Investigating Mechanisms of Nuclear mRNA Export

In eukaryotes, the segregation of the RNA synthesis/processing in the nucleus from the translation machinery in the cytoplasm has important implications for nuclear export and retention of mRNAs. This division ensures that only correctly processed mRNAs are exported to the cytoplasm to be translated into proteins, while misprocessed and aberrant transcripts are retained in the nucleus to be degraded by various nuclear surveillance mechanisms. In the first half of this work, I show that mature mRNAs containing the 5’ splice site (5’SS) motif are retained in the nucleus. Although a fraction of 5’SS motif containing mRNAs are rapidly degraded, those that escape decay are targeted to nuclear speckles. We show that ZFC3H1, a component of the nuclear exosome associated PolyAdenylation Exosome Targeting (PAXT) complex, is required for the nuclear retention and degradation of such transcripts. Similarly, we demonstrate that U1 snRNP, a component of the spliceosome that recognizes and binds to the 5’SS motif, mediates the nuclear retention of these aberrant mRNAs. We show that ZFC3H1 and U1-70K are not required for targeting 5’SS motif containing mRNAs to nuclear speckles, and these mRNAs phase-separate into this sub-nuclear compartment by some unknown process. Next, I perform RNA Frac-Seq and demonstrate that ZFC3H1 is required for the decay and nuclear retention of intronic polyadenylated transcripts, which naturally contain intact 5’SS motifs. In the second half of this thesis, I characterize the role of TPR, a nuclear pore basket protein, in mediating nuclear retention and export of mRNAs. While splicing has been shown to enhance nuclear mRNA export, it remains unclear whether RNAs generated from intronless genes use specific machinery for their export. We show that TPR is required for promoting the efficient export of intronless or intron-poor mRNAs and lncRNAs. Furthermore, we observe a strong correlation between transcripts generated from short pre-processed RNAs and TPR mediated nuclear export. My observations suggest that TPR acts in a late step of mRNA export and is most likely required for the mRNA docking with the nuclear pore. In summary, I show that mRNA export/nuclear retention processes are ‘two sides of the same coin’ and collectively mediates the export of bona fide mRNAs while simultaneously promotes the nuclear retention/degradation of misprocessed transcripts. Furthermore, the export of certain subtypes of RNAs (as exemplified by intronless genes) requires additional factors for their nuclear export, demonstrating that this process is more complex than previously envisioned.Ph.D