The structural landscape of Microprocessor-mediated processing of pri-let-7 miRNAs

Abstract

MicroRNA (miRNA) biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryoelectron microscopy (cryo-EM) and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has the structural plasticity to accommodate a range of pri-miRNAs. These structures revealed key features of the 5' UG sequence motif, more comprehensively represented as the "flipped U with paired N" (fUN) motif. Our analysis explains how cleavage of class-II pri-let-7 members harboring a bulged nucleotide generates a non-canonical precursor with a 1-nt 3' overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 contributes to MP fidelity by interacting with the CNNC motif and Drosha's Piwi/Argonaute/Zwille (PAZ)-like domain. Overall, this study sheds light on the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP.

Keywords: Microprocessor; RNAi; cryo-EM; let-7 miRNA; miRNA biogenesis; protein-RNA.

Figure-1-. Heme has a ubiquitous role in M²P².

(A) General architecture of a canonical pri-miRNA. The short sequence motifs and Drosha cleavage sites are shown. (B) Domain architecture of human Drosha (DR) and DGCR8 (DG). The DR^ΔN and DG^ΔN truncations used in this study are underlined. (C) Analytical SEC analysis for different MP heme-variants: MP^he, MP^hb & MP^apo. The UV280 and UV450 traces are shown in solid and dotted lines, respectively. Average A450/280 ratios and calculated heme/MP molar ratios are shown. (D, E) Qualitative cleavage assays, (F, G) plots for RNA substrate disappearance in M²P² in near-pre-steady state, and (H) the calculated cleavage rates for pri-miR-98 and (E) pri-let-7a1 by MP^apo, MP^hb and MP^he.

Figure-2-. Cryo-EM structure of MP^he in complex with class-II pri-let-7s

(A) 2D schematics of different class-II pri-let-7s used for cryoEM. Conserved sequence motifs and Drosha cleavage sites are shown. (B) Cartoon and space fill representation of the cryo-EM structures of MP^he-pri-let-7a1, (C) MP^he-pri-miR-98, and (D) MP^he-pri-let-7f1 complexes. (E) A zoom-in view of the 5’ catalytic site in MP^he-pri-let-7f1 map showing cryoEM density of the Ca²⁺ ion (green) and water molecules (red), (F) the conserved interactions in multiple pri-let-7 structures stabilizing the 5p +2 geometry via Q1144. (G) Pri-let-7a1 processing assay with MP^he-Q1144A mutant showing accumulation of 3’ nicked RNA products compared to MP^he.

Figure-3-. RNA-driven structural rearrangements in MP in the two pri-let-7 classes.

(A) 2D schematics of the pri-let-7a2, and the cryo-EM structure of the MP^he-pri-let-7a2 complex. (B) Drosha superimposition showing the conserved positioning of the RNA lower stem in different pri-let-7s. The difference in the (C) RNA positioning (D) Drosha dsRBD, (E) DGCR8-2 dsRBDI and DGCR8-2 dsRBD2 positioning in class-I (pri-miR-16-2 in blue) and class-II (pri-let-7a1 in red-orange) pri-miRNA bound MP^he structures. The repositioning in different components are shown with arrows and distances are also marked. Base-pairing patterns at the 5’ cleavage site in (G) class-I pri-let-7a2, and class-II (H) pri-let-7a1 (I) pri-let-7f1 and (J) pri-miR-98. The 5p +1nt (cyan) in pri-let-7a2 is paired with 3p +3nt, while it remains unpaired/bulged in class-II structures. Cleavage sites are highlighted with red arrows. (K) The local base twist and shift parameters for the 5p nucleotides around the 5’ cleavage site. The 5p +2 shows distorted geometry (highlighted in red) accommodating for the bulged 5p +1 nt. A schematic of the base-pairing patterns observed in class-I pre-let-7 (L) and class-II pri-let-7s (M) when bound to MP in their pre-catalytic state. Drosha cleavage sites are shown with red arrows. The bulged 5p +1 nt in class-II pri-let-7s stays unpaired and changes the upstream base-pairing register, allowing MP to generate pre-let-7 species with a 1 nt 3’ overhang.

Figure-4-. The 5’ UG or fUN motif?

