Involvement of Noncanonical Short RNAs in gene repression through the RNA-induced-silencing complex

ABSTRACT MicroRNAs have been studied for over two decades and found to impact extensively on various cellular functions like differentiation, proliferation and oncogenesis through regulation of gene expression using the Argonaute (Ago) containing RNA-induced silencing complex (RISC). In the cell, however, microRNAs co-exist with a nearly equal abundance of non-canonical short RNAs (ncsRNAs) that were not believed to enter the RISC. This has begun to change with our discovery that some members of the ncsRNAs, the tRNA derived fragments (tRFs) enter into RISC and silence gene expression, and others do not. The 18-26 base long tRF-3a molecules are derived from tRNAs by processes very different from the biogenesis of microRNAs, and yet repress gene expression by incorporation into Ago-RISC (RISC). In Aim 1 we will focus on specific tRF sub-classes, tRF-3b and tRF-1, that do not enter into Ago-RISC, to identify the surveillance pathways that keep short RNAs from dysregulating gene expression through RISC. We will study a methyltransferase that inactivates tRF-3b molecules by modifications on the RNA, a modification that is also regulated by demethylases that are inactivated by Isocitrate Dehydrogenase (IDH) mutations, seen in many cancers. We will also focus on an RNAse that degrades tRF-1 molecules to prevent them from entering into RISC and silencing gene expression. The results will reveal how the surveillance mechanisms work and how pathogenic or therapeutic alteration of the surveillance mechanisms will alter gene expression and improve RNA mediated therapy. In Aim 2 we will turn to ncsRNAs, exemplified by three tRF-3a molecules, that enter into RISC, silence gene expression and alter phenotypes of cancers and cancer cell-lines. We will test whether even in these cell line the tRF-3a molecules regulate gene expression by hijacking microRNA specific mechanisms and thus alter cellular phenotypes. We will also determine whether the ncsRNAs help or hinder microRNAs from doing their function. The field of short RNA mediated post-transcriptional gene regulation will be altered fundamentally by the recognition that microRNAs work in a complex milieu of other short RNAs that compete with or assist microRNAs, and that the cell has evolved mechanisms to protect the integrity of microRNA-mediated gene regulation.