Determining the role of RNA abasic sites in gene regulation

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RNA abasic sites are recently identified modifications in RNA that are generated by methylpurine glycosylase to regulate gene expression. They are abundant in RNA and they stabilize RNA yet we do not know much about how they form, their precursors, and their functions. DNA abasic sites were identified in the 1960s and those discoveries led to the elucidation of the base excision repair pathway in DNA damage. But RNA abasic sites were largely unknown, except those generated by the plant poison, ricin, in ribosomal RNA. While studying proteins that bind to the nucleic acid structure, R-loop, we identified RNA modification enzymes and methylpurine glycosylase as well as apurinic/apyrimidinic endonuclease I that were known to process DNA abasic sites. We have now shown that methylpurine glycosylase cleaves the glycosidic bond in RNA to form abasic sites and these reactions occur on RNA in RNA/DNA hybrids of R-loops. By mass spectrometry, we found that RNA abasic sites are rather abundant, there are tens to hundreds of thousands of them in each cell. We also found in an enhancer RNA of APOE, N6- methyladenosines are cleaved by methylpurine glycosylase to form RNA abasic sites on R- loops. The abasic sites then stabilize R-loops and pause RNA Polymerase II transcription. We show that this mechanism keeps the noncoding RNA poised to activate APOE expression in response to cellular demands. Given that we have early evidence for the role of RNA abasic sites in the regulation of noncoding RNA, it is necessary to better understand them. We propose to 1) examine the genome-wide effect of nucleic-acid-mediated pausing on noncoding RNA, 2) identify other glycosylases that form RNA abasic sites, and 3) determine what other modified RNA bases are precursors of RNA abasic sites. Together, these results will characterize RNA abasic sites by finding how they form and what they do.