RNA-DNA Sequence Differences Occur within Seconds following RNA exit PolII Active Sites and Are Responsive to Cellular Stress. V. G. Cheung1,3,4, I. X. Wang3, L. Core2, H. Kwak2, L. Brady5, A. Bruzel1,3, A. L. Richards5, M. Wu1,3, J. T. Lis2 1) Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA; 2) Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA; 3) Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104, USA; 4) Department of Pediatrics, University of Pennsylvania, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; 5) Cell and Molecular Biology Graduate Program, University of Pennsylvania, Philadelphia, PA 19104, USA.

   RNA and protein sequences are expected to be identical to the corresponding DNA sequences. However, there are known exceptions such as ADAR mediated RNA editing, and other types of RNA-DNA differences, RDDs, which we and others have reported. To understand how these RDDs arise and their biological implications, we are studying the mechanisms by which the RDDs are formed and their role in cellular responses to stress.
    First, we isolated nascent RNAs by PRO-seq and GRO-seq which are nuclear run-on assays followed by deep sequencing. We compared the sequences of these RNA with their corresponding DNA sequences and found all 12 types of RDDs, including transversions. We validated the presence of RDDs in nascent transcripts by examining chromatin-associated RNAs. The single nucleotide resolution of PRO-seq allows us to determine that RDDs are formed about 50 to 60 bases from the RNA polymerase II active sites. They are made after 5 capping and before splicing. These RDDs are found throughout the transcripts including the untranslated regions (3 and 5 UTRs), antisense RNAs associated with transcription start sites, introns and exons. The exonic sites are exported into the cytoplasm and translated into proteins.
    Second, to study the biological effects of RNA editing and RDDs, we found that their levels change in response to cellular stress. For example, the A-to-G editing levels in the SEC genes required for vesicle formation in the endoplasmic reticulum (ER) change following ER stress. In addition, ARAP1 that was found in genetic studies of type II diabetes to be significantly associated with fasting proinsulin level showed only modest changes in gene expression in response to insulin; however, the RDD level in ARAP1 increased significantly. Together these results show that RNA editing and RDDs play a previously unrecognized role in the maintenance of cellular homeostasis.
    In this presentation, I will describe our findings on when during transcription RDDs are formed and how RDD levels change in response to cellular stress.

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