and-gene

expression

. periments in Dmsopbih implicated a region near the dsx female 3’ splice site as the target of traand tra-2 protein action [74]. A series of tissue culture transfection experiments indicated that the tra and u-a-2 proteins, activate the female dale 3’ splice site by interacting with the regulatory site in the downstream 3’ exon, which consists of six repeats of a thirteen nucleotide sequence [75*=-77**]. These repeats were necessary for positive regulation [75**-77*=] and interacted directly with m-2 protein in vitro [75**]. Tra and tra-2 proteins also appeared to activate female-specific polyadenylation [75**] or a cryptic 3’ splice site [n**] in the absence of the regulated 3’ splice site. Recently, it has been shown that recombinant tra and tra-2 proteins can activate the u!s.x female as well as heterologous 3’ splice sites in a &x 13. base repeat sequence-dependent manner in vitro [78-l. It is possible that tra and tra-2 proteins both function to activate the c&x female-specific 3’ splice site by stabilizing an otherwise weak interaction between U2AF [53**] or other 3’ splice site recognition factors [52] and the rather poor polypyrimidine tract of the ds~ femalespecific intron. Alternatively, the tra and tra-2 proteins could act simply to alter the conformation of the &.Y pre-mRNA to improve its recognition as a substrate for splicing in females. Both these mechanisms are consistent with a role for these regulatory proteins early in intron recognition, splice site selection and spliceosome assembly, and are in line with the mammalian biochemical studies of the early steps of spliceosome assembly. Furthermore, both the dsv positive control and P element negative control require regulatory sequences in adjacent flanking exon sequences, rather than intron sequences. These results support the idea that exon sequences can influence splice site selection, possibly by forming mukiprotein-RNA complexes that might contain splicing factors as well as hnRNP proteins [ l**,l2**,25]. Cell biology

of pre-mRNA

splicing

The availability of antibodies to snRNP protein components and mammalian splicing factors, and the development of 2’ -O-methyl RNA oligonucleotides as probes for spliceosomal components has allowed their visualization and localization within mammalian nuclei and amphibian oocytes [79--85,86*=]. These studies suggest either that splicing takes place in discreet subnuclear locations or that spliceosome assembly occurs at delined ‘depots’ within mammalian nuclei. A series of antibody staining and in situ hybridization experiments in amphibian oocytes using antibodies to the snRNP Sm antigens and RNA probes for snRNAs has led to the observation of three types of ‘snurposomes’ (A, B and C) [86**]. Most importantly, the ‘B’ snurposome has all the components that are present in mammalian spiiceosomes, including the SC-35 spliting factor [85,86@*]. These studies raise the possibility that B snurposomes are sites of spliceosome assembly and that, following assembly, these particles may move along chromosomal fibers to sites of nascent transcription to carry out splicing. h mammalian cells, it has been known for many years that the snRNP Sm antigens appear as 20-50 bright

spots or ‘speckles’ in the nucleus as well as showing a diffusely stained background (see [79] >. More recently, it has been shown that these speckles also stain with antibodies to the SC-35 splicing factor [ 481. Using confocal and electron microscopy, the SC-35 antigen was shown to localize to interchromatin granules and perichromatin iibrils [49]. It is conceivable that the speckles are analogous to B snurposomes in amphibians because it has been suggested that the sites of transcription and the location of speckles within mammalian nuclei may be distinct [79], consistent with the idea that speckles may be sites of spliceosome assembly [48,49]. However, a recent report suggests that nacent transcripts of the mouse c-fa gene may be associated with the speckles [ 821. Interestingly, the Dmsopbi[a homolog of mammalian splicing factor SF2/ASF, B52 or SRp55, is associated with transcribed regions of Drosophziz polytene chromosomes [ 450,471. Thus, although speckles contain snRNPs and splicing factors, it has not been determined whether they represent sites of spliceosome assembly or splicing. The availability of biotinylated antisense 2’ -O-methyl RNA okgonucleoddes has provided a means of locakzing snRNA-containing organelles in mammalian nuclei by i?z situ hybridization [80,82&i]. In contrast with the results obtained with anti-Sm antibodies, in situ hybridization of mammalian cells either after fixation [83] or after direct microinjection of living cells (841 has identified a distinct pattern of 5-10 brightly staining ‘foci’. These foci are larger and fewer in number than the speckles observed with anti-SC-35 and Sm antibodies. They are also stained with antibody to the mammalian splicing factor U2AP [53**]. It has been suggested that they may correspond to coiled bodies, a subnuclear organelle characterized by electron microscopy [81]. The relationship between foci and speckles is not clear. However, a recent report using biotinylated antisense 2’.O-methyl RNA probes [80,81] shows that after longer periods of hybridization, speckles rather than foci are observed, indicating that the foci may only be detected by virtue of a more highly concentrated snRNA composition. Regardless of the relationship between speckles, foci and splicing, it is clear that snRNPs and splicing factors can be detected in a discreet rather than uniform distribution in mammalian nuclei and that these sites may represent either storage/assembly sites of spliceosomes and their constituents, or perhaps a morphologically distinct nuclear organelle in which splicing occurs.

