A genetic screen in C. elegans reveals roles for KIN17 and PRCC in maintaining 5' splice site identity.
A genetic screen in C. elegans reveals roles for KIN17 and PRCC in maintaining 5' splice site identity.
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DOI：
10.1371/journal.pgen.1010028
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发表时间：
2022-03
期刊：
影响因子：
4.5
通讯作者：
Zahler AM
中科院分区：
文献类型：
--
作者：
Suzuki JMNGL;Osterhoudt K;Cartwright-Acar CH;Gomez DR;Katzman S;Zahler AM
Pre-mRNA剪接是真核生物基因表达的重要步骤，由一系列动态的大分子蛋白/RNA复合物完成，这些复合物统称为剪接体。这一系列剪接体复合物定义、组装并催化内含子的去除。该过程中中间体的分子模型快照已经从低温电镜数据中创建，然而，剪接体中发生的动态变化的许多方面尚未完全了解。秀丽隐杆线虫遵循GU-AG剪接规则，几乎所有内含子都以5 ' GU开始，以3 ' AG结束。这些剪接位点在剪接周期的早期被识别出来，但随着周期的进行，随着催化位点的建立，pre-mRNA剪接位点的“保管权”从一个因子传递到另一个因子，剪接位点身份通过这些动态变化维持或重新建立的机制尚不清楚。我们在秀丽隐杆线虫中进行了遗传筛选，以寻找能够改变5 '剪接位点选择的因素。我们报道了KIN17和PRCC参与剪接位点选择，这是这两种蛋白中第一个被提出的功能性剪接作用。先前发现的隐式5 ‘剪接抑制因子促进远端隐式GU剪接位点，然而，KIN17和PRCC的突变反而促进了不寻常的近端5 ’剪接位点的使用，该位点定义了以UU开头的内含子，与GU供体相隔1nt。我们进行了高通量mRNA测序分析，发现PRCC的突变以及KIN17的突变在较小程度上改变了全基因组天然位点的替代5 '剪接位点的使用，通常促进了附近非共识位点的使用。我们的工作揭示了剪接体在复杂的组装过程中维持剪接位点身份的精细和粗糙机制。前信使RNA剪接是真核生物基因表达的重要调节因子，改变编码和非编码转录物的内容、框架和功能。我们对剪接体如何选择切割位置的理解主要集中在剪接位点的初步鉴定上。然而，我们的结果表明，剪接体在后续步骤中还依赖于其他组分来维持剪接供体位点的身份。我们目前正处于一场“分辨率革命”之中，停滞复合体的冷冻电镜快照越来越清晰，使研究人员能够可视化拼接周期中的时刻。这些模型具有启发性，但并不总是阐明高动态核糖核蛋白复合物的机制功能。因此，我们的实验室采用了一种互补的方法，利用多细胞动物遗传学的力量来获得剪接体的功能见解。利用秀丽隐杆线虫的遗传筛选，我们发现了两个蛋白KIN17和PRCC的新功能剪接作用。PRCC的突变尤其促进了本地位点附近的替代5 '剪接位点。这项工作提高了我们对剪接体如何维持剪切前mrna的身份的理解，从而了解基因如何在多细胞动物中表达和使用。
Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5’ GU and ending with 3’ AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and “custody” of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5’ splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5’ splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5’ splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC, and to a lesser extent KIN17, changed alternative 5’ splice site usage at native sites genome-wide, often promoting usage of nearby non-consensus sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process. Pre-messenger RNA splicing is an important regulator of eukaryotic gene expression, changing the content, frame, and functionality of both coding and non-coding transcripts. Our understanding of how the spliceosome chooses where to cut has focused on the initial identification of splice sites. However, our results suggest that the spliceosome also relies on other components in later steps to maintain the identity of the splice donor sites. We are currently in the midst of a “resolution revolution”, with ever-clearer cryo-EM snapshots of stalled complexes, allowing researchers to visualize moments in time in the splicing cycle. These models are illuminating, but do not always elucidate mechanistic functioning of a highly dynamic ribonucleoprotein complex. Therefore, our lab takes a complementary approach, using the power of genetics in a multicellular animal to gain functional insights into the spliceosome. Using a C.elegans genetic screen, we have found novel functional splicing roles for two proteins, KIN17 and PRCC. Mutations in PRCC in particular promote nearby alternative 5’ splice sites at native loci. This work improves our understanding of how the spliceosome maintains the identity of where to cut the pre-mRNA, and thus how genes are expressed and used in multicellular animals.
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影响因子：
5.3
作者：
Agafonov, Dmitry E.;Deckert, Jochen;Luehrmann, Reinhard
通讯作者：
Luehrmann, Reinhard
影响因子：
8
作者：
Carlier, Ludovic;Couprie, Joel;Zinn-Justin, Sophie
通讯作者：
Zinn-Justin, Sophie
影响因子：
1.2
作者：
Cingolani, Pablo;Platts, Adrian;Ruden, Douglas M.
通讯作者：
Ruden, Douglas M.
影响因子：
8.8
作者：
Darman, Rachel B.;Seiler, Michael;Buonamici, Silvia
通讯作者：
Buonamici, Silvia
DOI：
10.1002/wrna.1560
发表时间：
2020-01
期刊：
Wiley interdisciplinary reviews. RNA
影响因子：
--
作者：
通讯作者：
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