Elucidating the Role of Alternative RNA Export Promoting Signal Sequence Coding Regions in Potentiating Translation

Elucidating the Role of Alternative RNA Export Promoting Signal Sequence Coding Regions in Potentiating Translation Kohila Kulendra Doctor of Philosophy Graduate Department of Biochemistry University of Toronto 2015 Abstract In vertebrates, a sub-population of mRNAs encoding secreted, membrane-bound and mitochondrial proteins contain nucleotide elements that promote the alternative mRNA nuclear export (ALREX) of microinjected mRNAs. This RNA element is present within the signal sequence coding region (SSCR) or mitochondria targeting sequence coding region (MSCR) and located at the 5'end of the mRNA just after the start codon. It is typically depleted of adenines and present in genes that lack introns in their 5'UTRs. I demonstrated that ALREX-promoting elements can potentiate translation in the presence of nuclear factors such as Nup358/RanBP2. To determine which domains of RanBP2 are required to enhance translation, I expressed mutant and/or truncated versions of RanBP2 in cells depleted of the endogenous protein and tested the translation of various reporters. I found that the entire C-terminal portion of RanBP2, including its E3 sumo ligase domain, is not required to enhance translation. In contrast, an N-terminal region, which includes eight zinc fingers and a tetratricopeptide repeat region that can bind to ALREX-promoting SSCRs, is required. Incorporating point mutations in the N-terminal domain of RanBP2, which disrupt its ability to bind SSCR RNAs in vitro, also impaired RanBP2's ability to enhance the translation of a reporter mRNA in vivo. Furthermore, N-terminal domain mutations in RanBP2 are known to be associated with the autosomal dominant genetic disorder Acute Necrotizing Encephalopathy 1 (ANE1). In these individuals, influenza viral infection triggers a cytokine storm which leads to coma and death. I found that RanBP2 depletion promotes the translation of the interleukin 6 mRNA, which encodes a cytokine that is overproduced in ANE1 children during infection. Overall, the data suggests a novel mechanism by which RanBP2 may regulate the translation of mRNAs and thus impact human health.Ph.D