The ribonucleoprotein RNA-binding domain

consensus

sequence

A number of RNA-binding proteins, including the yeast poly A binding protein, hnRNP, snRNP and more recently Drosophila and mammalian splicing factors, contain an 80-90 amino acid domain, the RNP-CS, which is known to be responsible for mediating RNA binding (87’.,88,89]. This motif has since been found in a large number of proteins with diverse functions in RNA metabolism, including pre-mRNA splicing factors (see above). Analysis of several members of this family, such as the Ul snRNP 70K and A proteins, has shown

RNA

that the RNP-CS domain is indeed sufficient to bind RNA in the absence of other regions of the molecule [87**].

Rio

processing

for determining how splicing factors act to specify or alter splice site selection and spliceosome assembly. Conclusion

helix

A number of important advances in our understanding of basic and regulated pre-mRNA splicing have been made during the past year. In yeast, evidence has emerged that ATP hydrolysis and RNA unwinding may play key roles in the catalysis and fidelity of intron removal. In mammalian cells, several splicing factors have been purified, cloned and sequenced, and all show similarities to Drosophila regulators that contain RNP-CS RNA-binding domains and/or R/S dipeptide repeat domains. The availability of these molecular probes and in vitro splicing assays should lead to rapid insights regarding the biochemical basis of splice site selection and the RNA-protein and protein-protein interactions involved in the early steps of spliceosome assembly. These studies should also provide a mechanistic basis for understanding the action of RNA-binding proteins in RNA splicing and its regulation. Analysis of the structure of an RNP-CS type RNA-binding domain will provide a framework within which the mechanism of splicing factor action will be understood in the near future. Acknowledgements

Fig. 1. Schematic ribbon representation tein domain of the A protein, which antiparallel P-sheet (PI@4 and two the sheet.

of consists a-helices

the ribonucleoproof a four-stranded (A and B) behind

The ability of the isolated 90 amino acid RNP-CS domain of the Ul snRNP A protein to interact specifically with stem-loop II of 111 snRNA [90,91] has allowed a detailed structural analysis of this peptide, both by X-ray crystallography [92*] and by nuclear magnetic resonance [ 93’1. Although the structures determined by the two methods differ in detail, the overall secondary structural motifs are identical, consisting of a four-stranded antiparallel P-sheet with two ct-helices behind the sheet (Fig. 1). The most highly conserved regions within the RNP-CS domain are designated RNP-1 (KjRG F/YG:AFVX F) and RNP-2 (I,/1 F/t’V/I G/K N/G) [87**,88]. The RNP-1 and RNP-2 motifs contain hydrophobic amino acids that are brought together in the two central strands of the B-sheet. Based on a variety of mutagenesis and RNAbinding experiments [94] and because two aromatic amino acid residues in RNP-1 and RNP-2 can be covalently cross-linked to RNA [87**], it is presumed that the RNA lies across the p-sheet. Amino acids outside the P-sheet are also involved in RNA binding because a number of basic amino acids, including a conserved arginine residue that is essential for RNA binding, are located in loops adjacent to the surface of the p-sheet. Arginine ma) serve a critical role in the recognition of RNA by proteins as demonstrated for the human immunodeficiency virus Tat-tar interaction, where a single arginine residue in Tat forms an ‘arginine fork’ with two adjacent phosphates in the lur RNA [95**,96]. Thus, an understanding of the mechanism of RNA-protein recognition will be critical

I would like to thank my colleagues in the field for providing preprints of manuscripts and partkulariy MR Green, AR Krainer. T Maniatis. and R Reed for helpful discussions. Many thanks to DL Black for critical reading of the manuscript and to A Kron for help in its preparation. Work in my laboratory is supported hy the NIH CR01 HD28063-Ol ), the NSF (DMB 8757176), and the Lucille 1’. Markq Charitable Trust. I am supported by a Lucille P. hlarkey Scholar Axtrd in the Biomedical Sciences.

