A human U2 RNA mutant stalled in 3' end processing is impaired in nuclear import

Huang, Qian

doi:10.1093/nar/27.4.1025

Cited by 25 publications

(24 citation statements)

References 22 publications

(45 reference statements)

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“…3) (30) but also because the unprocessed transcripts are heterogeneous (15) and unlikely to be stable over the period of in vivo labeling (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Role of the C-Terminal Domain of RNA Polymerase II in U2 snRNA Transcription and 3′ Processing

Jacobs

Ogiwara

Weiner

2004

Molecular and Cellular Biology

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U small nuclear RNAs (snRNAs) and mRNAs are both transcribed by RNA polymerase II (Pol II), but the snRNAs have unusual TATA-less promoters and are neither spliced nor polyadenylated; instead, 3 processing is directed by a highly conserved 3 end formation signal that requires initiation from an snRNA promoter. Here we show that the C-terminal domain (CTD) of Pol II is required for efficient U2 snRNA transcription, as it is for mRNA transcription. However, CTD kinase inhibitors, such as 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), that block mRNA elongation do not affect U2 transcription, although 3 processing of the U2 primary transcript is impaired. We show further that U2 transcription is preferentially inhibited by low doses of UV irradiation or actinomycin D, which induce CTD kinase activity, and that UV inhibition can be rescued by treatment with DRB or H7. We propose that Pol II complexes transcribing snRNAs and mRNAs have distinct CTD phosphorylation patterns. mRNA promoters recruit factors including kinases that hyperphosphorylate the CTD, and the CTD in turn recruits proteins needed for mRNA splicing and polyadenylation. We predict that snRNA promoters recruit factors including a CTD kinase(s) whose snRNA-specific phosphorylation pattern recruits factors required for promoter-coupled 3 end formation.RNAs that encode proteins are transcribed by RNA polymerase II (Pol II) in almost all eukaryotes. In contrast, untranslated RNAs are transcribed by all three RNA polymerases: 5.8, 18, and 28S rRNA by Pol I; 5S rRNA, tRNA, and U6 small nuclear RNA (snRNA) by Pol III (56); and the other U snRNAs, which function in mRNA splicing and various RNA processing events, by Pol II (27). Kinetoplastid protozoa, a class of early diverging eukaryotes, are exceptions to these rules. Kinetoplastid snRNAs are transcribed not by Pol II but by Pol III (65), and certain mRNAs, including the immunologically important variant surface glycoprotein message, are hybrids of a U snRNA-like spliced leader transcribed by Pol II and a protein-coding mRNA body transcribed by Pol I (19).Although U snRNAs and mRNAs are both transcribed by Pol II in mammals, the genes are very different. U snRNA promoters have no TATA box and rely instead upon a dedicated U snRNA-specific promoter consisting of a highly conserved proximal sequence element (PSE) and an enhancer-like distal sequence element spaced one nucleosome apart (27). In addition, U snRNA genes are short (typically only a few hundred base pairs) and lack introns, whereas genes encoding mRNAs can span megabases and usually contain many introns. Also, U snRNA genes are typically present in multiple copies in higher eukaryotes-the human U1 and U2 genes are tandemly repeated (6, 40, 66, 68)-whereas most protein-coding genes are present in only one or a few copies per haploid genome.U snRNA processing also differs from mRNA processing. U snRNAs are neither spliced nor polyadenylated; instead, formation of the first U snRNA interme...

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“…3) (30) but also because the unprocessed transcripts are heterogeneous (15) and unlikely to be stable over the period of in vivo labeling (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Role of the C-Terminal Domain of RNA Polymerase II in U2 snRNA Transcription and 3′ Processing

Jacobs

Ogiwara

Weiner

2004

Molecular and Cellular Biology

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“…4,[7][8][9][10][11] However, the precise mechanisms of how WPRE increases vector titers and transgene expression in RVs are unknown. 4,12,13 Zaiss et al have shown that HIV-1-based LV vectors consistently expressed reporter genes athigher levels when compared to MLV-based RV vectors. 15 Their analysis showed that the differences in expression from RV and LV vectors could be attributed to inefficient transcript termination in RV vectors.…”

Section: Introductionmentioning

confidence: 99%

The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors

et al. 2007

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The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression from a variety of viral vectors, although the precise mechanism is not known. WPRE is most effective when placed downstream of the transgene, proximal to the polyadenylation signal. We hypothesized that WPRE likely reduces viral mRNA readthrough transcription by improving transcript termination, which in turn would increase viral titers and expression. Using a Cre-lox-mediated plasmid-based assay, we found significant readthrough transcription from g-retroviral vector (RV) long terminal repeat (wt RV-LTR) and RV LTR with a self-inactivating deletion (SIN RV-LTR). WPRE, when placed upstream of the RV LTRs, significantly reduced readthrough transcription. Readthrough, present at much lower levels with the SIN HIV-1 LV-LTR, was also reduced with WPRE. When placed in RV vectors, WPRE increased total RV genomic mRNA; and increased viral titers from transiently transfected 293T cells and stable PG13 producer cells by 7-to 15-fold. The mechanism of increased titers and expression was not due to increased nuclear mRNA export, increased rate of viral transcription or a significant increase in viral mRNA half-life. Our results showed that WPRE improved vector genomic transcript termination to increase titers and expression from RVs.

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“…Although Sm assembly onto U snRNAs can occur in vitro without SMN and its associated proteins (Raker et al 1996), recent work has shown that the SMN complex is required for proper assembly in vivo (Meister et al 2001;Pellizzoni et al 2002;Yong et al 2004). After assembly of the Sm core, the snRNA is subject to cap hypermethylation and 3′-end trimming, followed by import back into the nucleus, again under the aegis of the SMN complex (Huang and Pederson 1999;Massenet et al 2002;Mouaikel et al 2003;Narayanan et al 2002;Will and Luhrmann 2001).…”

Section: Introductionmentioning

confidence: 99%

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

Pillai

Azzouz

et al. 2005

Chromosoma

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Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of 'mature' snRNPs, or the reclamation of unassembled snRNP components.

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A human U2 RNA mutant stalled in 3' end processing is impaired in nuclear import

Cited by 25 publications

References 22 publications

Role of the C-Terminal Domain of RNA Polymerase II in U2 snRNA Transcription and 3′ Processing

Role of the C-Terminal Domain of RNA Polymerase II in U2 snRNA Transcription and 3′ Processing

The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

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