MicroRNAs (miRNAs) regulate the expression of a large number of protein-coding genes. Their primary transcripts (pri-miRNAs) have to undergo multiple processing steps to reach the functional form. Little is known about how the processing of miRNAs is modulated. Here we show that the RNA-binding protein DiGeorge critical region-8 (DGCR8), which is essential for the first processing step, is a heme-binding protein. The association with heme promotes dimerization of DGCR8. The heme-bound DGCR8 dimer seems to trimerize upon binding pri-miRNAs and is active in triggering pri-miRNA cleavage, whereas the heme-free monomer is much less active. A heme-binding region of DGCR8 inhibits the pri-miRNA-processing activity of the monomer. This putative autoinhibition is overcome by heme. Our finding that heme is involved in pri-miRNA processing suggests that the gene-regulation network of miRNAs and signal-transduction pathways involving heme might be connected.

Two classes of RNA polymerases transcribe RNA from promoters on DNA templates: promoter recognitioncompetent single polypeptides and multisubunit enzymes that require separable promoter recognition factors. Eukaryotic mitochondria utilize an unusual hybrid of these classes composed of a "core" RNA polymerase related to the single polypeptide enzymes plus a "specificity factor" necessary for promoter utilization. Using supercoiled or premelted templates, we have discovered that the yeast core mitochondrial RNA polymerase (Rpo41) has the intrinsic ability to initiate from promoters without its specificity factor (Mtf1). Rpo41 requires the mitochondrial promoter sequence (ATATAAGTA) for this activity. On premelted templates addition of Mtf1 actually inhibits the promoter selective activity of Rpo41. Mtf1 increases abortive relative to productive transcription by Rpo41, possibly by stabilizing the promoter complex and reducing escape into elongation. The requirement for Mtf1 on closed but not open templates indicates that Mtf1 facilitates melting but not recognition of promoters.The mitochondrial organelle in eukaryotic cells originated as an endosymbiotic bacterium that used a multisubunit RNA polymerase (RNAP) 1 like that found in eubacteria (1, 2). At some point the organelle replaced the several genes encoding the multisubunit RNAP with a gene for a single polypeptide RNAP (in yeast, Rpo41), related to that used by T7 phage (3). However, the core mitochondrial RNAP from yeast and mammals, unlike the self-sufficient phage RNAP, requires an additional "specificity" factor (Mtf1, mtTFB) for selective utilization of promoters (4, 5).Although Mtf1 structurally resembles RNA methyltransferases (6), functionally Mtf1 acts very much like bacterial RNAP sigma factors in that it associates with Rpo41 prior to DNA binding and dissociates after a short transcript is made (7). Also like sigma factors, Mtf1 appears to be important for promoter opening, based on the identification of Mtf1 mutations that result in altered promoter utilization in the context of the holo-RNAP, and the ability to correct these defects by supercoiling the DNA template (8, 9). However, our recent description of a mutation in Rpo41 with promoter utilization defects that also can be corrected by supercoiling (10) leaves open the possibility that the interactions of Mtf1 with Rpo41 may act to reveal the intrinsic ability of the core RNAP to recognize and initiate from mitochondrial promoters.In support of this idea, Rpo41 alone possesses some DNA sequence specificity, recognizing a non-selective template consisting of alternating AT residues but not any homopolymer sequences (11). This simple template has some similarity to the mitochondrial consensus promoter/initiation site, ATATA-AGTA, with the last A representing the ϩ1 nucleotide (nt) of the transcript (12). In this work we have used a highly purified recombinant form of the yeast mitochondrial RNAP to ask whether the core RNAP alone possesses selective transcription activity. We have found that...

