snRNAs: from gene architecture to RNA processing

TIBS - October 1984

435

Reviews snRNAs: from gene architecture to RNA processing lain W. Mattaj Functions for the snRNAs in the processing of precursor RNAs have long been proposed. Experimental evidence in support of these proposals is now accumulating. At the same time study of the genes coding for the snRNAs underlines the variability of gene structure among genes transcribed by RNA polymerase I1, the polymerase responsible for mRNA transcription. I-rlstory Small nuclear RNAs (snRNAs) are a group of short RNAs found in the nuclei of all eukaryotic cells, where they are complexed with specific proteins to form small nuclear ribonucleoproteins (snRNPs). These RNAs have been subdivided in three ways: (1) according to their structural characteristics, (2) according to the RNA polymerase responsible for their transcription, and (3) according to the proteins to which they bind ~,2. This last classification was made possible by the observation that some human patients suffering from autoimmune disorders produce antibodies which react with particular classes of snRNA 2. In the first part of this review I will discuss only two snRNAs in detail: UI-snRNA, thought to be involved in the splicing of introns from heterogeneous nuclear RNA (hnRNA), the precursor of messenger RNA (mRNA); and an snRNA which is still unnamed because, in contrast to other snRNAs, knowledge of its function (in the production of 3' termini on histone H3 mRNA) has preceded knowledge of its structure. U-snRNAs U-snRNAs were so named because they are U-rich. At least six U-RNAs (U1-U6) are found in all higher eukaryotes and there are 104-106 copies of each RNA in mammalian cells. Five of the RNAs (U1, U2, U4-U6) are nucleoplasmic and are precipitable with autoimmune antisera of the serotype Sm. The sixth, U3, is nucleolar and is not precipitable with Sm antisera L2. The antigenic determinants recognized by Sm antisera do not reside on the UsnRNAs themselves, but on proteins lain W. Mattaj is" at the Biocenter of the University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland.

associated with U-snRNAs in U-snRNPs. Seven to eight proteins have been consistently identified as belonging to UsnRNP particlesz. The protein composition of the particles containing individual U-snRNAs is still a matter of some uncertainty: However, it appears that some of the proteins (e.g. D) are common to all U-snRNP particles while others are confined to particles containing a particular U-snRNA 3. The heterogeneity of U-snRNP particles is also indicated by the fact that some auto-immune sera only precipitate one type of UsnRNP. An example is anti-(U1)RNP which, as its name suggests, only precipitates Ul-containing RNPs 2. The U-snRNAs, particularly U2, contain many modified bases and these are concentrated near the 5' end of the RNAs in all cases except U6L In addition U1-U5 have a unique trimethyl cap structure at their 5' ends 1. Since our knowledge of the structure and functions of U-snRNPs is at a relatively elementary level, it is impossible to say anything about the significance of these unusual base modifications. The presence of the trimethyl guanosine cap has enabled the development of a straightforward immunological method for the purification of U-snRNPs 4. Antibodies raised against trimethylguanosine can be immobilized and used to affinity purify U-snRNAs in the naked state or U-snRNPs. This methodology promises to be very useful in future preparation of U-snRNPs for functional study. U6snRNA does not have a trimethyl cap, and in the naked state will not bind to the anti-trimethylgnanosine antibody. However, when U-snRNPs were purified using the antibody, U6 was also found in the bound fraction4. This indicates that particle-bound U6 must be associated with another of the U-snRNAs, and recently U6 and U4 have been shown to be

present in a single U-snRNP particle 25'26. Within the cell nucleus U1, U2 and U4-U6 snRNAs have been shown by fractionation and cross-linkage studies L5 to be associated with hnRNA, the precursor of mRNA. U3, by similar methods, has been shown to be associated with pre-rRNA, the precursor of the 5.8S, 18S and 28S ribosomal RNAs t'5. This led to the suggestion that the UsnRNAs are involved in the processing of these precursors to their mature forms, or in the structure of hnRNP or pre-rRNP particles. Recent data for U I - s n R N A supports this hypothesis. The role of UI-snRNP in splicing Splicing is the process which occurs in the nucleus by which the intervening sequences (introns) are removed from hnRNA to produce the mature m R N A product. The first suggestions that U1snRNA might be involved in this process were stimulated by the observation that the 5' end of U I - s n R N A was complementary to (i.e. could base pair with) the conserved donor and acceptor sites at splice junctions2. Later it was shown that UI-snRNPs would bind specifically to the 5' splice junction of mouse [3-globin RNA in vitro and would protect the 5' splice junction from TI ribonuclease digestion6. U1snRNPs were more efficient in this protection assay than naked UI-snRNA, indicating that the protein components of the particleplay a role in stabilizing the interaction". In a more direct experimental approach to splicing, Yang eta/. 7 showed that antisera of both the anti-Sm and anti-(U1)RNP types would inhibit the splicing of adenovirus mRNA in nuclei isolated from adenovirus-infected cells. This implicated UI-snRNPs directly in the splicing process. Confirmation and extension of these results had to await the development of true in vitro splicing systems. With the advent of these systemss'9, it was demonstrated that (1) a subcellular fraction rich in U-snRNPs stimulated the in vitro splicing process 9 and (2) splicing in vitro was inhibited by incubation of the extracts with anti-Sm or anti-(U1)RNP antisera, but not by incubation with auto-immune antisera specific for RNP particles not of the UsnRNP type l°. Direct confirmation of the role of UI-snRNPs in splicing, by restoration of splicing activity to a depleted extract on re-addition of pure

