Regulation of mitochondrial activity and metastasis by dihydrouridine-modified tRNAs in renal cell carcinoma - PROJECT SUMMARY This project will investigate the impact of dihydrouridine (D) modifications in tRNA on a newly discovered post- transcriptional regulatory mechanism called P-site tRNA-mediated decay (PTMD), and the resulting effects on mitochondrial metabolism and cancer metastasis. PTMD is a pathway wherein specific arginyl-tRNAs recruit the CCR4-NOT complex to ribosomes translating mRNAs rich in CGG, CGA, and AGG arginine codons, resulting in reduced translation and accelerated turnover of these transcripts. Since mRNAs that encode components of mitochondrial ribosomes and the respiratory chain are rich in these codons, reduced efficiency of PTMD results in an increase in mitochondrial activity. Recently, activation of mitochondrial respiration was shown to drive metastasis in clear cell renal cell carcinoma (ccRCC), but how metastatic ccRCC cells upregulate mitochondrial activity remains unclear. Overexpression of multiple dihydrouridine synthase (DUS) enzymes that introduce D modifications at specific positions in tRNAs is also strongly associated with poor patient survival in ccRCC. Moreover, DUS overexpression is associated with upregulation of transcripts rich in CGG/CGA/AGG arginine codons, including those encoding mitochondrial proteins, indicative of PTMD impairment. Based on these findings, we hypothesize that DUS overexpression results in D hypermodification of tRNAs, reducing their ability to recruit the CCR4-NOT complex, and thereby increasing the translation and stability of CGG/CGA/AGG-rich mRNAs encoding components of mitochondrial ribosomes and the respiratory chain. In ccRCC, the increase in mitochondrial metabolism and respiration that results from this translational reprogramming is predicted to promote metastasis, resulting in poor patient outcomes. To investigate this hypothesis, our research program will examine how altering DUS enzyme activity affects tRNA D modifications, overall tRNA metabolism, and the PTMD pathway in human and mouse ccRCC cells, documenting the effects of both individual and combinatorial D modifications at multiple tRNA positions. Using ccRCC models, we will evaluate the impact of gain and loss of DUS enzyme activity, as well as mutations in the CCR4-NOT subunit CNOT3 that impair PTMD, on mitochondrial activity and metastasis in this malignancy. Cryo-electron microscopy (cryo-EM) will be used to investigate the molecular mechanisms whereby D hypermodification of tRNAs influences tRNA structure and CCR4-NOT recruitment. Altogether, these experiments will provide a mechanistic understanding of the impact of dihydrouridine modifications on PTMD and ccRCC pathogenesis, potentially revealing new therapeutic vulnerabilities in this malignancy. We have assembled a complementary team of experts in RNA biology, post- transcriptional regulation, metabolism, cancer biology, and structural biology to accomplish these aims, and we are eager to collaborate with other groups in the RNAMoDO program to investigate how additional RNA modifications impact normal physiology and cancer.
