Talk abstracts
Talk on Saturday 12:00-12:15pm submitted by Minli Ruan
Pseudouridine Synthase 7 localization influences mRNA modification landscape and stress tolerance
Minli Ruan (University of Michigan, Department of Biological Chemistry), Kristin S. Koutmou (University of Michigan, Department of Chemistry)
Abstract:
Pseudouridine (Ψ), one of the most abundant post-transcriptional RNA modifications, broadly affects RNA structure and function. Misregulation of Ψ incorporation caused by mutants of pseudouridine synthases (PUSs) is associated with a variety of human diseases, including human-inherited intellectual disorders. Recently, we have begun to gain insight into the function and regulation of PUS7; however substantial gaps in our knowledge remain regarding how PUS7 targets RNAs and the physiological consequences of this targeting. Unlike most other PUS enzymes, which either use an RNA guider or recognize RNA secondary structure, PUS7 chooses its substrates using a consensus UNUAR (N= A/G/C/U, R = A/G) sequence. While the consensus sequence UNUAR is highly abundant, less than 3% of RNAs within this sequence are modified under normal growth conditions. We hypothesized that PUS7 localization, not just inherent enzyme properties, largely determines RNA accessibility and restricts PUS7 substrates. I tested this hypothesis using EYFP labeled PUS7 and found stress conditions, such as heat shock, Co2+, and Ni2+, can cause PUS7 cytoplasmic relocalization from the nucleus in yeast. Furthermore, I developed yeast strains with different localization-tag fused PUS7 and used Nanopore Sequencing to determine their mRNA substrates. I found that yeast with cytoplasm localized PUS7 (either induced by localization tag or stresses) has a significantly increased number of mRNAs modified by PUS7. Additionally, yeast with cytoplasm localized PUS7 is more tolerant to metal stresses, accumulating top "hits" of RNAs with higher pseudouridylation levels in oxidoreductase activity (GO:0016491), including LPD1 and HFD1. These data suggest the possibility that PUS7 localization changes provide cells an avenue to alter translation in response to stress.
References:
1. Purchal, M. K.; Eyler, D. E.; Tardu, M.; Franco, M. K.; Korn, M. M.; Khan, T.; McNassor, R.; Giles, R.; Lev, K.; Sharma, H.; Monroe, J.; Mallik, L.; Koutmos, M.; Koutmou, K. S. Pseudouridine Synthase 7 Is an Opportunistic Enzyme That Binds and Modifies Substrates with Diverse Sequences and Structures. Proc. Natl. Acad. Sci. U. S. A. 2022, 119 (4), e2109708119.
2. Schwartz, S.; Bernstein, D. A.; Mumbach, M. R.; Jovanovic, M.; Herbst, R. H.; León-Ricardo, B. X.; Engreitz, J. M.; Guttman, M.; Satija, R.; Lander, E. S.; Fink, G.; Regev, A. Transcriptome-Wide Mapping Reveals Widespread Dynamic Regulated Pseudouridylation of NcRNA and MRNA. Cell 2014, 159 (1), 148–162.
Keywords: Pseudouridine synthase 7 (PUS7), Pseudouridine, Localization