(~ 1984, Elsevier Science Pubfishers B V , , Amsterdam

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436 UI-snRNPs should now be possible, given the biochemical3 and immunological4 methods available for U-snRNP purification. The obvious next question is: What are the other U-snRNPs doing? To attempt to answer this, one must keep in mind that all the experiments so far described have been looking at one aspect of m R N A maturation (splicing) in a few introns from two adenovirus genes. It is to be hoped that by looking at more genes, and at other steps in m R N A maturation, e.g. initiation and termination of transcription, 3' end processing and polyadenylation and transport of mature mRNAs from nucleus to cytoplasm, that the functions of the other U-snRNAs will be found. It is in one of the other aspects of mRNA maturation, the production of 3' termini, that we will find the action of another snRNA. A short RNA involved in producing mRNA 3' termini Studies of a cluster of sea urchin (Psammechinus miliaris) histone genes following microinjection of these genes into the nuclei of Xenopus laevis oocytes has contributed much to our understanding of RNA polymerase II transcription of eukaryotes. All of the genes present in this cluster, coding for histones H1, H2A, H2B, H3 and H4, are expressed in frog oocytes, but with greatly differing efficiencies1°. Of particular interest here is the observation that while the 5' termini of sea urchin histone H3 mRNA are correctly produced, processing of the H3 transcript to provide correct 3' termini is relatively inefficient12. This deficiency could be corrected by coinjecting a fraction prepared from sea urchin chromatin containing RNP particles with a sedimentation coefficient of 12S ~2. Two short RNAs, of 100 and 60 bases were shown to be abundant in this fraction 13. Purified sea urchin RNA in the size range 55-68 nucleotides was shown to be fully efficient in complementing the oocytes in the production of H3 mRNA 3' termini, demonstrating involvement of the 60 nucleotide RNA in this process. Apart from its length, nothing is known of the structure of this RNA, its classification as an snRNP 13 being based on two observations: (1) the purification proc.~lure for the 12S RNP particle indicates biochemical similarity to UsnRNP particles 12, and (2) the 60 nueleotide RNA is able to migrate from the cytoplasm (where it is deposited by microinjection) into the nucleus, its site of

T I B S - October 1984 action 13. The accumulation of snRNAs in the X. laevis oocyte nucleus after microinjection into the cytoplasm 14 h a s been shown to involve binding of the injected U-snRNAs to the protein components of U-snRNP particles. U-snRNP proteins are stored in the cytoplasm of X. laevis oocytes in the absence of U-snRNAs, ready for mobilization in early development ~5. Figure 1 shows current understanding of the functions of snRNP in precursor RNA maturation. r

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of restriction fragment are shown by Southern blot analysis using Ul-specific probes, to contain U1 coding sequences and (2) cloned DNA fragments each contain only one U1 gene. X. laevis contains still more copies of the U-snRNA genes, e.g. about 500-1000 copies of the U2-snRNA genes is, and in this species both U1- and U2-snRNA genes are organized in short tandem repeats of 1800 and 830 base pairs respectively lsa9. It has recently been reported that all of the human UI-snRNA I

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Composite mRNA gene showing the action of snRNP particles in RNA processing.

