The transcriptional cycle of HIV-1 in real-time and live cells

Boireau, Stéphanie; Maiuri, Paolo; Basyuk, Eugénia; Mata, Manuel de la; Knezevich, Anna; Pradet-Balade, Bérangère; Baecker, Volker; Kornblihtt, Alberto R.; Marcello, Alessandro; Bertrand, Édouard

doi:10.1084/jem20411oia25

Cited by 26 publications

(55 citation statements)

References 34 publications

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“…Then, the FRAP (fluorescence recovery after photobleaching) technique can be used, whereby the replacement of the bleached protein is mostly the result of a new transcription of the sequence and association with a free MS2-binding protein from the nucleoplasm. By use of this technique, together with mathematical modeling, the transcriptional elongation rate was estimated in single cells to be between 1.8 and 4.3 kb/min (37,38), which is in very good agreement with the biochemical measurements discussed above. By use of the same approach with versions of the reporter with the MS2 located in an intron, cotranscriptional splicing was supported by showing faster turnover of the intron with respect to the distal exon (39).…”

Section: Connections Between Transcription and Splicingsupporting

confidence: 79%

Regulation of Alternative Splicing Through Coupling with Transcription and Chromatin Structure

Naftelberg

Schor²,

Ast³

et al. 2015

Annu. Rev. Biochem.

392

354

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Alternative precursor messenger RNA (pre-mRNA) splicing plays a pivotal role in the flow of genetic information from DNA to proteins by expanding the coding capacity of genomes. Regulation of alternative splicing is as important as regulation of transcription to determine cell- and tissue-specific features, normal cell functioning, and responses of eukaryotic cells to external cues. Its importance is confirmed by the evolutionary conservation and diversification of alternative splicing and the fact that its deregulation causes hereditary disease and cancer. This review discusses the multiple layers of cotranscriptional regulation of alternative splicing in which chromatin structure, DNA methylation, histone marks, and nucleosome positioning play a fundamental role in providing a dynamic scaffold for interactions between the splicing and transcription machineries. We focus on evidence for how the kinetics of RNA polymerase II (RNAPII) elongation and the recruitment of splicing factors and adaptor proteins to chromatin components act in coordination to regulate alternative splicing.

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Section: Connections Between Transcription and Splicingsupporting

confidence: 79%

Regulation of Alternative Splicing Through Coupling with Transcription and Chromatin Structure

Naftelberg

Schor²,

Ast³

et al. 2015

Annu. Rev. Biochem.

392

354

View full text Add to dashboard Cite

show abstract

“…1). Similar transcription rates (0.31-0.78 kb min −1 ) were reported for a single cyclin D1 gene under endogenous or CMV promoter control 21 , whereas rates in the range of 2 to 4 kb min −1 have been estimated for different reporter genes using distinct kinetic modeling approaches 16,20 .…”

Section: An Assay To Image B-globin Transcription In Living Cellssupporting

confidence: 67%

“…The resulting expression plasmid pTRE-βWT-MS2exon was cotransfected with a puromycin resistance plasmid into U2OS Tet-On cells. We isolated and screened drug-resistant colonies by transient transfection with a plasmid expressing a fusion between MS2 coat protein, the red fluorescent protein mCherry and a nuclear localization signal (NLS) to target the chimeric protein to the nucleus 16 . We selected one clone (U2OS-βWT#9) for further analysis, because most cells contained a single, easily detectable nuclear focus of mCherry-MS2 upon induction with doxycycline.…”

Section: An Assay To Image B-globin Transcription In Living Cellsmentioning

confidence: 99%

“…Recovery of mCherry-MS2 fluorescence at the transcription site results from replacement of β-globin RNA stably bound to bleached mCherry-MS2 by newly synthesized RNA containing MS2 binding sites that recruit unbleached fluorescent fusion proteins from the nucleoplasm. Previous studies have measured rates of polymerase elongation from FRAP data under the assumption that fluorescence recovery results from the sequential addition of fluorescent molecules to newly synthesized RNA 16,20,21 . Following the approach described 21 , we found the mCherry-MS2 recovery curve to be best fit by a singleexponential curve, which is compatible with a 'reaction-dominant' model where slow transcription elongation is the rate-limiting step.…”

Section: An Assay To Image B-globin Transcription In Living Cellsmentioning

confidence: 99%

“…Cells plated on P35 dishes were cotransfected with mCherry-MS2 (ref. 16) and α-amanitin-resistant GFP-RNAPII (ref. 17) plasmids using the FuGENE6 reagent (Roche Biochemicals).…”

Section: Acknowledgmentsmentioning

confidence: 99%

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Spliceosome assembly is coupled to RNA polymerase II dynamics at the 3′ end of human genes

Martins

Rino

Carvalho

et al. 2011

Nat Struct Mol Biol

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In the nucleus of higher eukaryotes, maturation of mRNA precursors involves an orderly sequence of transcription-coupled interdependent steps. Transcription is well known to influence splicing, but how splicing may affect transcription remains unclear. Here we show that a splicing mutation that prevents recruitment of spliceosomal snRNPs to nascent transcripts causes co-transcriptional retention of unprocessed RNAs that remain associated with polymerases stalled predominantly at the 3' end of the gene. In contrast, treatment with spliceostatin A, which allows early spliceosome formation but destabilizes subsequent assembly of the catalytic complex, abolishes 3' end pausing of polymerases and induces leakage of unspliced transcripts to the nucleoplasm. Taken together, the data suggest that recruitment of splicing factors and correct assembly of the spliceosome are coupled to transcription termination, and this might ensure a proofreading mechanism that slows down release of unprocessed transcripts from the transcription site.

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Making ends meet: coordination between RNA 3′‐end processing and transcription initiation

2013

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RNA polymerase II (RNAPII)-mediated gene transcription initiates at promoters and ends at terminators. Transcription termination is intimately connected to 3'-end processing of the produced RNA and already when loaded at the promoter, RNAPII starts to become configured for this downstream event. Conversely, RNAPII is 'reset' as part of the 3'-end processing/termination event, thus preparing the enzyme for its next round of transcription--possibly on the same gene. There is both direct and circumstantial evidence for preferential recycling of RNAPII from the gene terminator back to its own promoter, which supposedly increases the efficiency of the transcription process under conditions where RNAPII levels are rate limiting. Here, we review differences and commonalities between initiation and 3'-end processing/termination processes on various types of RNAPII transcribed genes. In doing so, we discuss the requirements for efficient 3'-end processing/termination and how these may relate to proper recycling of RNAPII.

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The transcriptional cycle of HIV-1 in real-time and live cells

Cited by 26 publications

References 34 publications

Regulation of Alternative Splicing Through Coupling with Transcription and Chromatin Structure

Regulation of Alternative Splicing Through Coupling with Transcription and Chromatin Structure

Spliceosome assembly is coupled to RNA polymerase II dynamics at the 3′ end of human genes

Making ends meet: coordination between RNA 3′‐end processing and transcription initiation

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