Investigating the mechanisms that promote the nuclear retention of misprocessed mRNAs in human cells

研究促进人类细胞中错误加工的 mRNA 保留核的机制

• 批准号: RGPIN-2022-05270
• 负责人: Palazzo, Alexander
• 金额: $ 3.72万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744141
• 关键词: Investigating; mechanisms; promote; nuclear; retention

The eukaryotic cell is divided into two regions: the nucleoplasm where mRNA is synthesized and processed, and the cytoplasm where this mRNA is translated into protein. This division allows for quality control of mRNA processing, so that only properly processed mRNAs are translated into proteins while mis-processed mRNAs are retained in the nucleus and degraded. Our data indicates that nuclear speckles, which are phase-separated compartments present in the nucleus of most human cells, play a critical role in the retention and decay of certain mis-processed mRNAs. In this project we decipher the molecular mechanisms by which mis-processed mRNAs are sequestered in nuclear speckles, thus preventing their nuclear export. Aim 1) Deciphering the mechanism by which transcripts with unused 5' splice site (5'SS) motifs are retained within nuclear speckles. We have found that mis-processed mRNAs that are generated from cryptic 3' polyadenylation signals present in certain introns, contain 5'SS motifs that promote mRNA nuclear retention and decay (Lee, PLOS ONE 2015; Nuc Acid Res 2020). We have shown that these intronic polyadenylated (IPA) transcripts require both ZFC3H1 and the U1 snRNP to be retained in nuclear speckles (Lee, bioRxiv 2021). In this aim we will validate the role of nuclear speckles in mRNA nuclear retention, and identify additional factors required for this process. We will also explore the role of the m6A RNA modification in the nuclear retention of 5'SS motif-containing mRNAs. Aim 2) Dissecting how the 5'SS motif inhibits export in different RNA contexts. Using various reporters we will determine how the positioning of the 5'SS motif near certain mRNA landmarks affects nuclear retention. We will also explore how additional elements in unspliced introns impact nuclear localization and the protein factors that mediate their nuclear retention. Aim 3) Determine the role of GC-content in polyadenylation and nuclear retention. In preliminary data we have found that genes that generate IPA transcripts have introns with unusually high GC-content at their 5'end. Using reporter RNAs we will investigate how GC-content promotes the generation of IPAs and contributes to their nuclear retention. mRNA quality control helps to diminish the deleteriousness of splicing errors and transcriptional noise, and thus prevents the elimination of weak splicing sites, cryptic 3' cleavage sites, spurious transcriptional start sites and intergenic regions that generate junk RNA (Palazzo & Gregory PLOS Gen 2014; Palazzo & Lee Frontiers in Genetics 2015, 2018). This inefficient elimination of genetic elements promotes the evolution of novel alternative splicing events and new non-coding genes (Linquist et al., PLOS Genetics 2020; Palazzo & Koonin Cell 2020). Thus, by understanding how quality control processes operate, we ultimately learn about the forces that drive the evolution of the eukaryotic transcriptome and the complexification of the human genome.

真核细胞分为两个区域：核质，mRNA在其中合成和加工，以及细胞质，mRNA在其中翻译成蛋白质。这种分裂允许mRNA加工的质量控制，使得只有正确加工的mRNA被翻译成蛋白质，而错误加工的mRNA被保留在细胞核中并被降解。我们的数据表明，核斑点是存在于大多数人类细胞核中的相分离区室，在某些错误加工的mRNA的保留和衰变中起着关键作用。在这个项目中，我们破译了错误加工的mRNA被隔离在核斑点中的分子机制，从而阻止了它们的核输出。目的1）揭示5'剪接位点（5' SS）未使用的转录物保留在核斑点中的机制。我们已经发现，由某些内含子中存在的隐蔽3'多聚腺苷酸化信号产生的错误加工的mRNA含有促进mRNA核保留和衰变的5' SS基序（Lee，PLOS ONE 2015; Nuc Acid Res 2020）。我们已经证明，这些内含子聚腺苷酸化（IPA）转录物需要ZFC 3 H1和U1 snRNP两者才能保留在核斑点中（Lee，bioRxiv 2021）。在这个目标中，我们将验证核斑点在mRNA核保留中的作用，并确定此过程所需的其他因素。我们还将探讨m6 A RNA修饰在含5 'SS基序的mRNA的核保留中的作用。目的2）分析5 'SS基序在不同RNA环境中对输出的抑制作用。使用各种报告，我们将确定如何定位的5 'SS基序附近的某些mRNA的地标影响核保留。我们还将探讨未剪接内含子中的其他元素如何影响核定位以及介导其核保留的蛋白质因子。目的3）确定GC含量在多聚腺苷酸化和核保留中的作用。在初步数据中，我们发现产生IPA转录本的基因在其5 '端具有异常高GC含量的内含子。使用报告RNA，我们将研究GC含量如何促进IPAs的产生并有助于其核保留。mRNA质量控制有助于减少剪接错误和转录噪音的有害性，从而防止消除弱剪接位点，隐藏的3'切割位点，虚假的转录起始位点和产生垃圾RNA的基因间区域（Palazzo & Gregory PLOS Gen 2014; Palazzo & Lee Frontiers in Genetics 2015，2018）。遗传元件的这种无效消除促进了新的可变剪接事件和新的非编码基因的进化（Linquist等人，PLOS Genetics 2020; Palazzo & Koonin Cell 2020）。因此，通过了解质量控制过程如何运作，我们最终了解了驱动真核转录组进化和人类基因组复杂化的力量。