Investigating tRNA-Val-CAC-1-1: acting as a tRNA or tRF in Drosophila melanogaster

Abstract

Animal development is regulated by the organismal genes. These genes are broadly divided into protein coding and non-coding DNA. The latter in comprised of different functional groups, here we focus on investigating the role of transfer RNA (tRNA) in posttranscriptional gene regulation. Some tRNAs can function as tRNA fragments (tRFs). These RNAs act similarly to miRNAs by regulating mRNAs post transcriptionally. The tRNA:Val-CAC-1-1 (ValT) (CR31572) was computationally predicted as tRF in Drosophila melanogaster. One of the predicted 51 targets is Sprouty (Sty), a known negative regulator of the epidermal growth factor receptor (EGFR) signaling pathway. We performed experiments aiming to determine whether ValT acts as tRNA and/or tRF. Using genome engineering, CRISPR/Cas9, the following modifications were done in ValT, including eliminating either its normal tRNA or tRF function by changing the predicted tRF or anticodon domains. All substituted ValTs were homozygous viable. In delta ValT, we expected to see an increase in Sty, which in turn decreases EGFR signaling. Yet, the predicted eggshell phenotypes, which are sensitive to changes in EGFR signaling, were not consistent among the mutants, and no significant change was observed. Next, to achieve a better understanding of ValT functions, all transgenic lines were analyzed by RNAseq. We found groups of genes that were transcribed at different levels that were consistent across mutations in the tRF and others that were consistent across anticodon substitutions. These genes indicate potential targets for future studies into the function of ValT as a tRNA and a tRF.M.S.Includes bibliographical reference