UCLA

The RNA-binding protein DiGeorge Critical Region 8 (DGCR8) and its partner, the nuclease Drosha, are necessary for processing of primary microRNA (pri-miRNA) transcripts in animals. MicroRNAs, in turn, are essential pieces of the cell's regulatory machinery, where they serve to post-transcriptionally regulate gene expression through the RNA-Induced Silencing Complex. Work described in this dissertation concerns the interaction of DGCR8 with the cofactor heme, and shows how the activity of DGCR8 - and by extension, the activity of Drosha - is controlled by the presence of heme within the heme-binding domain. Biochemical and spectrophotometric assays are used to show that the heme in DGCR8 is in the Fe(III) form and is ligated to two cysteines, one from each subunit of the DGCR8 homodimer. The activity of DGCR8 is shown to depend on the oxidation state of the iron; Fe(II) is unable to activate processing or allow trimerization of DGCR8 on pri-miRNA. I also show that, from a screen of ten metalloporphyrins, only Co(III) protoporphyrin IX is able to both bind to DGCR8 and activate pri-miRNA processing. The implications of this work are discussed.