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

Abstract

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.

Structural basis of si RNA recognition by TRBP double‐stranded RNA binding domains

“…miRNA biogenesis is initiated in the nucleus with RNA polymerase II transcribing the primary miRNA (pri‐miRNA), a precursor containing one or several stem‐loop elements flanked by single‐stranded RNA regions (Bartel, ; Kim, ,b). The pri‐miRNA is processed by the microprocessor, a complex containing the ribonuclease III (RNase III) enzyme Drosha and the RNA‐binding protein DGCR8, resulting in the release of the pre‐miRNA (Denli et al , ; Gregory et al , ; Herbert et al , ; Kwon et al , ). Pre‐miRNA secondary structure typically consists of a hairpin of ~70 nucleotides with irregularities such as bulges and internal loops, and a characteristic two‐nucleotide overhang at its 3′ end.…”

The Drosophila Dicer-1 Partner Loquacious Enhances miRNA Processing from Hairpins with Unstable Structures at the Dicing Site

“…Drosha and its partner protein Pasha (DGCR8 in vertebrates) form the core of the Microprocessor complex, and Pasha is essential for recognition and precise cleavage of hairpin substrates (Denli et al, 2004; Gregory et al, 2004; Han et al, 2004; Herbert et al, 2016; Kwon et al, 2016; Martin et al, 2009; Nguyen et al, 2015). …”

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity

“…This hypothetical model of the Microprocessor complex implies that the Rhed region of DGCR8 dimers binds the apical stemloop junction of the pri-miRNA, with the DGCR8 dimers oriented in an asymmetrical fashion [13]. This model is reinforced by biochemical studies, which clearly implicate selective DGCR8 binding to the apical stem-loop junction in a ratio of 2:1:1 (DGCR8 dimer:Drosha:pri-miRNA substrate) [12]. Heme influences the binding of DGCR8 to its pri-miRNA substrate.…”

“…Drosha alone has no specific pri-miRNA cleavage activity, because it requires its partner DGCR8 for substrate recognition [6,7]. DGCR8 binds to key structural elements in pri-miRNAs; higher order structures of DGCR8 and Drosha appear to be required for Drosha-mediated cleavage [7][8][9][10][11][12][13][14].…”