A model in vitro system for co-transcriptional splicing.

Nucleic Acids Research
Yong YuRobin Reed

Abstract

A hallmark of metazoan RNA polymerase II transcripts is the presence of numerous small exons surrounded by large introns. Abundant evidence indicates that splicing to excise introns occurs co-transcriptionally, prior to release of the nascent transcript from RNAP II. Here, we established an efficient model system for co-transcriptional splicing in vitro. In this system, CMV-DNA constructs immobilized on beads generate RNAP II transcripts containing two exons and an intron. Consistent with previous work, our data indicate that elongating nascent transcripts are tethered to RNAP II on the immobilized DNA template. We show that nascent transcripts that reach full length, but are still attached to RNAP II, are efficiently spliced. When the nascent transcript is cleaved within the intron using RNase H, both the 5' and 3' cleavage fragments are detected in the bound fraction, where they undergo splicing. Together, our work establishes a system for co-transcriptional splicing in vitro, in which the spliceosome containing the 5' and 3' exons are tethered to RNAP II for splicing.

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Citations

Jul 1, 2015·Proceedings of the National Academy of Sciences of the United States of America·Yong Yu, Robin Reed
Apr 28, 2012·Gene·Marta MontesCarlos Suñé
Aug 9, 2011·Wiley Interdisciplinary Reviews. RNA·Amy Pandya-Jones
Mar 27, 2012·Wiley Interdisciplinary Reviews. RNA·Xavier Roca, Fedor V Karginov
Oct 15, 2013·Journal of Cellular Physiology·Giuliana NapolitanoBarbara Majello
May 3, 2011·Trends in Cell Biology·Fernando Carrillo OesterreichKarla M Neugebauer

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Methods Mentioned

BETA
PCR

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