Fe(III) heme sets an activation threshold for processing distinct groups of pri-miRNAs in mammalian cells

Abstract

The essential biological cofactor heme is synthesized in cells in the Fe(II) form. Oxidized Fe(III) heme is specifically required for processing primary transcripts of microRNAs (pri-miRNAs) by the RNA-binding protein DGCR8, a core component of the Microprocessor complex. It is unknown how readily available Fe(III) heme is in the largely reducing environment in human cells and how changes in cellular Fe(III) heme availability alter microRNA (miRNA) expression. Here we address the first question by characterizing DGCR8 mutants with various degrees of deficiency in heme-binding. We observed a strikingly simple correlation between Fe(III) heme affinity in vitro and the Microprocessor activity in HeLa cells, with the heme affinity threshold for activation estimated to be between 0.6-5 pM under typical cell culture conditions. The threshold is strongly influenced by cellular heme synthesis and uptake. We suggest that the threshold reflects a labile Fe(III) heme pool in cells. Based on our understanding of DGCR8 mutants, we reanalyzed recently reported miRNA sequencing data and conclude that heme is generally required for processing canonical pri-miRNAs, that heme modulates the specificity of Microprocessor, and that cellular heme level and differential DGCR8 heme occupancy alter the expression of distinct groups of miRNAs in a hierarchical fashion. Overall, our study provides the first glimpse of a labile Fe(III) heme pool important for a fundamental physiological function and reveal principles governing how Fe(III) heme modulates miRNA maturation at a genomic scale. We also discuss potential states and biological significance of the labile Fe(III) heme pool.

Porphyrin overdrive rewires pan-cancer cell metabolism

“…A total of 270 million molecules of heme are produced for every red blood cell (RBC) 1 , at a rate of over 2 million RBCs per second 2 . Heme, a porphyrin ring encaging most of the iron in humans, functions as an essential prosthetic group of numerous proteins with roles ranging from signal sensing, DNA binding, microRNA splicing and processing to enzymatic catalysis [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] . To ensure the enormous output of heme biosynthesis, the supply of substrates, intermediates and end-products in the pathway is tightly regulated and precisely balanced 7,22 .…”

Structures of microRNA-precursor apical junctions and loops reveal non-canonical base pairs important for processing

“…A UGU motif at the 5' end of the apical loop has been shown to enhance processing 23 . The heme cofactor is important for proper orientation of pri-miRNA hairpins in Microprocessor and for preferentially processing of distinct groups of pri-miRNAs, including those containing the UGU motif 24,25,26 . Most recently, cryo-EM structures of Microprocessor in complex with pri-miRNAs have revealed an extensive protein-RNA interface, but pri-miRNA apical junctions and loops and the Rhed domain could not be resolved 27,28 .…”