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and of 5’ splice

in Yeast.

snRNP-

Biodem

Sci

site recog-

Trends

Gene!

.

5.

SACHS

A: The Role of poly(A) in the Translation and Stability of mRNk Cwr Opi17 Cell Biol 1990, 2:1W2-1098.

6.

WICKENS

of poly(A) 153277-281.

M:

the

How

in

the

Messenger Got Its Tail: Addition Nucleus. Tre?ub Biocbem Sci 1990,

449

450

Nucleus

and gene expression

PROUDFOOT

N: Poly(A)

Signals.

10.

CECH TR: Self-splicing 1990. 59:543-568.

Il.

CECH TR: RNA 1990,64:667A69.

of Group

Editing:

the

641671-679.

CeN 1991.

MATI-AJ W: Splicing Studies and poly(A) RNA Processing and Transport. Corn2:52%538. MAQUAT LE: Nuclear mRNA Transport. 1991. 3:100+1012.

C11rr

Opirf

Cell

Rio/

A???zrr Ret, Rimhem

Smallest

Intron.

Cc//

12. ..

REED R: Protein Composition of Mammalian Spliceosomes Assembled in Vitro. Proc Nat/ Acad Sci i.‘.SA 1990, 87:8031*035. The protein complexity of purified mammalian spliceosomcs is revealed in elegant derail. 13.

SIIITH CW, PAITON JG. NXIA&GINARII in the Control of Gene Expression. 23~527-577.

MANtins T: Mechanisms of Alternative pre-mRNA Splicing. Scimce 1991, 25 1133-3-1. %is perspective gives an outline of recent mcqhanistic studies of regulated premRNA splicing. It compares JilTerent csamples of regulated splicing and describes the possible mmhanisms involved

16.

BLIRGE~ S, C~[~TO JR, GIUHRIE C: A Putative Protein Influences the Fidelity of Branchpoint in Yeast Splicing. Cell 1990, 60:705-71’. ~‘A%!MMAN

1991.

DA, STEIN/. JA 349:46316%

Alive

with

ENGEU~R!X~I’ J. \‘lxKt3.-blrilhlAN cific RNA Splicing in Yeast. L elegant set of genetic experiments of pre-mRNA splicing of yeast.

DEAD

ATP Binding Recognition

Proteins.

19.

PA-ITEIWN R. GI-~IIRIE C: A U-rich Tract Enhances Usage of an Alternative 3’ Splice Site in Yeast. C~J// 1991. 64:1X1-1X7.

30.

M~‘IJX.S MP. Shmii Cal. PA’I-I’ON JG. NAI)AI..GIN,UII) Tropomyosin Mutually Exclusive Exon Selection: tition Between Branchpoint/Polypyrimidine Tracts mines Exon Choice. Getrc>s /%>I, 1991, 5:6*2455.

31.

NI:I.\ON tih;. GKIZN XIR: Splice Site Selection and Ribonucleoprotein Complex Assembly During it1 Vitro pre-mRNA Splicing. <;(,I/<+ /I(,/’ 19HH. 2.3 I %3?9

37.

NEI.WS KK. GWES hlR. Mechanism for Activation During pre-mRNA Splicing. I ‘.\‘:I 1990. 87:6Lij625-!

33

LI~AH AL, EI~I~HOS I.P. Will:,vriJiy IM. EI’ERON IC. Hierarchy 5’ Splice Preference Determined in Vim .I .1/o/ Hid 21I:lO?-I Ii.

3-l.

CI%NIN(;II,L\I SA. Ei.sii AJ, IWii-I% Wl, EIWON ences of Separation and Adjacent Sequences of Alternative 5’ Splice Sites. ./ .Ilo/ Hid lo?l.

35. .

KIWNFH AR. Co~a,hy GC. Ko;r,\~ acterization of SF2. a Human Gcvrer Ik/’ 1‘990. 4: I 15% 1 1’1.

