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首页> 外文期刊>Molecular and Cellular Biology >Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing.
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Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing.

机译：RNA聚合酶II转录的新生RNA链的物理分离：共转录剪接的证据。

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In order to examine whether splicing can occur cotranscriptionally in mammalian nuclei, we mapped exon-intron boundaries on nascent RNA chains transcribed by RNA polymerase II. A procedure that allows fractionation of nuclei into a chromatin pellet containing DNA, histones, and ternary transcription complexes and a supernatant containing the bulk of the nonhistone proteins and RNAs that are released from their DNA templates was developed. The transcripts of the genes encoding DBP, a transcriptional activator protein, and HMG coenzyme A reductase recovered from the chromatin pellet and the supernatant were analyzed by S1 nuclease mapping. The large majority of the RNA molecules from the pellet appeared to be nascent transcripts, since, in contrast to the transcripts present in the supernatant, they were not cleaved at the polyadenylation site but rather contained heterogeneous 3' termini encompassing this site. Splicing intermediates could be detected among nascent and released transcripts, suggesting that splicing occurs both cotranscriptionally and posttranscriptionally. Our results also indicate that polyadenylation is not required for the splicing of the last DBP intron. In addition to allowing detailed structural analysis of nascent RNA chains, the physical isolation of nascent transcripts also yields reliable measurements of relative transcription rates.
机译：为了检查剪接是否可以在哺乳动物细胞核中共转录发生，我们将外显子-内含子边界定位在由RNA聚合酶II转录的新生RNA链上。开发了一种程序，可以将细胞核分离成含有DNA，组蛋白和三元转录复合物的染色质沉淀，以及含有从其DNA模板释放的大量非组蛋白和RNA的上清液。通过S1核酸酶作图分析了从染色质沉淀物和上清液中回收的编码DBP，转录激活蛋白和HMG辅酶A还原酶的基因的转录本。来自沉淀的大多数RNA分子似乎是新生的转录物，因为与存在于上清液中的转录物相反，它们在聚腺苷酸化位点处没有被切割，而是包含围绕该位点的异质3'末端。可以在新生和释放的转录本中检测到剪接中间体，表明剪接发生在共转录和转录后。我们的结果还表明，最后一个DBP内含子的剪接不需要聚腺苷酸化。除了可以对新生RNA链进行详细的结构分析外，新生转录本的物理分离还可以可靠地测量相对转录速率。

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