Cell, Vol. 25, 298-300, August 1981, Copyright 0 1981 by MIT

Snurps and Scyrps

Michael R. Lerner and Joan A. Steitz Department of Molecular Biophysics and Biochemistry Yale University New Haven, Connecticut 06510

Small nuclear and cytoplasmic ribonucleoproteins- snRNPs and scRNPs (pronounced snurps and scyrps)-are complexes of small RNAs with protein, found in eucaryotic cells. They fall into discrete classes, are abundant and are highly conserved across species. The idea that these particles might utilize intermolecular RNA-RNA interactions to pro- vide the precise recognition necessary for the accu- rate processing of RNA transcripts has suggested possible roles for snRNPs and scRNPs in the metab- olism of other RNA molecules. Already there exists evidence implicating one snRNP in the splicing of hnRNA.

Our current picture of snRNPs and scRNPs has emerged from the union of knowledge in two distinct fields-small RNAs (see Zieve, Cell 25, 296-297, 1981) and antigen-antibody systems associated with lupus erythematosus. Although small nuclear RNAs have been known to exist for some time, their func- tions have remained mysterious. Recently it has be- come apparent that different ones can be grouped according to the proteins they bind. The small ribo- nucleoprotein particles we focus on here fall into three classes and one subclass: the Sm snRNPs [with sub- class (Ul)RNP], the Ro scRNPs and the La snRNPs. Each class is specifically recognized by antibodies from different lupus patients (see Table).

Autoantibodies Lupus erythematosus, and the related diseases Sjogren’s syndrome, scleroderma, rheumatoid arthri- tis, polymyositis and dermatomyositis, are connective tissue disorders. A hallmark of these diseases is au- toantibody production against cellular components that are abundant and highly conserved, and thus poor immunogens otherwise. For instance, DNA is the best known antigen with which lupus antibodies react, but there are in fact many different lupus-associated antigen-antibody systems.

Between 1966 and 1974, four cellular antigens against which patients with lupus or Sjogren’s syn- drome make antibodies were detected by immunodif- fusion techniques (see Provost, J. Invest. Derm. 72, 110-l 13, 1979). These were dubbed Sm, “RNP,” Ro (also called SS-A) and La (also called SS-B or Ha). The two-letter designations are derived from patient names, for example, Sm stands for Smith. The Sm and “RNP” antigens were demonstrated to be nuclear by indirect immunofluorescence and cell fractionation, and were shown to be physically associated with each other by immunodiffusion. The cellular locations of Ro

and La were controversial. All antigens had molecular weights between 30,000 and 200,000, were present in a variety of mammalian tissues and were sensitive to trypsin. Only “RNP” and La were reported also to contain RNA, as assessed by sensitivity to ribonucle- ase.

Does the presence of antibodies of different speci- ficities in a patient’s blood correlate with specific clinical findings? Evidence so far indicates that auto- antibodies are not internalized by living cells in such a way that they interfere with specific metabolic proc- esses. Rather, many symptoms can be traced to the deposition of antigen-antibody complexes in tissues, leading to inflammatory responses. Cellular antigens in sera presumably arise from cell lysis. However, it is not clear why various immune complexes deposit in specific tissues. Detailed characterization of the Sm, “RNP,” Ro and La antigens may help resolve this issue. Some patients’ sera contain essentially mono- specific autoantibodies. These antibodies provide po- tent probes for studying the structure and function of the Sm, “RNP,” Ro and La antigens.

Sm snRNPs Contain U RNAs Lupus-associated anti-“RNP” antibodies specifically recognize small ribonucleoprotein particles that con- tain Ul RNA (Lerner and Steitz, PNAS 76, 5495 5499, 1979). We therefore propose renaming this antibody anti-(Ul)RNP. Ul RNA is stable and is the most abundant snRNA in mammalian cells, numbering about lo6 copies per nucleus (see Table for more details of all small RNAs.) The sequence of Ul RNA is known to be highly conserved among humans, rats, chickens and even fruit flies. In mouse cells, Ul re- sides in a complex with seven proteins, A through G, with molecular weights of 35,000 to 12,000 daltons. Recognition of the Ul snRNP by anti-(Ul)RNP re- quires protein. The (Ul )RNP antigenic determinant is conserved from man to insects, since the human antibody is capable of precipitating Ul -containing snRNPs from every intermediate species examined. Finally, the Ul RNA sequence adjacent to its 5’ cap exhibits complementarity to the consensus sequences for donor and acceptor splice junctions present in the hnRNA molecules of eucaryotes ranging from yeast to man (Lerner et al., Nature 283, 220-224, 1980; Rogers and Wall, PNAS 77, 1877-1879, 1980).

