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

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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.