SUMMARYThe methods of enzymatic and chemical treatment of end-labeled RNA were applied to the determination of the nucleotide sequence of chicken and man UlA RNA and to the reexamination of that of rat UlA RNA. The chemical method allowed the easy demonstration of the cap structure. All three RNA were 165 nucleotide long. Two hitherto non described modified pyrimidines were detected close to the 5' end. Only 9 base substitutions were observed from chicken to man indicating a high degree of conservation of UlA RNA through evolution. INTRODUCTIONMetabolically stable low molecular weight RNA were found in the nuclei of a variety of cell types (1-4). Four of these small RNA (4.5SI, U1A, U2 and U3B RNA) isolated from rat Novikoff Hepatoma cells were sequenced by Busch and coworkers (5-8). The interest for the small nuclear RNA was renewed by the finding that they may be hydrogen-bonded to premessenger RNA (9, 10) and that they were present in the ribonucleoproteins containing the premessenger RNA (11-14) which are assumed to be the site of splicing. Therefore, it was proposed that the small RNA might insure the proper alignment of premessenger RNA sequences for splicing (15)(16)(17). This idea was reinforced by the observation of at certain complementari:ty between a single sequence a the 5' end of UlA RNA and a consensus of the intron sequences adjacent to the splice point in premessenger RNA. As premessenger RNA from various animal species were used for the establishment of the consensus sequence and as only the published sequence of rat UlA RNA was available (6), the model implied a high conservation of the primary structure of UlA RNA through evolution (16). This prompted us to determine the complete nucleotide sequence of UlA RNA from 2 other animal species, chicken

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When U1 and U2 small nuclear ribonucleoproteins (snRNPs) purified by a procedure which preserves their immunoprecipitability by autoimmune antibodies (Hinterberger et al., J. Biol. Chem. 258:2604-2613, were submitted to extensive digestion with micrococcal nuclease, we found that their degradation pattern was sharply dependent upon magnesium concentration, indicating that they undergo a profound structural modification. At low Mg2+ (s5 mM), both particles only exhibit a core-resistant structure previously identified as being common to all but U6 snRNAs (Liautard et al., J. Mol. Biol. 162:623-643, 1982). At high Mg2+ (.7 mM), U1 and U2 snRNPs behave differently from one another. In U1 snRNP, most U1 snRNA sequence is protected, except for the 10 5'-terminal nucleotides presumably involved in splicing and a short sequence between nucleotides 102 and 108. Another region spanning nucleotides 60 to 79 is only weakly protected. This structural modification was demonstrated to be reversible. In U2 snRNP, the U2 snRNA sequence remains exposed in its 5' part up to nucleotide 92, and the 3'-terminal hairpin located outside the core structure becomes protected.All but U3 small nuclear RNAs (snRNAs) assemble in the cell with specific proteins to form small nuclear ribonucleoproteins (snRNPs) which are precipitable by sera from patients with systemic lupus erythematosus or mixed connective tissue disease. These U snRNPs (for a review, see C. Brunel, J. Sri-Widada, and P. Jeanteur, Progress in Molecular and Subcellular Biology, in press) interact with heterogenous nuclear RNPs (containing the heterogenous population of premessengers), which are now recognized as the real splicing substrate (8). Recent reports have strengthened the initial suggestions by Lerner et al. (13) and Rogers and Wall (20) that U1 snRNP is involved in splicing. Among these are the crucial demonstration that an in vitro splicing system is inhibited by monoclonal anti-Sm, patient anti-Sm, or anti-RNP antibodies but not by scleroderma-polymyositis overlap syndrome serum-precipitating U2 snRNP (18) and the biochemical evidence that U1 snRNP binds to an in vitro transcript containing splice sites (16) or that U1 snRNP stimulates in vitro splicing of adenoviral sequences (8).In the course of isolating U snRNPs from HeLa cells, we first purified a mixture containing Ul, Ul-, U4, U5, and U6 particles (5) and then purified the individual U1 snRNP (21). These particles have a common and simple protein complement which constitutes only the low-molecular-weight polypeptides (9 to 14 kilodaltons) among those immunoprecipitated by anti-RNP or anti-Sm antibodies. As such, they retained antigenicity with respect to anti-Sm but not anti-RNP (1) and were therefore designated as core snRNPs. Although certainly far from being functional, these core particles allowed us to identify the binding site for these common core proteins as being a single-stranded region containing the sequence A(U)nG, with n -3, bordered by two double-stranded stems (14). This site corresponds t...