Antibodies to Sm also recognize Ul snRNPs, but at a site physically distinct from the determinant bound by anti-(Ul)RNP. In addition, the Sm determinant is present on particles containing the snRNAs U2, U4, U5 or U6 (Lerner and Steitz, op. cit.). Like Ul , these RNAs are highly conserved and stable. While U2 is almost as abundant as Ul , the others are present in somewhat smaller numbers-about lo5 copies per nucleus. Curiously, anti-Sm precipitates the same pro- teins as anti-(Ul)RNP, suggesting that all U RNAs bind a common set of polypeptides. Like the (Ul )RNP determinant, the Sm determinant is conserved among

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Anti-(Ul)RNP

Anti-R0

Anti-La

Ribonucleoprotein Modified Antibody Class RNA Components Length 5’ Termini Nucleotides

Anti-Sm Sm snRNPs u2 196 m3Grw + u4 125 m3Gm f U5 118 m3GPPp + U6 106 ? + Ui 165 m3Gwp +

Ro scRNPs Mouse Yi -Y2 90-l 00 PPPS -

La snRNPs Many cellular RNAs 80-l 20 PPPk f including: rat 4.551 96 PPPB -

mouse or hamster 4.5s 94 PPPC -

Viral RNAs: VAI 160 PPPC - VAII 163 PPPB -

EBERI 167 PPPA -

EEERP 173 PPPA -

See Zieve (op. cit.) for more details on the structure and biosynthesis of small RNAs.

Table. Small RNPs

species from man to insects. Under conditions of physiological ionic strength, Sm snRNPs sediment at about lOS, except for those containing U5 RNA, which sediment significantly faster. Immunofluores- cence using a monoclonal anti-Sm antibody confirms the nuclear (but non-nucleolar) location of the Sm snRNPs (Lerner et al., PNAS 78, 2737-2741, 1981).

What are the functions of the Sm snRNPs? Ul- containing particles are probably elements active in the alignment of splice junctions during the conversion of hnRNA to mature mRNA. Conceivably they could also perform enzymatic functions in splicing, based on the precedent of RNAase P, a small ribonucleopro- tein that achieves the 5’ end processing of tRNA precursors in E. coli. Experiments with an in vitro system containing nuclei from HeLa cells infected with adenovirus (Yang et al., PNAS 78,1371-l 375,1981) reveal that both anti-(Ul)RNP and anti-Sm antibodies can inhibit the appearance of spliced mRNAs (but not the synthesis or polyadenylation of transcripts). Thus Ul snRNPs are required for splicing, for they are unique in that they carry the Sm and (Ul)RNP deter- minants simultaneously.

The functions of U2, U4, U.5 and U6 snRNPs are currently unknown. However, the fact that they pos- sess a common antigenic determinant with Ul parti- cles suggests that they too may be involved in the nuclear editing of RNA transcripts. Specific roles un- der consideration include the alignment of hnRNA splice junctions that deviate from the consensus se- quence, splicing or maturation of the 5’ or 3’ ends of tRNAs and cleavage of hnRNAs 3’ to the AAUAAA signal before polyadenylation.

Ro scRNPs Are Cytoplasmic RNA-Protein Complexes A second class of small ribonucleoproteins are those precipitated by anti-R0 antibodies (Lerner et al., Sci- ence 2 17, 400-402, 1981). Indirect immunofluores-

cence assigns these particles to the cytoplasm and further localizes them in what appear to be discrete organelles. The small RNAs in Ro scRNPs are distinct from those associated with Sm. The major ones in mouse cells are called Yl and Y2 (for cYtoplasmic, to distinguish them from the nuclear U RNA& They are quite stable, like the RNAs in the Sm snRNPs. Ro RNAs are significantly less abundant than the U RNAs, numbering less than 1 O5 copies per cell. They are not so highly conserved from species to species; HeLa cells have more different kinds of Ro RNAs than mouse cells. Little is known about the protein(s) re- quired for recognition by the anti-R0 antibody. Why small ribonucleoproteins might inhabit an organelle is a mystery. Speculations that certain organelles may be involved in mRNA turnover prompt the suggestion that Ro scRNPs are active in the accumulation or degradation of spent mRNAs in these loci.

