Transcription boundaries of U1 small nuclear RNA.

Abstract

Transcription-proximal stages of Ul small nuclear RNA biosynthesis were studied by 32p labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human Ul RNA gene. Transcription of Ul RNA was inhibited by >95% by at-amanitin at 1 ,ug/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature Ul RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of Ul RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled Ul RNA hybridization dropped off sharply within a very short region (-60 nucleotides) immediately downstream from the 3' end of mature Ul RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the Ul RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, Ul small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and Ul small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.Ul RNA is one of the stable, abundant, small nuclear RNAs characteristic of most eucaryotic cells (C. Brunel, J. Sri-Widada, and P. Jeanteur, Prog. Mol. Subcell. Biol., in press). The functional form of Ul RNA is a ribonucleoprotein (RNP) complex (Ul small nuclear RNP [snRNP]) that contains at least eight proteins (34, 42) and is immunoprecipitated by antisera from patients with certain autoimmune diseases (Brunel et al., in press). Several lines of evidence now point to an involvement of Ul snRNP in mRNA splicing (2,5,15,18,21,24,34,36,39,49).

Characterization of the mouse beta maj globin transcription termination region: a spacing sequence is required between the poly(A) signal sequence and multiple downstream termination elements.

“…The small nuclear RNAs, including Ul and U2, rely directly on transcription termination as the first step in the formation of their respective 3' ends (1,5,20,21,25,44). This direct termination-3' end formation mechanism contrasts with 3' end formation in mRNAs.…”

Upstream elements required for efficient transcription of a human U6 RNA gene resemble those of U1 and U2 genes even though a different polymerase is used.

“…The biosynthesis of snRNPs is itself complex, with several features different from mRNA transcription and processing. U1, U2, U4, and U5 RNAs are transcribed by polymerase II but, unlike most polymerase II transcripts (mRNA), are capped with trimethylguanosine (Ro-Choi et al 1976;Jensen et al 1979;Roop et al 1981;Kunkel and Pederson 1985). We and others have recently demonstrated that U6 RNA is transcribed by a different enzyme which, by the criteria of ~-amanitin sensitivity and competition for 5S gene transcription machinery, is likely to be RNA polymerase III (Kunkel et al 1986;Reddy et al 1987).…”

The highly conserved U small nuclear RNA 3'-end formation signal is quite tolerant to mutation.

“…Two pieces of evidence now indicate that the 3' ends of the snRNA precursors are formed by either termination of transcription or an RNA processing event directly coupled to transcription. First, run-on transcription in isolated nuclei does not proceed further than 60 nt beyond the 3' end of the mature Ul snRNA (24). Second, 3'-end formation of Ul precursors requires a compatible snRNA promoter (19,36).…”