U-snRNA genes Given the extreme conservation of UsnRNA structure and sequence throughout eukaryotic evolution1, one might have expected that the genes coding for these RNAs would also be uniform. However, studies on the U-snRNA genes of several organisms has brought to light great variability, both in the number and in the organization of these genes. Of the higher eukaryotes studied, the chicken has the fewest U1snRNA genes, having less than ten copies 16. Human beings have roughly 100 U1 genes 17. In both these cases the genes are assumed to be scattered in the genome because: (1) many different sizes

genes are on the same chromosome 2°. This would be consistent with the human U1 genes being organized in repeated units of too great a length to be detected by the methods so far employed, and could resolve the apparent species difference. Additionally human U2-snRNA genes have been shown to be organized in tandem arrays of about 6 kb repeat length21. While humans have only about 102 U1 genes they contain many U-snRNA pseudogenes, i.e. genes which either cannot be transcribed or if they were would give rise to abnormal transcripts. The ratio of pseudogenes to genes in humans is about 10:117. In X. laevis on the other hand

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T I B S - October 1984

arity between the X. laevis and human genes. The regions necessary for the transcription of the various U-snRNA genes contain two separate sequence homoloTranscription of U-snRNA genes The first U-snRNA gene transcrip- gies, thought to be functional in the tion studies were with cloned human initiation of transcription 19'23. Neither U I - s n R N A genes. Both whole-cell ex- of these sequences shows similarity to tracts and X. laevis oocytes were shown the standard elements, e.g. T A T A box, to be capable of transcribing U-snRNA C C A A T box, found in the 5' control genes and inhibitor studies showed that regions of most other R N A polymerase R N A polymerase II was the polymerase II-transcribed genes. responsible in each case 2~. A striking Starting 10-20 bp downstream from difference was observed in the human the 3' end of the coding region of U I - s n R N A transcripts obtained in the all higher eukaryotic U-snRNA genes two different systems. While the trans- so far sequenced there is a concripts seen in oocytes had the correct 5' served D N A sequence (Consensus: capped ends, those from in vitro extracts G 111NAAAAAA NAGA)part of which was T initiated 183 bp upstream from the nor- also found near the polyadenylation mal cap site 22. This led to the propo- signal in many m R N A coding genes Is. sition that the U I - s n R N A was first tran- This sequence was proposed to be inscribed as a precursor which was later volved in the formation of 3' termini on processed and capped post-transcrip- the U-snRNA transcripts 18. Deletions tionally. This was supported by the fact of parts of this sequence do indeed lead that it was possible to remove the D N A to a drastic reduction in the production sequences between positions - 6 bp and of correct 3' termini on transcripts from - 106 bp relative to .the UI-snRNA cap the X. laevis UI-snRNA gene (Mattaj, site without affecting the efficiency of I. W. and De Robertis, E. M., untranscription. In other R N A polymer- published results) showing that although ase II-transcribed genes these sequences different in sequence they may be would contain signals, e.g. T A T A box, analogous in function to the conserved necessa~ for transcription. Later studies 23 sequences found near to the 3' end of have confirmed that sequences essential histone mRNA coding sequences, which for the transcription of human U1- are involved in the production of histone snRNA genes lie between 203 and 230 m R N A 3' ends 24. bp upstream of the cap site. Post-transThe differences found between the criptional processing and capping of the control regions near to the 5' and 3' transcript appears, however, to have ends and those previously described for been ruled out by a number of experi- other R N A polymerase II-transcribed ments, the most definitive of which genes lead to my final point. If the signal shows that the cap structure of the tran- sequences on the genes are so different, script of the human UI-snRNA gene is how do the proteins necessary for translabelled in X. laevis oocytes when coin- cribing them differ? D o U-snRNA jected with [[3-32p]ATP but not with genes have transcription factors which [[3Yp]GTP -~ differ from those of other R N A polyThe picture presented by transcrip- merase II genes, or does the sub-unit ion studies of the X. laevis U I and U2 composition or state of modification snRNA genes is similar, hut differs in of R N A polymerase II itself change? detail. In both cases the sequences Further studies on the U-snRNA genes essential for transcription of the genes lie should answer these questions, and help closer to the cap site than was found in to explain the variety observed among humans. In the case of U1 less than 150 genes transcribed by the same polymerbp upstream j~ and in the case of U2 less ase. than 80 bp upstream (Mattaj, I. W. and Acknowledgement De Robertis, E. M., unpublished I would like to thank Professor E. M. results). Upstream elements affecting De Robertis for his continual advice and promoter efficiency are present in these encouragement, and my laboratory colcases, which might indicate basic simil- leagues, particularly Susanne Lienhard there is no evidence so far for pseudogenes among the 1000 or so copies each of U1- and U2-snRNA genes t8'19.

and Rolf Zeller, who have made working with U-snRNPs such a pleasure.

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