UCLA

MicroRNAs (miRNAs) are essential regulatory molecules that function to block translation as part of the miRNA-Induced Silencing Complex. Mature miRNAs are produced through a series of cleavage steps following transcription of the primary miRNA transcript (pri-miRNA). pri-miRNAs are recognized and cleaved by the Microprocessor complex that is composed of the RNA-binding protein DiGeorge Critical Region gene 8 (DGCR8) and the ribonuclease III enzyme Drosha. Biochemical characterization of recombinant DGCR8 expressed in E. coli indicated a Fe(III) heme cofactor. However, it is not clear whether this interaction is biologically relevant. Work described in this dissertation concerns the development of a live-cell fluorescence-based assay for measuring the cleavage efficiency of pri-miRNAs. I extensively validate the assay and show that it faithfully indicates intracellular activity of DGCR8. I then use this assay to answer a series of questions about pri-miRNA processing that in the past was not easily addressed in mammalian cells. I show that heme is required for DGCR8 activity in cells. DGCR8 binds this cofactor through its RNA-binding heme domain (Rhed) that also directly contacts the pri-miRNA hairpin. My work indicates that the RNA-binding interface is important for pri-miRNA processing in cells. Biochemical screens of metalloporphyrins indicated that Co(III) protoporphyrin IX (PPIX) binds and activates DGCR8. My work clearly indicates that Co(III) activates pri-miRNA processing in HeLa cells. I have also studied a variety of Fe(III) protoporphyrins with modifications at the vinyl group on the ring. Only the porphyrins with small modifications, such as Fe(III) mesoprotoporphyrin IX and 2,4-dimethyldeuteroproporphyrin IX, retain the high potency of activating processing, suggesting that the vinyl group is involved in contacting DGCR8. Finally, by testing the activity of DGCR8 mutants with reduced affinity for heme to various degrees, and under various heme stressed cell culture conditions, I estimate that cellular Fe(III) heme availability is very low, likely to be in the picomolar range. This estimate is consistent with the high affinity of DGCR8 for Fe(III) heme. A detailed description of how to perform the cellular miRNA processing assay is also included. My study demonstrates that the cellular pri-miRNA processing assay is a powerful tool to understand the mechanism of pri-miRNA processing and how processing may be regulated by small molecules.