A model in vitro system for co-transcriptional splicing

被引:8
|
作者
Yu, Yong [1 ]
Das, Rita [1 ]
Folco, Eric G. [1 ]
Reed, Robin [1 ]
机构
[1] Harvard Univ, Sch Med, Dept Cell Biol, Boston, MA 02115 USA
基金
美国国家卫生研究院;
关键词
RNA-POLYMERASE-II; MESSENGER-RNA; PREINITIATION COMPLEX; ELONGATION; INITIATION; CLEAVAGE; GENE; POLYADENYLATION; RECRUITMENT; TEMPLATES;
D O I
10.1093/nar/gkq620
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:7570 / 7578
页数:9
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