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. We developed an epithelial transport assay using renal proximal tubule cells and examined the effect of dysregulation of mitochondrial RNA polymerase pausing on proximal tubule function by stabilizing by the mitochondrial quadruplexes. Loss of mitochondrial ATP generation due to impaired mitochondrial RNA polymerase transcription resulted in impaired mitochondrial transport function, linking regulation of mitochondrial RNA polymerase transcription by nucleic acid structure to cellular function. 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 . At some genes in response to osmotic stress, the stable R-loops are resolved allowing full length transcription and increased gene expression following osmotic stress. Using an osmotic stress model, we have identified a transcription factor which releases promoter-proximal paused polymerase to regulate the expression of tonicity-responsive genes. We are extending this work to study how the combination of osmotic stress and heavy metal exposure results in attenuation of tonicity responsive gene expression. Through these studies we will gain more granular understanding of how dynamic control of nucleic acid structure can lead to regulated changes in the extent of RNA polymerase pausing II and gene expression.