MicroRNA Regulation of DNA Repair
Mammalian cells have developed an elaborate DNA damage response (DDR) system and DNA repair machinery, which play a critical role in development of resistance to DNA damaging agents. The precise regulation of DDR and DNA repair is crucial for cell survival and its abrogation often results in genetic instability. We are interested in determining the role microRNAs (miRNAs) play in regulation of DDR and DNA repair. MiRNAs are short, non-coding RNA molecules. Mature single-stranded miRNAs cooperate with Argonaute proteins to form the RNA-induced silencing complex and guide this complex to their target mRNAs through partial sequence homology, which subsequently causes block of translation and/or mRNA degradation. Some miRNAs function as oncogenes or tumor suppressors by modulating biological processes such as cell cycle control, proliferation, differentiation and apoptosis. However, only a few reports describe miRNAs that regulate DDR pathways.
We have developed cell-based screening methods and identified several microRNAs that regulate the accumulation of DNA repair proteins at damage sites. We are now investigating how these microRNAs modulate DNA repair and cellular sensitivity to DNA damaging agents. We are also interested in how deregulation of these microRNAs can contribute to tumorigenesis.
Regulation of MicroRNA BiogenesisMiRNA biogenesis is initiated in the nucleus. MiRNA genes are located in intergenic and intragenic regions of the genome. They are transcribed by RNA polymerase II or III to produce long mono- or polycistronic primary miRNA (pri-miRNAs) transcripts that form a hairpin-like secondary structure. The hairpins are subsequently excised by the microprocessor complex, which consists of Drosha, an RNase III endonuclease, and DGCR8, a double-strand RNA binding protein, to generate ~70nt precursor mirNAs. Subsequently, precursor miRNAs bind to a nuclear export protein, exportin-5, and are translocated to the cytoplasm where they are cleaved near the terminal loop by another RNase III, Dicer, and its binding partners TRBP and PACT, to generate a ~22nt miRNA duplex. The strand that is less thermodynamically stable of the miRNA duplex is preferentially loaded into the miRNA-induced silencing complex (miRISC), which consists of Dicer, TRBP, PACT and Argonaute proteins. The strand loaded into the miRISC complex is called the guide strand, which directs the miRISC complex to its mRNA target. The other strand, the passenger strand, is subsequently degraded by an unknown mechanism. The miRNA/miRISC complex mainly binds to the 3' untranslated region of the target mRNA in a sequence-dependent manner. The sequence complementarity of the miRNA seed sequence (5' region of the miRNA centered on nt 2-7) to its target mRNA is sufficient for translational repression. In a rare event, in which there is perfect complementarity between the miRNA and its target mRNA, the mRNA is cleaved by the endonuclease, Argonaute 2, a cofactor of the miRISC complex.
Expression of miRNAs can be regulated by 1) transcriptional activation or repression, 2) post-transcriptional modifications, 3) processing and 4) degradation/decay. We are interested in understanding of the relationship between the miRNA biogenesis pathway and the signaling pathways that regulate them. Thus, elucidating the signaling system, including the identification of the modified target, the modifications, and modifiers, is an important step in understanding how the miRNA biogenesis pathway is regulated in response to cellular stress and DNA damage.