DiGeorge critical region 8 (DGCR8) is essential for maturation of microRNAs (miRNAs) in animals. In the cleavage of primary transcripts of miRNAs (pri-miRNAs) by the Drosha nuclease, the DGCR8 protein directly binds and recognizes pri-miRNAs through a mechanism currently controversial. Our previous data suggest that DGCR8 trimerizes upon cooperative binding to pri-mir-30a. However, a separate study proposed a model in which a DGCR8 molecule contacts one or two pri-miRNA molecules using its two double-stranded RNA binding domains. Here, we extensively characterized the interaction between DGCR8 and pri-miRNAs using biochemical and structural methods. First, a strong correlation was observed between the association of DGCR8 with pri-mir-30a and the rate of pri-miRNA processing in vitro. Second, we show that the high binding cooperativity allows DGCR8 to distinguish pri-miRNAs from a nonspecific competitor with subtle differences in dissociation constants. The highly cooperative binding of DGCR8 to a pri-miRNA is mediated by the formation of higher-order structures, most likely a trimer of DGCR8 dimers, on the pri-miRNA. These properties are not limited to its interaction with pri-mir-30a. Furthermore, the amphipathic C-terminal helix of DGCR8 is important both for trimerization of DGCR8 on pri-miRNAs and for the cleavage of pri-miRNAs by Drosha. Finally, our three-dimensional model from electron tomography analysis of the negatively stained DGCR8-pri-mir-30a complex directly supports the trimerization model. Our study provides a molecular basis for recognition of pri-miRNAs by DGCR8. We further propose that the higher-order structures of the DGCR8-pri-miRNA complexes trigger the cleavage of pri-miRNAs by Drosha.

The yeast mitochondrial RNA polymerase (RNAP) is composed of the core RNAP, Rpo41, and the mitochondrial transcription factor, Mtf1. Both are required for mitochondrial transcription, but how the two proteins interact to create a functional, promoter-selective holoenzyme is still unknown. Rpo41 is similar to the single polypeptide bacteriophage T7RNAP, which does not require additional factors for promoter-selective initiation but whose activity is modulated during infection by association with T7 lysozyme. In this study we used the co-crystal structure of T7RNAP and T7 lysozyme as a model to define a potential Mtf1 interaction surface on Rpo41, making site-directed mutations in Rpo41 at positions predicted to reside at the same location as the T7RNAP/T7 lysozyme interface. We identified Rpo41 mutant E1224A as having reduced interactions with Mtf1 in a two-hybrid assay and a temperature-sensitive petite phenotype in vivo. Although the E1224A mutant has full activity in a non-selective in vitro transcription assay, it is temperature-sensitive for selective transcription from linear DNA templates containing the 14S rRNA, COX2, and tRNAcys mitochondrial promoters. The tRNAcys promoter defect can be rescued by template supercoiling but not by addition of a dinucleotide primer. The fact that mutation of Rpo41 results in selective transcription defects indicates that the core RNAP, like T7RNAP, plays an important role in promoter utilization.Mitochondria contain a separate genome (mtDNA) 1 that encodes several mRNAs and the rRNAs and tRNAs required for their translation into components of the oxidative phosphorylation system (1-3). These mitochondrial transcripts are synthesized by an RNA polymerase (RNAP) distinct from those found in the nucleus of the eukaryotic cell (2). In the budding yeast Saccharomyces cerevisiae, two well characterized nuclear genes, RPO41, encoding the mitochondrial core RNAP, and MTF1, encoding a required mitochondrial transcription factor, interact to form the functional mitochondrial RNAP holoenzyme (mtRNAP) (4, 5). Homologues of Rpo41 have been identified in many organisms (6), and apparent Mtf1 (sc-mtTFB) homologues required for selective mitochondrial transcription have been described recently in humans and mice (7-9). Therefore it is clear that more complex eukaryotes also use a multisubunit mtRNAP to express genes from the mtDNA.The origins of the multi-subunit mtRNAP are still not clear. Rpo41 shares extensive amino acid similarity with RNAP from bacteriophage T7 and T3 (4). However, these phage-encoded single polypeptide RNAPs do not require additional factors for promoter recognition (10). The recent crystal structure determination of Mtf1 has revealed a significant structural similarity with the family of RNA methyltransferases (11). Furthermore, Mtf1 and its functional homologues from multicellular organisms all share amino acid sequence similarity with RNA methyltransferases (7-9, 11). In fact, one of the human Mtf1 homologues has been shown to have methyltransferase activity (...

No abstract