La snRNPs Contain Cellular or Viral RNAs Complexed with Cellular Protein The third class of small ribonucleoproteins are snRNPs possessing the La determinant (Lerner et al., Science 27 7, 400-402, 1981). Upon electrophoresis on polyacrylamide gels, the RNAs in these particles yield a highly banded pattern that ranges from about 80 to 120 nucleotides. Fingerprint analysis indicates that they are nonetheless discrete. Two cellular mem- bers have been sequenced: 4.5SI from Novikoff hep- atoma (rat) and 4.5s from mouse and hamster cells. The latter was initially found associated with poly(A)- containing RNA and is partially homologous to certain highly repetitive, interspersed sequences in mamma- lian genomes called Alu family DNA (Jelinek et al., PNAS 77, 1398-1402, 1980). La RNAs, like the Ro RNAs, are not as highly conserved as the U RNAs; while present in every mammalian cell examined, their gel spectra and fingerprints differ from species to species. They are significantly less stable than the U

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or Ro RNAs. In addition to the many cellular La RNAs, at least four viral members of this class exist: VAI and VAII encoded by adenovirus, and EBERl and EBER2 (Epstein Barr-encoded RNA) found in EBV-trans- formed cells (Lerner et al., Science 27 7, 400-402, 1981; Lerner et al., PNAS 78, 805-809, 1981). Re- constitution experiments demonstrate that the viral VA and EBER RNAs are precipitated by anti-La because they are bound by cellular protein(s) carrying the La determinant (Rosa et al., Mol. Cell. Biol., in press). La particles are primarily localized in the nucleus by indirect immunofluorescence, although they seem to leak out easily during biochemical fractionation of cells.

What could be the cellular role of La snRNPs? We know that VAI RNA can bind to spliced adenovirus fiber message and to cDNA copies of fiber mRNA, but not to genomic DNA coding for fiber (Mathews, Nature 285, 575-577, 1980). The last differs from the first two in that genomic DNA contains intervening se- quences, suggesting that VAI functions either in splic- ing or in export of fiber messages from the nucleus. The observation that hamster 4.5s RNA is bound to poly(A)-containing RNA has led to similar suggestions concerning its function. A most attractive idea is that La snRNPs are vehicles that can exit from the nu- cleus; specific interaction with a particular La RNA could thereby allow a newly fashioned mRNA to pig- gyback its way to the cytoplasm. However, alternative roles for the La particles in RNA synthesis or even in DNA replication should still be entertained.

Current Range of RNPs Precipitated by Autoantibodies Recently we undertook a survey to determine the range of RNAs precipitable by sera from patients (Hardin et al., Arth, Rheum. 245, 58, 1981). Of 240 patients, 50 had anti+1 )RNP, anti-Sm, anti-Ro, anti- La or some combination of these. Other patients whose sera precipitated 5s and 5.8s as well as large RNAs were observed; this pattern is diagnostic for

antiribosome antibodies, previously reported in pa- tients with lupus erythematosus. In addition, several new antigen-antibody systems were uncovered. One serum precipitates 5s rRNA exclusively, while others react with different subsets of tRNA-sized molecules. So far our investigations have shown that these new antibodies do not recognize naked RNAs; hence we are again dealing with specific small RNA-protein complexes, with functions unknown. This leaves only two well characterized small RNAs that have not been identified in discrete small ribonucleoproteins precip- itable by antibodies associated with connective tissue disease-nucleolar U3 and viral 7s.

We have only begun to appreciate the diversity and the functional contributions of snRNPs and scRNPs to eucaryotic cell metabolism. The autoantibodies pro- vide for the first time a way of classifying most of the major small RNAs, at least on the basis of the pro- tein(s) they bind. In general, the ribonucleoprotein classes we describe are nonoverlapping, in that each kind of small RNA molecule is quantitatively and ex- clusively found in only one type of particle. This is particularly clear for the U RNAs, which are often found associated with hnRNP but are even then con- tained in snRNPs precipitable by the anti-(Ul )RNP or anti-Sm antibodies, or both. Hints of an exception to this rule come from observations that RNAs in other classes may sometimes bear the La determinant, per- haps in relation to their deposition in or brief passage through the cell cytoplasm. Although the human anti- bodies do not recognize snRNPs and scRNPs from the full range of eucaryotic species, it seems obvious from our growing knowledge of small RNAs in plants and lower eucaryotes that comparable particles exist. Such conservation is compatible with the idea that these small ribonucleoproteins arose at an early stage in the evolution of eucaryotic gene expression. Estab- lishing whether snRNPs and scRNPs are in fact in- volved in the biogenesis or catabolism of other RNA molecules would significantly enhance our under- standing of eucaryotic cells.