Chromatin Associated Small RNA Show Evidence Of Processing After Promoter Proximal RNA Polymerase II Pausing

Paused Pol II has been implicated in the generation of short non-coding RNAs, such as transcription start site RNAs (TSSa RNAs) and transcription initiation RNAs (tiRNAs), which are found within or around the 5’ ends of gene promoters. The generation of these RNAs (which I termed “processed RNAs”) is known to occur in the nucleus; however, whether their biogenesis is co-transcriptional or post-transcriptional is unknown. These RNAs have been proposed to be remnants of 5’ to 3’ processing and protected by paused Pol II. Processed RNAs may represent a novel class of small RNAs derived from promoter-proximal transcription termination. I hypothesized that processed RNAs are co-transcriptionally generated from RNAs associated with paused Pol II. In order to distinguish between a co-transcriptional or post-transcriptional event, I fractionated the nucleus into the chromatin fraction and the nucleoplasmic fraction. If these processed RNAs were generated co-transcriptionally, they would be present in the chromatin fraction along with the chromatin-bound paused Pol II complex, and if they were generated post-transcriptionally, they would be present in the nucleoplasmic fraction. I demonstrated that processed RNAs associate with the chromatin fraction, indicating that they are co-transcriptionally generated. I further developed a modified version of PRO-Seq (PRO-Start and processed PRO-Seq) to confirm that processed RNAs are generated from paused Pol II. In the literature, it is unclear whether the generation of these RNAs is independent of backtracking. To test whether backtracking was involved in the biogenesis of processed RNAs, I compared the 3’ end locations of the processed RNAs and short capped RNAs and found that most genes do not demonstrate backtracking. Alternatively, to study processing from the 5’ ends, I investigated the role of XRN2, a nuclear 5’ to 3’ exoribonuclease, in the generation of processed RNAs. I discovered that XRN2 depletion resulted in an enrichment of these processed RNAs, but the results indicated other proteins may also be involved. Currently, these processed RNAs are proposed to play a role in gene repression. Future experiments will focus on identifying and demonstrating the functional roles of these transcripts