Processing of microRNA primary transcripts requires heme in mammalian cells.
DiGeorge syndrome critical region gene 8 (DGCR8) is the RNA-binding partner protein of the nuclease Drosha. DGCR8 and Drosha recognize and cleave primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical microRNAs (miRNAs) in animals. We previously reported that human, frog, and starfish DGCR8 bind heme when expressed in Escherichia coli and that Fe(III) heme activates apoDGCR8 in reconstituted pri-miRNA processing assays. However, the physiological relevance of heme in miRNA maturation has not been clear. Here, we present a live-cell pri-miRNA processing assay that produces robust signals and faithfully indicates DGCR8 and Drosha activities. We demonstrate that all known heme-binding-deficient DGCR8 mutants are defective in pri-miRNA processing in HeLa cells. DGCR8 contains a previously uncharacterized heme-binding motif, "IPCL," that is also required for its activity. Heme availability and biosynthesis in HeLa cells positively affect pri-miRNA processing and production of mature miRNA. These results establish an essential function for heme in pri-miRNA processing in mammalian cells. Our study suggests that abnormal heme biosynthesis and degradation may contribute to diseases via miRNA-mediated gene regulation networks.
Interdepartmental Program in Molecular, Cellular, and Integrative Physiology, Department of Biological Chemistry, David Geffen School of Medicine, Department of Chemistry and Biochemistry, and Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095.
Sara H
SH
Weitz
Ming
M
Gong
Ian
I
Barr
Shimon
S
Weiss
Feng
F
Guo
D000818:Animals; D001483:Base Sequence; D005453:Fluorescence; D017930:Genes, Reporter; D006367:HeLa Cells; D006418:Heme / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D008322:Mammals; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D059372:Nucleotide Motifs / Q000235:genetics; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics*; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D043244:Ribonuclease III / Q000378:metabolism
C474043:DGCR8 protein, human; D035683:MicroRNAs; D050505:Mutant Proteins; D011506:Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D006418:Heme; C517918:DROSHA protein, human; D043244:Ribonuclease III
RNA processing; fluorescence microscopy; iron; noncoding RNA; protoporphyrin IX