cis-regulation of RNA polymerase pausing

National Institute Of Environmental Health Sciences

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Abstract

The Transcriptional Responses in Disease group is focused on understanding the contribution of nucleic acid structure in transcription regulation in human cells. DNA and RNA can form non-canonical secondary structures such as guanine quadruplexes and R-loops which can alter the movement of RNA polymerase during transcription elongation. To examine the interaction between RNA, DNA, and the RNA polymerase during transcription we turned to a mitochondria transcription system to and found mitochondrial RNA polymerase pauses after synthesis of guanine rich RNAs. These guanine rich RNAs can undergo non-canonical base pairing and fold into quadruplexes. We found that guanine quadruplex stabilization with a small molecule is sufficient to pause the mitochondrial RNA polymerase during transcription, alter mitochondrial gene expression, and impair ATP production. The reduced ATP generation is sufficient to decreased proximal tubule function, linking nucleic acid mediated transcription regulation to overall cellular function. We are extending these results to investigate how guanine quadruplex dynamics are altered in acute kidney injury. We find the cis-regulators which contribute to the control of RNA polymerase pausing, including pausing in guanine-rich regions are shared are shared between pause sites in the promoter and gene body of protein-coding genes in the nucleus as well as the mitochondria. Despite shared cis-regulatory elements at pause sites in different locations in the genome, there is specificity to pause-release in response to exogenous stressors. We are elucidating how protein factors interact with the cis regulatory elements to dynamically tune the extent of RNA polymerase pausing. We uncovered a mechanism where the stability of R-loop, a three stranded structure where the nascent RNA hybridizes with the template DNA displacing the non-template DNA strand, regulates the extent of RNA polymerase pausing. When the R-loop is stabilized by formation of abasic RNA it pauses RNA Pol II during transcription elongation. We uncovered this mode of regulation is central to tune the activity of some enhancer RNAs and critical for regulation of genes such at APOE. In turn we are investigating how dysregulation of APOE expression in renal epithelial can contribute to kidney disease. Using an osmotic stress model, we have identified a conserved transcription factor which releases promoter-proximal paused polymerase to regulate the expression of tonicity-responsive genes. We are extending this work to study how RNA polymerase pausing is regulated in response to environmental exposures including heavy metals and pesticides. Through this work we aim to better understand interactions between sequence mediated cis pausing regulators and trans-acting protein factors.