/Q&or

.%XWX.R B. GtITHRIE C: PRPl6 is an RNA-dependent ATFQse that Interacts Transiently with the Spliceosome. rVattrre 1991, 349:49-i-399. This paper demonstrates RNA-dependent ATPase activit) for the PRP16 protein and identilies the role of PRPl6 in yeast pre-mRNA splicing.

17. ..

18. .

COMPANY M. AKENAS J. AHELWN J: Requirement of the Helicase-like Protein PRP22 for Release of Messenger From Spliceosomes. A’rrture 1991, .ti9:+87--l93. Biochemical analysis of the PRP 22 protein in ytasr splicmg. 19.

20.

S’I’RA~~SSEJ, GL~THIU~ C: A Cold-sensitive tant is a Member of the RNA Helicase Dell 1991. 5:629&l. CIiEN J-II. dependent with Two Res 1990.

RNA RNA

mRNA Splicing MuGene Family. Ge,~es

LIN RJ: The Yeast PRP2 Protein, a Putative RNAATPase. Shares Extensive Sequence Homolop;) Other pre-mRNA Splicing Factors. Nuckic .&id< 18:6+#7.

SHANNON KW, GLITHRIE C: Suppressors of a U4 snRNA Mutation Define a Novel U6 snRh’P Protein with RNA-binding Motifs. Grrre.< gels 1991. 5:T73-7x5.

22.

SERAPHIN B. ROSHA~H is not Required for pre-mRNA Complex U2 cnRNA. FJIROJ

23.

GOGLIEL V, k0 X, PA’hloNn BC. Influence 3’ Splice Site as Well Genes Dev 1991. 5:143C-l-138.

M: The Yeast Branchpoint the Formation of a Stable and is Recognized in the 1991. 10:20%1216.

25.

Sequence Ul snRNAAbsence of

ROWASH M: Ul snRNP Can as 5’ Splice Site Selection.

BARABINO S, BIINCO\VE BJ, RYDER U. Targeted snRNP Depletion Reveals Mammalian Ul snRNP in Spliceosome 63:29%302. A novel method of snRNP depletion, using nucleotides, was used to probe the function of pre-mRNA splicing. This is a powerful technical

36. ..

Gli II. hlANlIy JI; A Protein tive Splicing of SV& Early 62:25-31. see I39**l

B: ‘1. CompeDeter-

Cryptic Splice Site /‘,vc :Vcct/ Aicrd Sci

for 1990.

IC: Itltluon the Use 217265-281.

I): Purihcation and Charpre-mRNA Splicing Factor.

Factor, ASF. Controls pre-mRNA it1 Vifro.

AhemaCd/ 19’90.

37.

KRAINER AR, MAYI~IM A. Kwnh: 1). 131~~s G: Functional Expression of Cloned Human Splicing Factor SF2: Homology to RNA-binding Proteins, Ul 70K. and Drosophila Splicing Regulators. Cc,// 1991, 66 X13-835. See I39**]. ..

21.

24. ..

K. Roel,t% GS: Meiosis-spe&I/ 1991, 66:1?+1268. identilied :I rcgulatov pathnqy

ENC FI. WARNER JR: Structural Basis for the Regulation of 2x. . Splicing of a Yeast Messenger RNA. Cc,// 1991, 65:797-80-1. This study clemonstrdtes ho~v RNA structure can play :I role in the con. trol of pre-mRNA splicing.

B: Alternative Splicing .+fjrtr Ret, Gcvrc/ 1989.

14.

15.

N~X%IAN A. NOM!AN C: Mutations in Yeast US snRNA Alter the Specificity of 5’ Splice Site Cleavage. CeN 1991. 65:115-123. Describes the use of yeast genetics to isolate suppressor mutations of 5’ splice site mutations. 27.

I Introns.

World’s

26. .

Tails: an Update on Opirr Ce// Rio/ 1990,

SPROAT BS. L\~IOND Al: an Additional Role for Assembly. Cc// 1990, 2’.O-methyl RNA oligo Ul snRNP in mammalian advance for the field.

MICHALID S. REED A: An ATP-independent Complex Commits pre-mRNA to the Spliceosome Assembly Pathway. Getles Deu 1991, 5:253+2546.

3x. ..

GE II. ZI:O I’. MANILY JI.: Primary Structure of the Human Splicing Factor ASF Reveals Similarities with Drosophila Regulators. Cc~li 1991,66:373-381.

