TheDrosophilaU1 and U6 Gene Proximal Sequence Elements Act as Important Determinants of the RNA Polymerase Specificity of Small Nuclear RNA Gene Promotersin Vitroandin Vivo

Two distinct chicken U4 RNA genes have been cloned and characterized. They are closely linked within 465 base pairs of each other and have the same transcriptional orientation. The downstream U4 homology is a true gene, based on the criteria that it is colinear with chicken U4B RNA and is expressed when injected into Xenopus laevis oocytes. The upstream U4 homology, however, contains seven base substitutions relative to U4B RNA. This sequence may be a nonexpressed pseudogene, but the pattern of base substitutions suggests that it more probably encodes a variant yet functional U4 RNA product not yet characterized at the RNA level. In support of this, the two U4 genes have regions of homology with each other in their 5'-flanking DNA at two positions known to be essential for the efficient expression of vertebrate U1 and U2 small nuclear RNA genes. In the case of U1 and U2 RNA genes, the more distal region (located near position-200 with respect to the RNA cap site) is known to function as a transcriptional enhancer. Although this region is highly conserved in overall structure and sequence among U1 and U2 RNA genes, it is much less conserved in the chicken U4 RNA genes reported here. Interestingly, short sequence elements present in the -200 region of the U4 RNA genes are inverted (i.e., on the complementary strand) relative to their usual orientation upstream of U1 and U2 RNA genes. Thus, the -200 region of the U4 RNA genes may represent a natural evolutionary occurrence of an enhancer sequence inversion.

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Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.2019 ◽

1995 ◽

Vol 15 (4) ◽

pp. 2019-2027 ◽

Cited By ~ 73

Author(s):

J B Yoon ◽

S Murphy ◽

L Bai ◽

Z Wang ◽

R G Roeder

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Small Nuclear Rna ◽

Class Iii ◽

Sequence Element ◽

Pol Ii ◽

Proximal Sequence Element ◽

Nuclear Rna ◽

Class Iii Genes ◽

Pol Iii

The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP-TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific as gene-specific factors for PSE-and B-box-containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the from used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.

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Integrator, a Multiprotein Mediator of Small Nuclear RNA Processing, Associates with the C-Terminal Repeat of RNA Polymerase II

Cell ◽

10.1016/j.cell.2005.08.019 ◽

2005 ◽

Vol 123 (2) ◽

pp. 265-276 ◽

Cited By ~ 254

Author(s):

David Baillat ◽

Mohamed-Ali Hakimi ◽

Anders M. Näär ◽

Ali Shilatifard ◽

Neil Cooch ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Processing ◽

Terminal Repeat ◽

Small Nuclear Rna ◽

Nuclear Rna ◽

Nuclear Rna Processing

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The General Transcription Factors IIA, IIB, IIF, and IIE Are Required for RNA Polymerase II Transcription from the Human U1 Small Nuclear RNA Promoter

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.2130 ◽

1999 ◽

Vol 19 (3) ◽

pp. 2130-2141 ◽

Cited By ~ 39

Author(s):

T. C. Kuhlman ◽

H. Cho ◽

D. Reinberg ◽

N. Hernandez

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Small Nuclear Rna ◽

Initiation Complex ◽

General Transcription Factors ◽

Nuclear Rna ◽

Transcription Initiation Complex ◽

Rna Polymerase Ii Transcription

ABSTRACT RNA polymerase II transcribes the mRNA-encoding genes and the majority of the small nuclear RNA (snRNA) genes. The formation of a minimal functional transcription initiation complex on a TATA-box-containing mRNA promoter has been well characterized and involves the ordered assembly of a number of general transcription factors (GTFs), all of which have been either cloned or purified to near homogeneity. In the human RNA polymerase II snRNA promoters, a single element, the proximal sequence element (PSE), is sufficient to direct basal levels of transcription in vitro. The PSE is recognized by the basal transcription complex SNAPc. SNAPc, which is not required for transcription from mRNA-type RNA polymerase II promoters such as the adenovirus type 2 major late (Ad2ML) promoter, is thought to recruit TATA binding protein (TBP) and nucleate the assembly of the snRNA transcription initiation complex, but little is known about which GTFs other than TBP are required. Here we show that the GTFs IIA, IIB, IIF, and IIE are required for efficient RNA polymerase II transcription from snRNA promoters. Thus, although the factors that recognize the core elements of RNA polymerase II mRNA and snRNA-type promoters differ, they mediate the recruitment of many common GTFs.

