RSR-2, the Caenorhabditis elegans ortholog of human spliceosomal component SRm300/SRRM2, regulates development by influencing the transcriptional machinery.
RSR-2, the Caenorhabditis elegans ortholog of human spliceosomal component SRm300/SRRM2, regulates development by influencing the transcriptional machinery.
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DOI：
10.1371/journal.pgen.1003543
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发表时间：
2013-06
期刊：
影响因子：
4.5
通讯作者：
Cerón J
中科院分区：
文献类型：
--
作者：
Fontrodona L;Porta-de-la-Riva M;Morán T;Niu W;Díaz M;Aristizábal-Corrales D;Villanueva A;Schwartz S Jr;Reinke V;Cerón J
剪接体的蛋白质组分在真核生物中高度保守，可以影响基因表达过程的几个步骤。RSR-2是人类剪接体蛋白SRm300/SRRM2的秀丽线虫同源基因，与酵母同源基因Cwc21p相比，RSR-2对生存是必不可少的。我们利用突变体和RNA干扰技术(RNAi)研究了RSR-2在线虫中的功能，并通过遗传上位性分析发现RSR-2位于种系性别决定途径中。有趣的是，对RSR-2(RNAi)动物的转录组分析没有发现明显的剪接缺陷，而是全球转录水平略有下降。我们进一步研究了这一转录效应，观察到RSR-2与DNA在胚系核中共定位，并与染色质共沉淀，显示出类似于RNA聚合酶II(RNAPII)的ChIP-Seq图谱。根据一个新的转录功能，我们证明了RSR-2对染色质的募集是不依赖剪接的，并且RSR-2与RNAPII相互作用并影响RNAPII的磷酸化状态。蛋白质组学分析发现，与RSR-2相关的蛋白质参与了不同的基因表达步骤，包括RNA代谢和转录，其中PRP-8和PRP-19是最强的相互作用伙伴。PRP-8是剪接体的核心成分，PRP-19是PRP19复合体的核心成分，它与RNAPII相互作用，是充分转录活动所必需的。综上所述，我们的研究认为RSR-2是一种多功能蛋白，它在转录中的作用影响线虫的发育。众所周知，剪接是以共转录的方式发生的，但在发育中的多细胞生物体中，剪接和转录之间的功能耦合尚未被仔细研究。我们利用柔韧线虫的遗传学和基因组学来证明RSR-2和转录之间的功能关系。RSR-2的酵母和人类同源基因是剪接体的组成部分。虽然我们发现RSR-2与剪接体中的蛋白质相互作用，但RNAi对RSR-2的适度抑制并没有显著影响剪接，而是导致了对生殖系性别决定至关重要的转录水平的下降。我们对剪接体成分影响转录的这种悖论的研究发现了一系列将RSR-2与转录联系起来的证据：(I)RSR-2免疫共沉淀物的染色质类似于RNAPII的芯片序列轮廓；(Ii)RSR-2存在于无内含子基因中；(Iii)RSR-2(RNAi)全局改变RNAPII沿基因的分布及其磷酸化状态；(Iv)RSR-2免疫共沉淀物与RNAPII相互作用；以及(V)RSR-2与PRP-19相互作用，PRP-19是有效转录活动所需的剪接体成分。我们的发现提出了一个有趣的问题：一些剪接体成分的适度改变会在多大程度上通过干扰剪接或转录来影响基因表达过程？
Protein components of the spliceosome are highly conserved in eukaryotes and can influence several steps of the gene expression process. RSR-2, the Caenorhabditis elegans ortholog of the human spliceosomal protein SRm300/SRRM2, is essential for viability, in contrast to the yeast ortholog Cwc21p. We took advantage of mutants and RNA interference (RNAi) to study rsr-2 functions in C. elegans, and through genetic epistasis analysis found that rsr-2 is within the germline sex determination pathway. Intriguingly, transcriptome analyses of rsr-2(RNAi) animals did not reveal appreciable splicing defects but instead a slight global decrease in transcript levels. We further investigated this effect in transcription and observed that RSR-2 colocalizes with DNA in germline nuclei and coprecipitates with chromatin, displaying a ChIP-Seq profile similar to that obtained for the RNA Polymerase II (RNAPII). Consistent with a novel transcription function we demonstrate that the recruitment of RSR-2 to chromatin is splicing-independent and that RSR-2 interacts with RNAPII and affects RNAPII phosphorylation states. Proteomic analyses identified proteins associated with RSR-2 that are involved in different gene expression steps, including RNA metabolism and transcription with PRP-8 and PRP-19 being the strongest interacting partners. PRP-8 is a core component of the spliceosome and PRP-19 is the core component of the PRP19 complex, which interacts with RNAPII and is necessary for full transcriptional activity. Taken together, our study proposes that RSR-2 is a multifunctional protein whose role in transcription influences C. elegans development. It is well known that splicing occurs cotranscriptionally but the functional coupling between splicing and transcription has not been studied carefully in the context of a multicellular organism in development. We took advantage of the amenable C. elegans genetics and genomics to demonstrate a functional relationship between RSR-2, whose yeast and human orthologs are components of the spliceosome, and transcription. Although we found that RSR-2 interacts with proteins present in the spliceosome, moderate inhibition of rsr-2 by RNAi did not significantly affect splicing, but rather caused a decrease in transcript levels that was critical for germline sex determination. Our investigation on such a paradox of a spliceosomal component affecting transcription resulted in several lines of evidence linking RSR-2 with transcription: (i) RSR-2 immunoprecipitates chromatin resembling the ChIP-Seq profile of RNAPII, (ii) RSR-2 is present in intronless genes, (iii) rsr-2(RNAi) globally modifies the distribution of RNAPII along genes and its phosphorylation state, (iv) RSR-2 coimmunoprecipitates with RNAPII, and (v) RSR-2 interacts with PRP-19, which is a component of the spliceosome required for efficient transcriptional activity. Our findings raise an intriguing question: to what extent does a moderate alteration in some spliceosome components affect the gene expression process by perturbing splicing or transcription?
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影响因子：
14.9
作者：
Contrino S;Smith RN;Butano D;Carr A;Hu F;Lyne R;Rutherford K;Kalderimis A;Sullivan J;Carbon S;Kephart ET;Lloyd P;Stinson EO;Washington NL;Perry MD;Ruzanov P;Zha Z;Lewis SE;Stein LD;Micklem G
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Micklem G
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