The mechanistic basis for the coupling between transcription, splicing and polyadenylation machineries. Frank Willi Rigo Nusser

ISBN: 9780549979760

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119 pages


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The mechanistic basis for the coupling between transcription, splicing and polyadenylation machineries.  by  Frank Willi Rigo Nusser

The mechanistic basis for the coupling between transcription, splicing and polyadenylation machineries. by Frank Willi Rigo Nusser
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Capping, splicing and cleavage/polyadenylation of pre-mRNA in eukaryotes is carried out by molecular machineries whose activities are functionally interconnected both with the transcriptional apparatus and with each other. To study how this couplingMoreCapping, splicing and cleavage/polyadenylation of pre-mRNA in eukaryotes is carried out by molecular machineries whose activities are functionally interconnected both with the transcriptional apparatus and with each other. To study how this coupling occurs I solved a long-standing problem in the field by developing a HeLa nuclear extract-based in vitro system that simultaneously supports all of these interconnected activities.-In this system, both last-intron splicing and cleavage/polyadenylation are functionally coupled to transcription via the tether of nascent RNA that extends from the terminal exon to the transcribing polymerase downstream.

RNA polymerase II pull-down and immobilized template experiments suggest that the role of the tether is to hold the poly(A) signal close to the polymerase during the early stages of processing complex assembly until the complex is sufficiently mature to remain stably associated with the polymerase on its own.-Communication between the 3 splice site and the poly(A) site across the terminal exon is established within minutes of their transcription, and multiple steps leading up to 3-end processing of this exon can be distinguished.

First, the 3 splice site establishes connections to enhance cleavage/polyadenylation, while the nascent cleavage/polyadenylation apparatus (CPA) makes reciprocal functional connections to enhance splicing. Then, commitment to poly(A) site cleavage itself occurs and the connections of the CPA to the transcribing polymerase are strengthened. Finally, the chemical steps in processing of the terminal exon take place, beginning with poly(A) site cleavage, continuing with polyadenylation of the 3 end, and then finishing with splicing of the last intron.-The nascent transcripts that become attached to the polymerase during assembly of the CPA, are not released even after cleavage at the poly(A) site.

These cleaved transcripts are anchored to the polymerase at their 3 ends by the CPA or, when introns are present, by the larger 3 terminal exon definition complex (EDC), which consists of splicing factors complexed with the CPA. Poly(A) addition releases the RNA from the polymerase when the RNA is anchored only by the CPA. When anchored by the EDC, poly(A) addition remains a requirement, but it triggers release only after being licensed by splicing.



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