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Each of the conserved sequence elements flanking the cleavage site of mammalian histone pre-mRNAs has a distinct role in the 3'-end processing reaction

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.3105-3108.1989 ◽

1989 ◽

Vol 9 (7) ◽

pp. 3105-3108

Author(s):

K L Mowry ◽

R Oh ◽

J A Steitz

Keyword(s):

Small Nuclear Rna ◽

Hairpin Loop ◽

Apparent Contrast ◽

Conserved Sequence ◽

Histone 3 ◽

Nuclear Rna ◽

Sequence Elements ◽

Loop Element ◽

Processing Site

To study the substrate requirements for the histone 3'-end processing reaction of mammalian histone pre-mRNAs, we created a set of mutations in the sequences flanking the processing site of a mouse H3 gene. We found that deletion of the downstream purine-rich element hypothesized to interact with U7 small nuclear RNA abolishes in vitro 3'-end processing. Somewhat surprisingly, however, mutations in the hairpin loop element which destabilize or destroy the secondary structure reduce but do not abolish 3'-end processing. This is in apparent contrast to results obtained for the sea urchin system, where both sequence elements appear to be absolutely required for 3'-end formation.

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Architecture of the U5 small nuclear RNA

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.2180-2190.1994 ◽

1994 ◽

Vol 14 (3) ◽

pp. 2180-2190

Author(s):

D N Frank ◽

H Roiha ◽

C Guthrie

Keyword(s):

Secondary Structure ◽

Comparative Sequence Analysis ◽

Fungal Species ◽

Deletion Analysis ◽

Small Nuclear Rna ◽

Stem Loop ◽

Internal Loop ◽

Nuclear Rna ◽

Sequence Elements ◽

U5 Snrna

We have used comparative sequence analysis and deletion analysis to examine the secondary structure of the U5 small nuclear RNA (snRNA), an essential component of the pre-mRNA splicing apparatus. The secondary structure of Saccharomyces cerevisiae U5 snRNA was studied in detail, while sequences from six other fungal species were included in the phylogenetic analysis. Our results indicate that fungal U5 snRNAs, like their counterparts from other taxa, can be folded into a secondary structure characterized by a highly conserved stem-loop (stem-loop 1) that is flanked by a moderately conserved internal loop (internal loop 1). In addition, several of the fungal U5 snRNAs include a novel stem-loop structure (ca. 30 nucleotides) that is adjacent to stem-loop 1. By deletion analysis of the S. cerevisiae snRNA, we have demonstrated that the minimal U5 snRNA that can complement the lethal phenotype of a U5 gene disruption consists of (i) stem-loop 1, (ii) internal loop 1, (iii) a stem-closing internal loop 1, and (iv) the conserved Sm protein binding site. Remarkably, all essential, U5-specific primary sequence elements are encoded by a 39-nucleotide domain consisting of stem-loop 1 and internal loop 1. This domain must, therefore, contain all U5-specific sequences that are essential for splicing activity, including binding sites for U5-specific proteins.

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Transcription boundaries of U1 small nuclear RNA

Molecular and Cellular Biology ◽

10.1128/mcb.5.9.2332-2340.1985 ◽

1985 ◽

Vol 5 (9) ◽

pp. 2332-2340

Author(s):

G R Kunkel ◽

T Pederson

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Small Nuclear Rna ◽

Cell Nuclei ◽

Sm Proteins ◽

Isolated Nuclei ◽

Nuclear Rna ◽

Rna Biosynthesis ◽

Labeled Rna ◽

Alpha Amanitin

Transcription-proximal stages of U1 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 U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/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 U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 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 U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 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, U1 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 U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.

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