515A Poster - 06. Regulation of gene expression
Thursday April 07, 2:00 PM - 4:00 PM

Authors:
Jennifer Dumouchel ¹; Kimberly Rose Madhwani ²; Caley Hogan ³; Jenna Lentini ⁴; Kevin Welle ⁵; Dragony Fu ⁴; Kate O'Connor-Giles ^6,7

Affiliations:
1) Therapeutic Sciences Graduate Program, Brown University, Providence, RI; 2) Neuroscience Graduate Program, Brown University, Providence, RI; 3) Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI; 4) Department of Biology, Center for RNA Biology, University of Rochester, Rochester, NY; 5) Mass Spectrometry Resource Laboratory, University of Rochester Medical Center, Rochester, NY; 6) Department of Neuroscience, Brown University, Providence, RI; 7) Carney Institute for Brain Science, Providence, RI

Keywords:
y. posttranslational modifications; f. neuromuscular junction

Dynamic regulation of neuronal genes during transcription and translation is required for proper nervous system development and function. Investigation of transfer RNA (tRNA) modifying enzymes has highlighted the importance of chemical modifications for tRNA stability and protein synthesis. Yeast tRNA methyltransferase 9 (Trm9) methylates wobble uridines in anticodon loops to reinforce cognate codon-anticodon pairings. In metazoa, two Trm9 paralogs, ALKBH8 and TRMT9B have been identified. We identified TRMT9B in an RNAi screen as a negative regulator of synaptic growth, and find that it also plays a key role in regulating neurotransmission. While TRMT9B has been studied for its role as a tumor suppressor, its biochemical role has remained unknown. To study the role of the expanded family, we have generated mutants in both paralogs and assessed changes in bulk tRNA chemical modifications by mass spectrometry. Wobble uridine methylation is absent in ALKBH8 mutants, consistent with studies in mammals. In contrast, we do not observe deficits in wobble uridine methylation in TRMT9B mutants under basal conditions. Thus, TRMT9B may catalyze wobble uridine modification of a small subset of tRNA isodecoders not detectable by bulk nucleotide analysis or evolved a new function. Wild-type, but not methyltransferase-dead, transgenes rescue synaptic overgrowth in TRMT9B mutants, indicating that TRMT9B functions through a methyltransferase-dependent mechanism. Unbiased homology modeling of TRMT9B’s methyltransferase domain revealed a strong conservation of secondary structure with the reported yeast Trm9 X-ray crystallography structure; consistent with a role as a methyltransferase. To assess TRMT9B function, we turned to transcriptomic and proteomic studies, and will present our latest findings. We investigated transcriptome and proteome changes in TRMT9B null adult fly heads by RNA sequencing and high resolution mass spectrometry, respectively. Differentially expressed genes and proteins are consistent with TRMT9B’s role at synapses and prior studies of yeast Trm9, which modulates translation of specific mRNAs in response to DNA damage and oxidative stress. Together, our studies reveal a novel role for tRNA methyltransferase family member TRMT9B in the regulation of synaptic growth and neuronal function.