Processing of microRNA primary transcripts requires heme in mammalian cells

Weitz, Sara H.; Gong, Ming; Barr, Ian; Weiss, Shimon; Guo, Feng

doi:10.1073/pnas.1309915111

Cited by 77 publications

(77 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, previous studies concluded that heme binds DGCR8 across its dimerization interface and that heme availability affects pri-miRNA processing both in vivo and in vitro (Faller et al 2007;Senturia et al 2010;Weitz et al 2014). …”

Section: Discussionmentioning

confidence: 98%

A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting

Herbert

Sarkar

Mills

et al. 2015

RNA

View full text Add to dashboard Cite

During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stemloop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins.

show abstract

Section: Discussionmentioning

confidence: 98%

A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting

Herbert

Sarkar

Mills

et al. 2015

RNA

View full text Add to dashboard Cite

show abstract

“…To test these possibilities, we resorted to a widely used in vivo reporter vector for pri-miRNA processing ( Fig. 2D; Mori et al 2014;Weitz et al 2014). Specifically, miRNA hairpins or their mutants were cloned into the 3 ′ UTR of mCherry in a dual GFP/mCherry vector so that pri-miRNA processing will destabilize mCherry mRNA, with more efficient processing leading to a higher ratio of GFP/ mCherry signals by flow cytometry (see example in Supplemental Fig.…”

Section: Systematic Comparison Of Hairpin Features For Mammalian Pri-mentioning

confidence: 99%

Novel determinants of mammalian primary microRNA processing revealed by systematic evaluation of hairpin-containing transcripts and human genetic variation

et al. 2017

View full text Add to dashboard Cite

Mature microRNAs (miRNAs) are processed from hairpin-containing primary miRNAs (pri-miRNAs). However, rules that distinguish pri-miRNAs from other hairpin-containing transcripts in the genome are incompletely understood. By developing a computational pipeline to systematically evaluate 30 structural and sequence features of mammalian RNA hairpins, we report several new rules that are preferentially utilized in miRNA hairpins and govern efficient pri-miRNA processing. We propose that a hairpin stem length of 36 ± 3 nt is optimal for pri-miRNA processing. We identify two bulge-depleted regions on the miRNA stem, located ∼16-21 nt and ∼28-32 nt from the base of the stem, that are less tolerant of unpaired bases. We further show that the CNNC primary sequence motif selectively enhances the processing of optimal-length hairpins. We predict that a small but significant fraction of human single-nucleotide polymorphisms (SNPs) alter pri-miRNA processing, and confirm several predictions experimentally including a disease-causing mutation. Our study enhances the rules governing mammalian pri-miRNA processing and suggests a diverse impact of human genetic variation on miRNA biogenesis.

show abstract

“…In the cytosolic compartment binding of miRNAs to target mRNAs interferes with translation or mRNA stability. A critical complex partner of DROSHA is DGCR8 (DiGeorge syndrome critical region gene 8 protein) which possesses a heme-binding domain relevant for substrate recognition (Quick-Cleveland et al, 2014;Weitz et al, 2014), and hence links mitochondrial iron supply via heme formation to the entire regulatory miRNA network (Fig. 4).…”

Section: Posttranscriptional Iron Regulation Involving Microrna-210 Amentioning

confidence: 99%

Compartmentalization of iron between mitochondria and the cytosol and its regulation

Mühlenhoff

Hoffmann

Richter

et al. 2015

European Journal of Cell Biology

View full text Add to dashboard Cite

Processing of microRNA primary transcripts requires heme in mammalian cells

Cited by 77 publications

References 44 publications

A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting

A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting

Novel determinants of mammalian primary microRNA processing revealed by systematic evaluation of hairpin-containing transcripts and human genetic variation

Compartmentalization of iron between mitochondria and the cytosol and its regulation

Contact Info

Product

Resources

About