3'UTR-mediated protein-protein interactions determine protein functions

项目摘要 生物体的生物复杂性是如何实现的，在很大程度上是未知的。大多数细胞过程 蛋白质通过与其他蛋白质的相互作用来实现。有人认为， 决定蛋白质间相互作用的基因是由蛋白质本身编码的。因此，最初 令人惊讶地发现，蛋白质编码基因的数量和编码区长度保持不变， 在从蠕虫到人类的进化过程中相当稳定。然而，产生这些基因的数量 替代的3'非翻译区（3' UTR）增加了一倍，3 'UTR长度增加了十倍， 从蠕虫进化到人类此外，我们最近发现3 'UTR可以介导蛋白质- 蛋白质相互作用我们发现长的3 'UTR可以作为结合RNA结合蛋白的支架， 其将效应蛋白募集到翻译位点。在翻译过程中，效应蛋白被 从mRNA转移到新生蛋白质，导致形成蛋白质复合物， 需要长的3 'UTR的存在。 这一发现的普遍化表明，在替代3 'UTR的情况下，短UTR的翻译可能是一个很好的选择。 3 'UTR同种型产生“裸”蛋白，其基于随机的蛋白质相互作用寻找其蛋白质相互作用伴侣。 遇到并将与其周围具有最高亲和力的伴侣结合。的平移期间 然而，较长3’UTR同种型RNA结合蛋白充当多种效应子集合的募集者 蛋白质，包括分子伴侣，以实现替代蛋白质折叠，酶，添加替代后， 翻译修饰，或与新生蛋白相互作用以形成 替代蛋白质复合物。因此，我们认为3 'UTR可以显著增加 蛋白质-蛋白质相互作用伙伴，并可能大大多样化蛋白质功能。 关于效应蛋白从RNA转移到新生蛋白的机制，我们 假设被许多RNA结合蛋白结合的具有较长3 'UTR的mRNA可以成核 RNA颗粒的疏水环境似乎促进静电相互作用，从而使 效应蛋白的转移。为了鉴定由3 'UTR募集的蛋白质相互作用物，我们 开发了一种叫做UTR-co-IP的方法。我们将用GFP融合蛋白的cDNA构建体进行转染， 候选基因的编码区不含3 'UTR，或其相应的短或长3' UTR。 将通过免疫共沉淀获得GFP结合蛋白，并使用质谱法定量。 这将是研究替代性3 'UTR是否可能导致 生物复杂性通过3 'UTR介导的RNA颗粒形成， 区室化，通过招募结合伴侣，它们增加协同性， 替代性3 'UTR的产生促进了蛋白质的多功能性。

PROJECT SUMMARY It is largely unknown how biological complexity of organisms is achieved. Most cellular processes are carried out by proteins through interaction with other proteins. It has been thought that the information that determines protein-protein interactions is encoded within the protein itself. Therefore, it was initially surprising to find that the number of protein-encoding genes and the coding region length have remained fairly constant during evolution from worms to humans. However, the number of genes that produce alternative 3' untranslated regions (3'UTRs) has doubled and 3'UTR length has increased ten-fold during evolution from worms to humans. Furthermore, we recently discovered that 3'UTRs can mediate protein- protein interactions. We found that long 3'UTRs can act as scaffolds that bind RNA-binding proteins, which recruit effector proteins to the site of translation. During translation, the effector protein is transferred from the mRNA to the nascent protein, resulting in the formation of a protein complex that requires the presence of the long 3'UTR. Generalization of this finding suggests that, in the case of alternative 3'UTRs, translation of the short 3'UTR isoform generates the `naked' protein, which finds its protein interaction partners based on random encounters and will bind to the partner with the highest affinity in its surroundings. During translation of the long 3'UTR isoform, however, RNA-binding proteins serve as recruiters for a diverse set of effector proteins, including chaperones, to achieve alternative protein folds, enzymes that add alternative post- translational modifications, or protein binding partners that interact with the nascent protein to form alternative protein complexes. Thus, we propose that 3'UTRs can substantially increase the number of protein-protein interaction partners and may considerably diversify protein functions. With respect to the mechanism of transfer of effector proteins from the RNA to the nascent protein, we hypothesize that mRNAs with long 3'UTRs that are bound by many RNA-binding proteins may nucleate RNA granules whose hydrophobic milieu seems to facilitate electrostatic interactions, thus enabling the transfer of effector proteins. To identify the protein interactors that are recruited by 3'UTRs, we are developing a method called UTR-co-IP. We will transfect cDNA constructs of GFP fusions with the coding region of a candidate gene that either contains no 3'UTR, or its corresponding short or long 3'UTR. GFP-bound proteins will be obtained by co-immunoprecipitation and quantified using mass spectrometry. This will be the first step in investigating if alternative 3'UTRs may contribute to the emergence of biological complexity. Through 3'UTR-mediated RNA granule formation, they enable compartmentalization, through recruitment of binding partners they increase cooperativity, and through the generation of alternative 3'UTRs they facilitate multi-functionality of proteins.