Synthesis of low molecular weight RNA components A, C and D by polymerase II in α-amanitin-resistant hamster cells

Jensen, Elisabeth Gram; Hellung‐Larsen, Per; Frederiksen, Sune

doi:10.1093/nar/6.1.321

Cited by 64 publications

(20 citation statements)

References 24 publications

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“…An almost complete suppression of Ul RNA labeling was observed when in vitro transcription was carried out in the presence of a-amanitin at a concentration of 1 ,ug/ml (Fig. 2), consistent with previous evidence that Ul RNA is transcribed by RNA polymerase II (19,40). The most interesting result in Fig.…”

Section: Resultssupporting

confidence: 91%

“…Human cells contain approximately 30 Ul RNA genes per haploid genome (27), clustered on chromosome 1 (26). Ul RNA is synthesized by RNA polymerase 11 (19,40) but, although the 5' sequences flanking several cloned human Ul genes are highly conserved (27,31), they lack the canonical promoter sequences characteristic of other polymerase II-transcribed genes (4). Moreover, the transcription unit of Ul RNA has not been clearly defined.…”

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confidence: 99%

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Transcription boundaries of U1 small nuclear RNA.

Kunkel¹,

Pederson²

1985

Mol. Cell. Biol.

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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).

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Section: Resultssupporting

confidence: 91%

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confidence: 99%

Transcription boundaries of U1 small nuclear RNA.

Kunkel¹,

Pederson²

1985

Mol. Cell. Biol.

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“…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).…”

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confidence: 99%

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.

Kunkel¹,

Pederson²

1988

Genes Dev.

154

142

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U6 small nuclear RNA is transcribed by a different polymerase than U1-U5 RNAs, likely to be RNA polymerase III. Transcription from human U6 gene deletion-substitution templates in a HeLa S100 extract delineated the 5' border of a control element lying between 67 and 43 bp upstream from the initiation site. This region matches the location of, and shows considerable sequence similarity with, the proximal control element of U1 and U2 RNA genes, which are transcribed by RNA polymerase II. Transfection of human 293 cells with 5'-flanking deletion-substitution mutants of a U6 maxigene revealed a dominant control element between 245 and 149 bp upstream of the transcription start site. An octamer motif was found in this region in an inverted orientation relative to that of the human U1 and U2 RNA gene enhancers but in the same orientation as a human U4 RNA gene, the transcript of which functions together with U6 RNA in a single small nuclear ribonucleoprotein (snRNP) particle. The human U2 gene enhancer joined to the U6 maxigene was able to functionally replace the U6 distal control element(s).

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“…Like other RNA polymerase II transcripts, such as mRNA, snRNAs are capped, and their synthesis is inhibited by a-amanitin (8). Unlike most polymerase II transcripts, snRNAs are metabolically stable, are not polyadenylated, and contain an unusual 2,2,7-trimethyl guanosine cap structure instead of the 7-methyl guanosine cap found on mRNA (for a review, see reference 31).…”

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confidence: 99%

Orientation-dependent transcriptional activator upstream of a human U2 snRNA gene.

Ares¹,

Mangin²,

Weiner³

1985

Mol. Cell. Biol.

114

116

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We examined the structure of the promoter for the human U2 snRNA gene, a strong RNA polymerase II transcription unit without an obvious TATA box. A set of 5' deletions was constructed and assayed for the ability to direct initiation of U2 snRNA after microinjection into Xenopus oocytes. Sequences between positions -295 and -218 contain an activator element which stimulates accurate initiation by 20-to 50-fold, although as few as 62 base pairs of 5' flanking sequence are sufficient to direct the accurate initiation of U2 RNA. When the activator was recloned in the proper orientation at either of two different upstream locations, the use of the normal U2 start site was stimulated. Inversion of the element destroyed the stimulation of accurate U2 initiation, but initiation at aberrant upstream start sites was enhanced by the element in both orientations. A 4-base-pair deletion that destroyed the activity of the element lies within a sequence (region III) which is highly conserved among U2 genes from different organisms. Mutations in the activator also affected the ability of the U2 template to compete with a wild-type Ul gene in coinjection experiments. We propose that the element enhances the efficiency of transcription in part by facilitating the association of a limiting factor with transcription complexes. Human Ul snRNA genes possess a region homologous to U2 region III, and we suggest that upstream activator elements may be a general feature of snRNA promoters.

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Synthesis of low molecular weight RNA components A, C and D by polymerase II in α-amanitin-resistant hamster cells

Cited by 64 publications

References 24 publications

Transcription boundaries of U1 small nuclear RNA.

Transcription boundaries of U1 small nuclear RNA.

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.

Orientation-dependent transcriptional activator upstream of a human U2 snRNA gene.

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