Dissecting the mechanisms underlying RNA processing and function of non-coding RNAs derived by SINE elements.

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Background: More than 97% of human and mouse genomes do not encode for proteins (non-coding genome) and until recently, a large part of this non-coding genome was referred as "junk DNA" that was believed to have no functional importance. More than 50% of the non-coding genome consists of repetitive elements, with small interspersed nuclear elements (SINEs) making the largest group of these elements. Interestingly, a large number of these SINE elements are transcribed into non-coding RNAs (SINE RNAs). However, the function of these SINE RNAs remains largely unknown. Specific classes of SINE RNAs, such as the B2 and Alu RNAs, have been recently shown to inhibit transcription of specific target genes by binding RNA Polymerase II. In a previous study, we revealed that after application of environmental stimuli, these RNAs are extensively processed and destabilized, which results in their inability to bind and inhibit RNA Polymerase II and thus, it adds an additional layer of regulation of expression of target genes. However, the mechanism of processing of SINE RNAs and the factors regulating this process remain unclear.******Objectives: My proposed research program aims to dissect the mechanisms underlying RNA processing of SINE RNAs. It will focus on 1) identifying the factors that trigger this processing and regulate RNA processing rate, 2) identifying and studying the function of the RNA products of the SINE RNA processing in regulation of cellular stress response, including both linear and circular RNA fragments, and 3) investigating how changes in the sequence of SINE RNAs affect their processing, to understand better the sequence requirements for this process.******Research Approach: To achieve these aims, my trainees and I will employ and customize a series of RNA genomics and bioinformatics approaches, such as library construction approaches for RNA sequencing and bioinformatics pipelines adapted to the study of repetitive, short and circular RNAs. These approaches include our previously developed short-RNA sequencing for estimation of the SINE RNA processing as well as RNA-sequencing and circRNA-sequencing for identification of linear and circular RNAs, respectively. The proposed research program will also employ RNA biochemistry and cell culture techniques to test in vitro and in cells the interaction between SINE RNAs and a number of proteins that control SINE RNA processing.******Impact: This research program will shed light to the function of a part of the non-coding genome previously believed to be "junk DNA". It will allow for a better understanding of the under-estimated until now role of SINE RNAs in response to cellular stress and regulation of gene expression that are critical for cell function.