Title: Pushing DM15 Boundaries: Investigating the functional influence of conserved regions adjacent to DM15 domain
Abstract:
La-related protein 1 (LARP1) is a eukaryotic cytoplasmic RNA-binding protein that is a target of the TOR pathway, a master protein kinase whose actions in the TORC1 complex regulate metabolic pathways including protein production. It does so, in part, by regulating the translation of TOP mRNAs, or transcripts that contain a 5’ terminal oligopyrimidine (TOP) tract and encode the ribosomal proteins themselves. Despite decades of research on 5’TOP mRNA translation, we are only beginning to understand the molecular bases for their translation regulation.
Recent research suggests that human LARP1 plays a role in stabilizing TOP mRNA transcripts in their repressed form via binding to the 5’cap and TOP motif using its DM15 domain. The 3 HEAT-like repeats of the DM15 domain are sufficient and necessary for recognition and regulation of TOP mRNA metabolism in a TOR-dependent manner in all systems studied to date. However, the established DM15 domain is N-terminally flanked by a highly conserved, flexible region, termed conserved region 2 (CR2), containing TOR-sensitive residues in nearly all LARP1 homologs; phosphomimetic modification of all these TOR-targeted residues within the CR2 region selectively weakens the affinity of the DM15 region for TOP mRNA targets in-vitro. To uncover how this extension contributes to the RNA-binding mechanism of the LARP1 DM15 region, I will conduct biochemical assays with wild-type LARP1 extended DM15 (CR2-DM15), and with CR2-DM15 containing phosphomimetic and phosphonull mutants. I hypothesize that the extent of phosphorylation of the CR2 region will tune DM15 affinity for TOP mRNA targets.
Additionally, plant LARP1 proteins exhibit a partial fourth HEAT-like repeat and interact with mRNAs implicated in plant cell stress. I hypothesize this extended fold alters the mechanistic interaction with cap and 5’ pyrimidine tracts commonly found in the 5’ UTRs of these mRNAs. Interestingly, plant LARP1 has been implicated in the mRNA decay response to heat-stress, though the mechanistic contribution of the RNA-binding DM15 domain remains to be understood. I will conduct in-vitro biochemical assays and structural experiments to assess the mechanism and structural differences of plant DM15-mRNA binding.
Berman Lab
Friday, April 19th, 2024
12:00PM
Langley A219B