Final Report Summary - RNAMEDTGS (RNA-mediated Transcriptional Gene Silencing in Humans) This project has revealed an important role for nuclear RNA surveillance factors in gene expression in humans. We showed that factors such as MTR4, ZFC3H1 and ZCCHC8, are repressors of HIV-1 transcription and are implicated in viral latency. At the genome-wide level, analysis of human nuclear RNA surveillance factors revealed an important role in the 3D architecture of the nucleus. The project also identified novel mechanisms by which Microprocessor modulates gene expression, through processing of intron-embedded miRNAs. We showed that modulation of an intronic miRNA, miR-3173 within Dicer pre-mRNA modulates expression of DICER protein. This has important effects in ovarian cancer that is associated with low expression of DICER. Our data furthermore indicate that the processing of numerous human miRNAs may be regulated in a similar manner. Finally, we identified protein factors associated with a highly transcribed locus using the unbiased technique PICh (proteomics of isolated chromatin fragments). This analysis confirmed the presence of TGS factors such as Mtr4, and termination factors Xrn2 associated with the gene in its transcriptionally repressed state. The analysis furthermore revealed the unanticipated recruitment of numerous factors in the DNA damage response, particularly Mediator of DNA Damage Checkpoint 1 (MDC1), that are specifically associated with the locus during active transcription. MDC1 was found to physically interact with numerous factors implicated in transcription and RNA processing. Our analysis has revealed MDC1-associated MRN complex is highly associated with actively transcribing genes. Altogether, our data support a model by which association of the MRN complex with the transcriptional machinery constitutively scans active genes for transcription-induced DNA damage to preserve the integrity of the coding genome.