KRAINEH AR, CohwAY Splicing Factor IF2 Activating Proximal These papers [350,36**~39**] chemical and gene cloning pre-mRNA splicing factors.

39. ..

GC. KoLuc I>: The Essential pre-mRNA Influences 5’ Splice Site Selection by Sites. Cc,// 19‘X), 62:35-12. describe :I tour de force in the hio analysis of one of the tirst mammalian

40. .

MAYXIIA 4 KKAINER AR: Regulation of Alternative pre-mRNA Splicing by hnRNPA1 and Splicing Factor SF2. Cell 1992. 60~36%376. Describes the identilication and purilication of hnRNPA1 as a splicing factor. 41. .

HARXR JE. MANUil JL: A Novel Protein for Llse of Distal Alternative 5’ Splice Cdl Rio1 1991. 115945-5953. This paper describes the biochemical identification that shifts proximal to distal 5’ splice site usage. 42.

LI H. BIN(;IIA~! s@‘~ and Ira

PM: Arginine/.Serine-rich RNA Processing Regulators

Factor is Required Sites in Vitro. MO/ of a splicing

activity

Domains Target

of the Proteins

RNA

to a Subnuclear 1991, 67:335-342.

Compartment

Implicated

in Splicing.

Cell

ZAMOKE PD, GIUXN MR: Cloning and Domain Structure of 43. .. Mammalian Splicing Factor U2AF. Nul~rre 1992. 355:60%614. This paper shows that the R/S domain of 112AF is essential for splicing activity in rli!ro. Clearly demonstrates that the WS domain plays a functional role in splicing rather than acting simply a a nuclrar localization signal. 44.

Ron1 MB. Y%II\~~~LHAv. STOLK JAI A Conserved Family of Nuclear Phosphoproteins Localized to Sites of Polymerase II Transcription. J Cell Hiol 1991, 115:5X7-596. This paper describes the epitopes recognizci by a monoclonJ1 nntibody and the Itxtiization of the protein to tmnscriptively active regions 46. .

MAKWA A, ;~\IIIJ% AM. IGAINI~R AR, Rcml MB: Two Members of a Conserved Family of Nuclear Phospho Proteins are Involved in General and Alternative prc-mRNA Splicing. Proc Null Acud Sci l!.Srl 1992. 89:1301-130+. This pap descrilxs the identilicntion and analysis of a consented in RNPCS-contzining phosphoprotein family and it.s role in RNA splicing. 47.

CIIA~IPIJN DT. FILWZII tion of a Drosophila of Transcriptionally 5:1611-1621.

M, SA~I~IUTIW.H I I. 115 JT: CharacterizaProtein Associated with Boundaries Active Chromatin. Gc>,re.< /In, 1991.

4x.

FII X-D. MANIA’I~~ T: Factor Required for Mammalian some Assembly is Localized to Discrete Regions Nucleus. Nurrrre 1990, 343:-137-4-1 I.

-19.

SIJ~~CTOR DL. 131 X D. MANIATS T: Associations Between Distinct pre-mRNA Splicing Components and the Cell Nucleus. h.,1Jh’O ./ 1991. 10:3467-3481.

Spliceoin the

50. .

FII X-D. MANIATIS T: Isolation of a complementary DNA that encodes the mammalian splicing factor SC35. Scietrcr 1992. 256:535-538. Char.irtcdzation of the gent for splicing factor SC35 sholvs that II has RNIKS and R’S domains. 51.

As’l’ G, GUJ)ICI,U-r I), Ol’tw Novel Splicing Factor is an Large Nuclear Ribonucleoprotein J 1‘991. 10:42k32.

III. SIJEKIJN~; J, SIV%IJ~~; R: A Integral Component of 200s (InRNP) Particles. rZllR0

52.

KRAAII% A, IVANS 11. Three Protein Factors (SFI. IIZAF) Function in Pre-splicing Complex Formation dition to snRNPs. /i\/fjO .I 1’991, 10:503-l 509.

SF3 and in Ad-

?AXIORE PD. GREI;N MR: Biochemical Characterization of U2 snRNP Auxiliary Factor: an Essential Pre-mRNA Splicing Factor with a Novel lntranuclear Distribution. E,IIRO .I 1991, 10:207-21-l. This study analyzes the subunit activiv of PILAF and demonstmtes ph! logenetic conservation of 112AF activiv in Dru~ophilcr. It also shows that li2AF is localized to nuclear foci.

i9.

TACKI: R. G~IUDIS C: Alternative Splicing in the Neural Cell Adhesion Molecule pre-mRNA Regulation of Exon 18 Skipping Depends on the 5’ Splice Site. Genes Del, 1991. 5:1416-1429.

Ci.

D‘ORYAI. RC. I)‘AI~BENTON. CARUA Y. SIRAND-PUGNET P, GAUEG~ M. BROW E, MARE J: RNA Secondary Structure Repression of a Muscle-specific Exon in HeLa Cell Nuclear Extracts. Scieuce 1991, 252:1823-1828.

61.

Gw W. MIUJJGAN GJ. Wo&vsimv 5, HEU;hlAN DM: Alternative Splicing of p- Tropomyosin pre-mRNA: cis-acting Elements and Cellular Factors that Block the Use of a Skeletal Muscle Exon in Nonmuscle Cells. Gene.~ Del* 1991. 5:2096-2107.

62.

BAKER BS: Sex wo:521-52+.

6.3.

BELL LR. Hotbwi~ JI, Sciierx P, CUNE TW: Positive Autoregulation of Sex-lethal by Alternative Splicing Maintains the Female Determined State in Drosophila CeJJ 1991. 55.229-239.

6-h

IWIT Ii. tllstli~ihm K. SAUVOTO li, SHihlllIu Y: Binding of the Drosopbih Sex-/etbul Gene Product to the Alternative Splice Site of Transformer Primary Transcript. Nullrye 1990, 344:+61+63.

6.

Rio IX. Element

66.

Rio DC: Regulation of Drosophila Tretiuk Gerirl 1991, 7282-287.

see 55. .

These splice

GII. A, S~IARIJ PA. JA~~IS~N SF, GARCIA-BIANCO tion of cDNAs Encoding the Polypyrimidine Protein. Get2e.s De/l 1991, 5:122+1236. 155*1.

MA: CharacterizaTract-binding

PAI-~~N JG. MASER SA, TEAII~SI’ P. NA~.A.-GINARII 13: Characterization and Molecular Cloning of Polypyrimidine Tractbinding Protein: a Component of a Complex Necessary for pre-mRNA Splicing. (;L’)~e.c fIc~/’ 1991. 5:1237-1251. papers [5+*,55*1 characterize the gent encoding the pPTB 3’ site recognition protein.

56.

K~IO H-C, N&SIN I’ll, Gtwww’s~tr PJ: Control of Alternative Splicing by the Differential Bud of Cl1 Small Nuclear Ribonucleoprotein Particles. Science 1991, 25 1: lO15- 1050.

57.

GKAHOVVSKI PJ. NA.%I FH. Kilo I-l.c. I~IIRCI~ R: Combinatorial Splicing of Exon Pairs by Two-site Binding of UI Small Ribonucleoprotein Particle. jllol Ccl/ Rio/ 1991, 11:5913-5928.

Molecular Transposition.

Flies:

the

Splice

of

Life.

Narlrre

Sites Exon

1989.

Mechanisms Regulating Drosophila /i?z)z11 Kerr Ge?lef 1990, 24:513-578. P Element

P

Transposition.

67. .

SIE~EI. CW, RIO DC: Regulated Splicing of the Drosophila P Transposable Element Third lntron in Vitro Somatic Repression. Science 1990, 248:200-1208. This paper describes how a biochemical complementation assay allowed detection of somatic Dru~opMa RNA-binding proteins that function to repress splicing of the P element third intron. 6x .

CI IAIK AC, %otJxla,u S. TSENG JC. L.wl FA: Identification of a cis-acting Sequence Required for Germ Line-specific Splicing of the P Element ORFZ-ORF3 Intron. ,\,Jo/ Cell Eiol 1991, 11:I53~15-16. This study uses P element tmnsfom~tion to identify &acting RNA sequences that goveem germline specifici! of P element third intron pm-mRNA splicing. 69.

T%NG

JC, %OI.IAWN S, CIWN AC, L~SKI FA: Splicing of the P Element ORF2-ORF3 Intron is Inhibited in a Human CeU Extract. rt1eL-h Der* 1991, 35:65-72.

Drosophilu

53 ..

54. .

