The RNA Tether from the Poly(A) Signal to the Polymerase Mediates Coupling of Transcription to Cleavage and Polyadenylation

Rigo, Frank; Kazerouninia, Amir; Nag, Anita; Martinson, Harold G.

doi:10.1016/j.molcel.2005.09.026

Cited by 35 publications

(86 citation statements)

References 45 publications

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“…In the past few years successful attempts were made to develop systems that coupled transcription and capping (Moteki and Price 2002), as well as transcription and polyadenylation (Adamson et al 2005;Rigo et al 2005). In vitro systems that combine transcription and splicing have also been described (Ghosh and GarciaBlanco 2000;Ibrahim et al 2005;Das et al 2006).…”

Section: Resultsmentioning

confidence: 99%

Concurrent splicing and transcription are not sufficient to enhance splicing efficiency

Lazarev

Manley²

2007

RNA

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The concept of a tight integration of transcription and splicing of mRNA precursors has been supported with increasing evidence in recent years. However, the mechanism and functional consequences of this integration remain largely unknown. We have examined how these processes impact upon one another when they occur together in HeLa nuclear extract. While both processes do in fact occur in parallel reactions in the extracts, we found no evidence that one process affects the other, under a variety of conditions tested. For example, neither the kinetics nor efficiency of splicing is significantly enhanced by de novo RNA polymerase II-mediated transcription, relative to that of presynthesized RNA added exogenously to the extract. Our results indicate that the act of transcription by RNA polymerase II in vitro is not sufficient to enhance splicing of the newly made RNA.

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Section: Resultsmentioning

confidence: 99%

Concurrent splicing and transcription are not sufficient to enhance splicing efficiency

Lazarev

Manley²

2007

RNA

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“…Previous studies have provided detailed mechanistic insights into the coupling between transcription and capping, and significant advances have recently been made in understanding the mechanisms that couple transcription and polyadenylation (Yonaha andProudfoot 1999, 2000;Adamson et al 2005;Rigo et al 2005). By establishing an efficient in vitro system for coupling transcription to splicing, and showing that functional coupling of these two steps in gene expression occurs during spliceosome assembly, it should now be possible to identify the factors involved.…”

Section: Discussionmentioning

confidence: 99%

“…This functional coupling is thought to serve as an important timing mechanism, which is required to prevent degradation of the nascent transcript. The recent development of efficient in vitro systems for functional coupling of RNAP II transcription to 3Ј-end cleavage/polyadenylation has also led to insights into the mechanisms involved in this coupling event (Yonaha andProudfoot 1999, 2000;Adamson et al 2005;Rigo et al 2005). In particular, the nascent pre-mRNA itself is thought to play a key role in establishing an interaction between RNAP II and the 3Ј-end cleavage machinery (Rigo et al 2005).…”

mentioning

confidence: 99%

Functional coupling of RNAP II transcription to spliceosome assembly

Das¹,

Dufu²,

Romney³

et al. 2006

Genes Dev.

104

117

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The pathway of gene expression in higher eukaryotes involves a highly complex network of physical and functional interactions among the different machines involved in each step of the pathway. Here we established an efficient in vitro system to determine how RNA polymerase II (RNAP II) transcription is functionally coupled to pre-mRNA splicing. Strikingly, our data show that nascent premessenger RNA (pre-mRNA) synthesized by RNAP II is immediately and quantitatively directed into the spliceosome assembly pathway. In contrast, nascent pre-mRNA synthesized by T7 RNA polymerase is quantitatively assembled into the nonspecific H complex, which consists of heterogeneous nuclear ribonucleoprotein (hnRNP) proteins and is inhibitory for spliceosome assembly. Consequently, RNAP II transcription results in a dramatic increase in both the kinetics of splicing and overall yield of spliced mRNA relative to that observed for T7 transcription. We conclude that RNAP II mediates the functional coupling of transcription to splicing by directing the nascent pre-mRNA into spliceosome assembly, thereby bypassing interaction of the pre-mRNA with the inhibitory hnRNP proteins.

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“…(D) Size exclusion of transcripts depends on the tether to the polymerase. Transcription was carried out in the presence of a DNA oligonucleotide that directs RNase H to cut the transcript about 800 nt downstream of the promoter (Rigo et al 2005). Size-exclusion chromatography was then carried out to separate the ternary complexes from the released RNA.…”

Section: The G/u-rich Region Is Not Required For Poly(a)-dependent Pamentioning

confidence: 99%

The conserved AAUAAA hexamer of the poly(A) signal can act alone to trigger a stable decrease in RNA polymerase II transcription velocity

Nag¹,

Narsinh²,

Kazerouninia³

et al. 2006

RNA

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In vivo the poly(A) signal not only directs 39-end processing but also controls the rate and extent of transcription. Thus, upon crossing the poly(A) signal RNA polymerase II first pauses and then terminates. We show that the G/U-rich region of the poly(A) signal, although required for termination in vivo, is not required for poly(A)-dependent pausing either in vivo or in vitro. Consistent with this, neither CstF, which recognizes the G/U-rich element, nor the polymerase CTD, which binds CstF, is required for pausing. The only part of the poly(A) signal required to direct the polymerase to pause is the AAUAAA hexamer. The effect of the hexamer on the polymerase is long lasting-in many situations polymerases over 1 kb downstream of the hexamer continue to exhibit delayed progress down the template in vivo. The hexamer is the first part of the poly(A) signal to emerge from the polymerase and may play a role independent of the rest of the poly(A) signal in paving the way for subsequent events such as 39-end processing and termination of transcription.

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The RNA Tether from the Poly(A) Signal to the Polymerase Mediates Coupling of Transcription to Cleavage and Polyadenylation

Cited by 35 publications

References 45 publications

Concurrent splicing and transcription are not sufficient to enhance splicing efficiency

Concurrent splicing and transcription are not sufficient to enhance splicing efficiency

Functional coupling of RNAP II transcription to spliceosome assembly

The conserved AAUAAA hexamer of the poly(A) signal can act alone to trigger a stable decrease in RNA polymerase II transcription velocity

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