A link between nuclear RNA surveillance, the human exosome and RNA polymerase II transcriptional termination

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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Pause Sites Promote Transcriptional Termination of Mammalian RNA Polymerase II

Molecular and Cellular Biology ◽

10.1128/mcb.26.10.3986-3996.2006 ◽

2006 ◽

Vol 26 (10) ◽

pp. 3986-3996 ◽

Cited By ~ 111

Author(s):

Natalia Gromak ◽

Steven West ◽

Nick J. Proudfoot

Keyword(s):

Steady State ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Cleavage ◽

Actin Gene ◽

General Role ◽

Pol Ii ◽

Transcriptional Termination ◽

A Site ◽

Pause Site

ABSTRACT Polymerase II (Pol II) transcriptional termination depends on two independent genetic elements: poly(A) signals and downstream terminator sequences. The latter may either promote cotranscriptional RNA cleavage or pause elongating Pol II. We demonstrate that the previously characterized MAZ4 pause element promotes Pol II termination downstream of a poly(A) signal, dependent on both the proximity of the pause site and poly(A) signal and the strength of the poly(A) signal. The 5′→3′ exonuclease Xrn2 facilitates this pause-dependent termination by degrading the 3′ product of poly(A) site cleavage. The human β-actin gene also possesses poly(A) site proximal pause sequences, which like MAZ4 are G rich and promote transcriptional termination. Xrn2 depletion causes an increase in both steady-state RNA and Pol II levels downstream of the β-actin poly(A) site. Taken together, we provide new insights into the mechanism of pause site-mediated termination and establish a general role for the 5′→3′ exonuclease Xrn2 in Pol II termination.

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Out or decay: fate determination of nuclear RNAs

Essays in Biochemistry ◽

10.1042/ebc20200005 ◽

2020 ◽

Vol 64 (6) ◽

pp. 895-905 ◽

Cited By ~ 2

Author(s):

Jianshu Wang ◽

Hong Cheng

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Fate Determination ◽

Biological Relevance ◽

Nuclear Rna ◽

Nuclear Degradation ◽

Processing Steps ◽

Mammalian System

Abstract In eukaryotes, RNAs newly synthesized by RNA polymerase II (RNAPII) undergo several processing steps prior to transport to the cytoplasm. It has long been known that RNAs with defects in processing or export are removed in the nucleus. Recent studies revealed that RNAs without apparent defects are also subjected to nuclear degradation, indicating that nuclear RNA fate is determined in a more complex and dynamic way than previously thought. Nuclear RNA sorting directly determines the quality and quantity of RNA pools for future translation and thus is of significant importance. In this essay, we will summarize recent studies on this topic, mainly focusing on findings in mammalian system, and discuss about important remaining questions and possible biological relevance for nuclear RNA fate determination.

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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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Heterogeneous nuclear RNA-ribonucleoprotein F binds to DNA via an oligo(dG)-motif and is associated with RNA polymerase II

Genes to Cells ◽

10.1046/j.1365-2443.1999.00295.x ◽

1999 ◽

Vol 4 (12) ◽

pp. 707-719 ◽

Cited By ~ 15

Author(s):

Tatsushi Yoshida ◽

Kenji Kokura ◽

Yasutaka Makino ◽

Vincent Ossipow ◽

Taka-aki Tamura

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nuclear Rna

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Oct-1 and Oct-2 potentiate functional interactions of a transcription factor with the proximal sequence element of small nuclear RNA genes

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3247-3261.1992 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3247-3261

Author(s):

S Murphy ◽

J B Yoon ◽

T Gerster ◽

R G Roeder

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Polymerase Iii ◽

Small Nuclear Rna ◽

Sequence Element ◽

Proximal Sequence Element ◽

Pou Domain ◽

Polymerase Iii ◽

Nuclear Rna

The promoters of both RNA polymerase II- and RNA polymerase III-transcribed small nuclear RNA (snRNA) genes contain an essential and highly conserved proximal sequence element (PSE) approximately 55 bp upstream from the transcription start site. In addition, the upstream enhancers of all snRNA genes contain binding sites for octamer-binding transcription factors (Octs), and functional studies have indicated that the PSE and octamer elements work cooperatively. The present study has identified and characterized a novel transcription factor (designated PTF) which specifically binds to the PSE sequence of both RNA polymerase II- and RNA polymerase III-transcribed snRNA genes. PTF binding is markedly potentiated by Oct binding to an adjacent octamer site. This potentiation is effected by Oct-1, Oct-2, or the conserved POU domain of these factors. In agreement with these results and despite the independent binding of Octs to the promoter, PTF and Oct-1 enhance transcription from the 7SK promoter in an interdependent manner. Moreover, the POU domain of Oct-1 is sufficient for significant in vitro activity in the presence of PTF. These results suggest that essential activation domains reside in PTF and that the potentiation of PTF binding by Octs plays a key role in the function of octamer-containing snRNA gene enhancers.

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