451

BLACK DL: Does Steric Block the Splicing of in Non-neuronal Cell?

in

Interference Between Splice a Short c-src Neuron-specific Genes Del@ 1991, 5:38*02.

Rio

58.

R~IH MB. M~v~~IN C, GAIJ. JG A Monoclonal Antibody that Recognizes a Phosphorylated Epitope Stains Lampbrush Chromosome Loops and Small Granules in the Amphibian Germinal Vessicle. J Cell Hi01 1990. 111:2217-2223.

45. .

processing

‘0. ..

SIEIW. CW. FKESCO LD. RIO DC: The Mechanism of Somatic Inhibition of Drosophila P Element Pre-mRNA Splicing: Multiprotein Complexes at an Exon Pseudo-5’ Splice Site Control Ill snRNP Binding. GL’)I~.~ Del, 1992. in press. Describes how RNA-protein interactions can influence (11 snRNP-5’ splice site recognition t(> control premRNA splicing. ‘I.

BINC;II~ PM, f∨ of Gene Expression 1988, 4:13-l-138.

-2.

AWEIN H. Transcripts

TB. IMIXIS I, ZKH.%~ : On/off Regulation at the Level of Splicing. Trenrls Genet

MANWTIS T, NOTFIINGER R: Alternatively-spliced of the Sex-determining Gene rra-2 of Encode Functional Proteins of Different Sizes.

Drosophila ElJUO J 1990, 9~361F-3629. 73.

--I.

MATI’OS W, BAKER BS: Autoregulation Transcripts from the transformer-2 Gene Gwres Del9 1991. 5~786796.

of of

Splicing

of

Drosophila.

NACOSHI RN, Bl\t(~a BS: Regulation of Sex-specific RNA Splicing at the Drosophila doublesex Gene: cis-acting Mutations in Exon Sequences after Sex-specific RNA Splicing Patterns. Gems Ders 1990, 4:8’+97.

452

Nucleus

and

gene

expression

. HEDIIY ML, MANIATIS T: Sex-specific Splicing and Polyadenylation of dsx pre-mRNA Requires a Sequence that Binds Specifically to tra-2 Protein in Vitro. Cell 1991. 65579586. This paper demonsrrates &vsplicing in cell culture and the interaction of bacterial tra-2 protein with &Y pre.niRNA. 75. ..

87. ..

KENAN DJ. Q~IERY CC, KEENE JD: RNA Recognition: Towards Identifying Determinants of Specilicity. Tren& Biohem Sci 1991, 16:214-220. An up-to-date review of RNI’CS type RNA.binding proteins. 88.

MA~~‘AJ IW: A Binding in Splicing, snRNPs.

H9.

BANIXI~~I.IS RJ. SW’ANSON Proteins as Developmental 3:-13143’.

pre-mRNA Genes De!

90.

SCHERIX D. BOEIENS W. CAN VENHOOJ u(q, DATHAN NA. Hmhr J. MAlTAJ IW’: identification of the RNA Binding Segment of Human UlA Protein and Delinition of Its Binding Site on Ul snRNA EllBO J 1989. 8:-1163-i170.

TLAN M. MANIA’~~ T: Positive control of pre-mRNA splicing in vitro. Science 1991 256:237-L-+0. %is important paper describes an in &ro assq system li)r the stud! of rLsr pre-mRNA splicing nith rtzombinant tra and tr,l-2 proteins.

91.

L~Il%-FaE~r!Rh![ini C, QLI~RY CC, KIXNI; JD: Quantitative Determination that One of the Two Potential RNA Binding Domains of the A Protein Component of the Ul Small Nuclear Ribonucleoprotein Complex Binds with High Affinity to Stem-loop II of II1 RNA 1%~ A&/ .+I& Sci l[SA 1990. 87:63934397.

76. ..

This

HOSHIJIMA K. INOLIE K. HICLICHI 1, SAKA~%OTO Ii. Slllhrt~ib~ Y: Control of doublesex Alternative Splicing by trans. former and tmnsfowner-2 in Drosophila. Science 1991, 252:833-836. paper describes regulation of rirs regulation in cell culture.

77. ..

RYDER LC. BAKER BS: Regulation of doublesex Processing Occurs by 3’ Splice Site Activation. 1991, 5:2071-2085. A very careful analysis of ICY regulation in cell culture. 78.

79.

