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

真核细胞分为两个区域：核质和细胞质，核质是合成和加工信使核糖核酸的地方，细胞质是信使核糖核酸转化为蛋白质的地方。这种划分允许对信使核糖核酸的处理进行质量控制，因此只有正确处理的信使核糖核酸被翻译成蛋白质，而错误处理的信使核糖核酸保留在细胞核中并被降解。我们的数据表明，核斑点，即存在于大多数人类细胞细胞核中的相分离的间隔，在某些错误处理的mRNAs的保留和衰退中发挥着关键作用。在这个项目中，我们破译了错误处理的mRNAs被隔离在核斑点中从而阻止它们核出口的分子机制。目的1)破译含有未使用的5‘剪接位点(5’SS)基序的转录本保留在核斑点内的机制。我们发现，由某些内含子中隐含的3‘聚腺苷酸化信号产生的错误处理的mRNAs，含有促进mRNA核保留和衰退的5’SS基序(Lee，PLOS One 2015；Nuc ACID RES 2020)。我们已经证明，这些内含子多腺化(IPA)转录本需要ZFC3H1和U1 SnRNP都保留在核斑点中(Lee，BioRxiv 2021)。在这一目标中，我们将验证核斑点在mRNA核保持中的作用，并确定这一过程所需的其他因素。我们还将探索m6A RNA修饰在含有5‘SS基序的mRNA核保留中的作用。目的2)剖析5‘SS基序如何在不同的RNA环境中抑制输出。利用不同的记者，我们将确定5‘SS基序在某些mRNA标志物附近的位置如何影响核保留。我们还将探索非剪接内含子中的额外元素如何影响核定位，以及介导其核保留的蛋白质因素。目的3)确定GC含量在多聚腺苷化和核滞留中的作用。在初步数据中，我们发现产生IPA转录本的基因在其5‘端具有异常高的GC含量的内含子。使用报告RNA，我们将调查GC含量如何促进IPA的生成，并有助于它们的核保持。信使核糖核酸质量控制有助于减少剪接错误和转录噪声的危害，从而防止消除产生垃圾RNA的弱剪接位点、隐蔽的3‘切割位点、虚假的转录起始点和基因间区(Palazzo&Gregory PLOS Gen 2014；Palazzo&Lee Frontiers in Genetics 2015,2018)。这种对遗传元件的低效消除促进了新的选择性剪接事件和新的非编码基因的进化(Linquist等人，PLOS Genetics，2020；Palazzo&Koning Cell 2020)。因此，通过了解质量控制过程是如何运作的，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。

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.