U1 small nuclear ribonucleoproteins are required early during spliceosome assembly.

Abstract

Ul small nuclear ribonucleoproteins (snRNPs) are required for in vitro splicing of pre-mRNA. Sequences within Ul RNA hybridize to, and thus recognize, 5' splice junctions. We have investigated the mechanism of association of Ul snRNPs with the splicesome. Ul-specific antibodies detected Ul association with precursor RNA early during assembly. Removal of the 5' terminal sequences of Ul RNA by oligo-directed cleavage or removal of Ul snRNPs by immunoprecipitation prior to the addition of precursor RNA depressed the association of all snRNPs with precursor RNA as detected by immunoprecipitation of splicing complexes by either Sm or Ul-specific antibodies. Assembly of the spliceosome as monitored by gel electrophoresis was also depressed after cleavage of Ul RNA. The dependency of Sm precipitability of precursor RNA upon the presence of Ul snRNPs suggests that Ul snRNPs participate in the early recognition of substrate RNAs by U2 to U6 snRNPs. Although removal of the 5'-terminal sequences of Ul depressed Ul snRNP association with precursor RNA, it did not eliminate it, suggesting semistable association of Ul snRNPs with the assembling spliceosome in the absence of Ul RNA hybridization. This association was not dependent upon 5' splice junction sequences but was dependent upon 3' intronic sequences, indicating that Ul snRNPs interact with factors recognizing 3' intronic sequences. Mutual dependence of 5' and 3' recognition factors suggests significant snRNP-snRNP communication during early assembly.Small nuclear ribonucleoproteins (snRNPs) recognize consensus sequence elements within pre-mRNA and target those sequences for cleavage and/or ligation by the splicing machinery (1, 3,4,6,7,9,(14)(15)(16). Ul and U2 snRNPs bind to splice junctions and branch point sequences, respectively; binding protects the consensus sequences from exogenous ribonuclease digestion (6,7,16). An Sm-reactive element, probably U5 snRNPs, recognizes 3' splice junction sequences and protects the 3' junction and adjacent polypyrimidine track against ribonuclease digestion (10, 23). The mechanism whereby individual snRNPs recognize their respective target sequences is under intensive investigation. Both splicing activity and protection of 5' splice junctions of Ul snRNPs require the sequences of Ul RNA complementary to 5' junction sequences (6,7,15,24), leading to the suggestion that hybridization of U RNAs to precursor RNA is required for association of snRNPs with the spliceosome.Studies of in vitro assembly of exogenous precursor RNA into spliceosomes (2,5,9,11,13,(19)(20)(21) indicate that assembly of exogenous precursor RNA into the active spliceosome is dependent on the presence of the consensus sequences recognized by U snRNPs. Substrates lacking all consensus sequences, however, assemble into RNP complexes, indicating that snRNPs may recognize splicing consensus sequences in precursor RNAs already complexed with heterogeneous nuclear RNP (hnRNP) RNP polypeptides (8,22). Smaller assemblies are produced by using substrates lacking 3' splic...

In vitro polyadenylation is stimulated by the presence of an upstream intron.

“…These two sequences splice and polyadenylate very efficiently when assayed in vitro as isolated sites (Sheets et al 1987;Zarkower andWickens 1987, 1988;Zillmann et al 1987Zillmann et al , 1988Ryner et al 1987). In fact, these are probably the two most active in vitro precursor RNAs yet described.…”

“…The chimeric DNA constructed for this study (MXSVL) consists of 217 nucleotides from the major late transcription unit of human adenovirus containing two exons and a single intron (MX) fused within exon 2 to a standard polyadenylation cassette of 237 nucleotides from the late transcription unit of SV40 (SVL). Precursor RNAs made from only the splicing or polyadenylation portion of the chimeric gene splice or polyadenylate well, respectively, in in vitro HeLa cell extracts (Sperry and Berget 1986;Zillmann et al 1987). MX DNA contains no known polyadenylation signals, and the SVL cassette contains no splicing signals.…”

“…The MXSVL chimeric RNA was constructed by fusion of 217 nucleotides from the MINX precursor RNA to 237 nucleotides from the SV40 late poly(A) site at the BamHI site in the SP64 polylinker, terminating the MINX construct and the natural BamHI site from the SV40 late region (Zillmann et al 1987). Three nucleotides of polylinker separate the first half of exon 2 from the adenovirus major late region in MINX from the beginning of the SV40 late region.…”

The organization of 3' splice-site sequences in mammalian introns.

“…Several different factors appear to interact with sequences at the 3' splice site (Kramer 1988), including three different heteronuclear RNP (hnRNP) proteins (Swanson and Dreyfuss 1988), U1 snRNP (Zillman et al 1987), and two different proteins, designated U2AF and intron-binding protein (IBP). U2AF is thought to mediate U2 snRNP binding at the branch site (Ruskin et al 1988), whereas IBP may mediate U5 snRNP binding at the 3' splice site (Gerke et al 1986;Tazi et al 1986).…”

Presplicing complex formation requires two proteins and U2 snRNP.

“…The Ul small nuclear ribonucleoprotein particle (snRNP) binds to the 5' splice site in an ATP-and temperature-independent fashion (Mount et al 1983;Black et al 1985;Chabot and Steitz 1987). This binding occurs via base pairing of Ul RNA with the 5' splice site (Zhuang and Weiner 1986), and additional mechanisms for early interactions with the pre-mRNA may exist (Zillmann et al 1987).…”