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Studies of the ncRNA binding site on RNA polymerase II and the sequence determinants of B2 RNA function as a repressor of transcription
Upon cell stress, the production of mRNA by RNA polymerase II (Pol II) is downregulated. In mouse cells B2 RNA, a ~180 nt non-coding RNA transcribed from retrotransposon elements, binds Pol II and represses transcription in response to heat shock. The studies detailed in this thesis examine the ncRNA binding site on Pol II, and the sequence determinants of B2 RNA that mediate repression of transcription in vitro. To identify the surface on Pol II to which B2 RNA binds, I developed a reversible crosslinking MS/MS assay coupled to a label free quantitation method. The crosslinking assay identified 36 Pol II peptides that crosslinked to B2 RNA(81-130), a region of B2 RNA that is fully functional. The label free quantitation revealed four of these peptides accounted for 92% of the total intensity. I mapped these highly enriched peptides onto a conserved yeast crystal structure and found the peptides are not only located in the DNA binding cleft and active site region of Pol II, but the peptides themselves also play key functional roles in the transcription reaction. To identify the sequence determinants of B2 RNA that allow it to repress transcription in vitro, I undertook a mutational strategy. Guided by an experimentally determined secondary structural model, I made a series of deletions from the 5\u27 and 3\u27 ends of B2 RNA, which revealed four different regions that function in repression of transcription in vitro. Deleting each of these regions showed that the unstructured region, 99-113, had the greatest effect on transcriptional repression in vitro. Moreover, deleting two unstructured regions, 99-113 and 153-178, from full length B2 RNA resulted in an RNA that no longer repressed transcription in vitro. Electrophoretic mobility shift assays (EMSAs) revealed that removing these transcriptionally repressive regions did not lead to a loss of Pol II binding by the ncRNAs. Hence, B2 RNA contains separate regions for binding Pol II and repressing transcription
DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration
Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness