51 Epigenetic regulation of ribosomal RNA transcription and processing by the tumor suppressor SETD2

Abstract Background The methyltransferase SETD2 was originally identified as a tumor suppressor in clear cell renal cell carcinoma (ccRCC), and ccRCC with SETD2 mutations have increased metastatic potential and poor progression free survival. A known epigenetic regulator, SETD2 interacts with actively transcribing RNA polymerase II (RNAPII) and tri-methylates Histone 3 at Lysine 36 (H3K36me3), a modification that correlates with active gene transcription and is recognized by mRNA methylation and splicing factors. Despite these known roles in mRNA transcription, loss of SETD2 does not alter genic transcription, raising the question of whether or how SETD2 regulates RNA biology. Methods Given that SETD2 influences RNA processing, we sought to identify RNA-protein complexes that are regulated by SETD2. To do this in an unbiased and high-throughput manner, we used orthogonal organic phase separation (OOPS) coupled with mass spectrometry to identify changes in RNA binding proteins and pathways between control and SETD2-knockout cells. Results Corroborating previous findings, we identified that RNA binding proteins governing mRNA splicing and cellular metabolism were dysregulated in SETD2 mutant cells. However, the most robust changes in SETD2 knockout cells occurred in pathways regulated ribosome biogenesis (See Figure, orange highlights), resulting in a significant decrease in binding of ribosomal RNA (rRNA) processing factors to RNA. We confirm these alterations lead to defects in rRNA transcription, processing, and ribosome biogenesis. We identify that loss of SETD2 catalytic activity phenocopies these rRNA processing defects, suggesting H3K36me3 is essential for rRNA processing. Disruptions occur to both the large (60S) and small (40S) ribosome subunits, modifying protein translation. Lastly, we identify synthetic lethality between SETD2 deletion or inhibition and small molecules that interfere with rRNA transcription. Conclusions While the majority of research emphasizes the role SETD2 in the regulation of mRNA, our data identify that SETD2 and its catalytic activity play a critical role in the transcription and processing of rRNA. Importantly, rRNA comprises 80-90% of cellular RNA yet is typically overlooked and understudied. Next-generation RNA sequencing or transcriptional assays investigate mRNA or genic transcription while ignoring rRNA, demonstrating why SETD2-mediated rRNA processing has been unexplored. This may also have important implications for prospective clinical trials that utilized RNA-sequencing to identify optimal therapeutic regimens. Our work also identified that SETD2-mutated cells are more sensitive to compounds that inhibit rRNA transcription, potentially uncovering a new therapeutic vulnerability of ccRCC with mono- and bi-allelic loss of SETD2. Alterations in ribosome biogenesis leads to epithelial-to-mesenchymal transition and metastasis in many tumor types, and our future goal will be to investigate the role of ribosomes and rRNA transcription in transformation and metastasis in ccRCC as well as the molecular mechanisms connecting SETD2 to ribosome fidelity. DOD CDMRP Funding: yes