SPECTOR DL: Higher Order Nuclear Organization: dimensional Distribution of Small Ribonucleoprotein ticles. Pmc N&l Acad Sci 1iSA 1990. 87:1+7-151.

80.

HUANG S, SI’IX’I’OR Present in Nuclear 89:30>308.

81.

C~~WO.FON~IXO The snRNP-rich ies. J Cell Biol

ThreePar-

DL Ul and Ul Small Nuclear RNAs are Speckles. 13~~ ;Vni/ .~LzI~/ .%I l’.Y,l 1992.

h4. PIYPEHKOK R. CARVNHO Foci in Mammalian Nuclei 1992, in press.

MT, Lur1o~13 AL Are Coiled Bo&

82.

H~IANC S. SPECTOR Dl; Nacent pre-mRNA sociated with Nuclear Regions Enriched Genes Det’ 1991. 5:228%2302.

83.

CAIU.!O-FONSECA M. TOILER\IT D. PEPPEIUXX R, B.AR.AI~I~~:SML MERIXS A, BR~INNER C. &U~OKI; P, GREEN M. HIIRT E, ~~IONI> AI: Mammalian Nuclei Contain Foci which Are Highly Enriched in Components of the pre-mRNA Splicing Machinery. EURO J 1991. 10:195-206.

Transcripts in Splicing

Are A+ Factors.

84.

C.%WO-FONSIXA M. PIYP[~~(OK R. SPROAI’ 135. ANSORC~~ W. !?WAN.SON MS. hlONI) Al: In Vito Detection of snRNP-rich Organelles in the Nuclei of Mammalian Cells. E\lI~O./ 1991, 10:1863-18373.

85.

Wo %. MtUWM’ C, CAllAN HG. GAII. JG: Small Ribonucleoproteins and Heterogeneous Nuclear Ribonucleoproteins with Amphibian Germinal Vessicle: Loops, Spheres, and Snurposomes. J Cell Hiol 1991. 113:t65-#3.

86. Ghll. JC: Spliceosomes and Snurposomes. .Scrr,rcc~ 1~991. .. 252:149’+1500. This perspective summarizes a comparative study of sn~~i’-containing organelles in amphibia and mammalian cells.

Consensus: RNA Protein and Sex. Ce// 1989, 57:1-3. MS, DIU%XI~S Regulators.

Interactions

G: RNA-binding Gefrc>s Der, 1989.

92. .

NA<;AI K. OIIIIRIIXX C, JI;<~EN TH, LI J, E\‘AN~ PR: Crystal Structure of the RNA-binding Domain of the Ul Small Nuclear Ribonucleoprotein A. Nnf~~tz~ 1990. 348:ili-520. This paper describes the S ray structure dercrnmination of the II 1 A RNI’CS RNA-binding domain. 93. .

HoIw~~~ DW. Q~IIX’I. CC, GOIIIFN BL \X’tiI-rli SW, KIXNI~ JD RNA-binding Domain of the A Protein Component of the Ul Smaller Nuclear Ribonucleoprotein Analyzed by NMR Spectroscopy is Structurally Similar to Ribosomal Proteins. I’rtx .Vrrtl :lirrrl Sci 1 ‘S.-l 199 1, 88~24%2499. This paper describes rhe NXIR slructure determination of the 1’1 A RNPCS RNA.binJing domain. 9-1.

JEYXU T-II. OL’RKIIXX C. Trio CH. PKrrcti.\W) C. NA(;AI K: ldentification of Molecular Contacts Between the UlA Small Ribonucleoprotein and Ul RNA. E\If
95. ..

CAI-WAS 13J. TIIX)R 13. I~KANCAIANA S. 131KWN D. FRAMXI. AD: Arginine-mediated RNA Recognition: the Arginine Fork. Sci etrcr 1991. 252:l 16--l 1’1 Suggt%s a mcxiel of protein--RNA recognition via intenmtions betwcrn argininr amino acid side chaim and RNA phosphates. which ma! have general implications for peptide, prorein-RNA inremctions. 96.

GKIXS hlR: 1 Z-ii-2-1-.

RNA

Bent

for

Recognition.

C~rrr

No/

1991,

DC Rio, Depannlem of hlolecular and Cell Biolofl. Divisions of Genrrits Biochemists and Molecular Biolo~, 401 Darker Hall, Universiv of California. Berkelq, California 947720. USA.