A zoomed-in view of the interactions observed in the UG (or fUN) motif in (A) pri-miR-98, (B) pri-let-7a1, (C) pri-let-7a2 and (D) pri-let-7f1 with residues (green sticks) from, Drosha’s wedge, dsRBD, and the belt-helix. The U flips out (underscored) while N establishes the 1^st base-pairs in the RNA lower stem. (E) Conserved structural features characteristic of this motif.

Figure-5-. SRSF3 assists Drosha processing of pri-let-7s.

(A) Qualitative cleavage assays of pri-let-7c and pri-let-7c^CNNC/UUUU by MP^he, showing the effect of SRSF3 on pre-let-7c product formation (green arrowhead) and unproductive cleavage products (red arrowhead). (B) Plot for pri-let-7c substrate disappearance (C) inverse processing and (D) pre-let-7c appearance in near-pre-steady state, showing the effect of SRSF3 in M²P². As formed pre-let-7c product decreases overtime, its traces are shown as connected lines only. (E) Plot for pri-let-7a1 cleavage and (F) pre-let-7a1 appearance in near-pre-steady state, showing SRSF3 effect on M²P². (G) The calculated rates for pri-let-7s processing with SRSF3.

Figure-6-. The MP^he-pri-let-7f1-SRSF3 quaternary complex

(A) Cryo-EM structure of MP^he-pri-let-7f1-SRSF3 in the pre-catalytic state shown in cartoon and (B) surface representation. The SRSF3 RRM domain (yellow) is bound to 3p CNNC motif in pri-let-7f1 (pink) and is docked onto Drosha’s PAZ-like domain (grey). The thumb peptide is clearly visible and interleaved between the two RNA strands. (C) The pri-let-7f1 3p ssRNA region in the MP^he (dark grey) and MP^he-SRSF3 structures (pink) shown in two orientations. The 3p strand in the MP^he structure (dark grey) would clash with the SRSF3 thumb peptide. In the MP^he-SRSF3 bound structure, the CNNC motif (green) passes through the electrostatically charged channel formed by the SRSF3 RRM domain and thumb. Mapping of the electrostatics on the SRSF3 surface is shown (−5 (red) to +5 kT (blue)). (D) Molecular interactions between the CNNC motif (green sticks) and SRSF3 RRM domain (beige-colored sticks). Direct H-bonding interactions are shown as red dotted lines. (E) In-vitro M²P² of pri-let-7c with different SRSF3 mutations/truncations. Only the RRM-thumb restores pre-let-7c (marked) levels to SRSF3-WT level. (F) Plot for the pri-let-7c inverse processing and (G) pre-let-7c formation in near-pre-steady state, showing the effect of SRSF3 mutations/truncations in M²P². As formed pre-let-7c product decreases overtime, its traces are shown as connected lines only. SRSF3-M-1, M-2, and M-1+2 indicate SRSF3 with mutations in C¹, C⁴, or C¹+C⁴ nucleotide-stabilizing residues in the CNNC motif, respectively. RRM and RRM thumb denotes SRSF3^1-84 and SRSF3^1-90 truncations. SRSF3-M-I1, M-I2, and M-I1+2 are SRSF3 with mutations in the Drosha interface 1, 2, or 1+2, respectively.

Figure-7-. The SRSF3-Drosha PAZ-like domain interface

(A) A cutaway view of the Drosha-SRSF3 interface showing SRSF3 (yellow) perfectly nestled into the PAZ-like domain scaffold (shown as an electrostatic surface). (B) The interaction between SRSF3 (yellow sticks) and the PAZ-like domain (sky blue sticks), clusters into two regions, interface-1 and interface-2 (black dotted ovals) with complementary charges between the two proteins. (C) In-vitro processing of pri-let-7c using Drosha-mutations in the Drosha-SRSF3 interface. Mutations in Interface-1, 2 or 1+2 show less or no pre-let-7c product during the time course. (D-E) Northern analysis of miRNA processing in HEK293T Drosha-KO cells rescued by wt or mutant Drosha constructs. Cells were co-transfected with expression constructs for CNNC-bearing pri-let-7g (D) or pri-miR-16 (E), or counterparts with mutations in CNNC motifs; non-CNNC pri-miR-24 serves as a negative control. Mutations in the SRSF3-interacting interface of Drosha reduce pri-miRNA processing for CNNC-bearing miRNAs, as clearly indicated by the reduction of pre-miRNA hairpin species (dotted boxes). These Drosha mutants also exhibit impaired accumulation of mature let-7g and miR-16 (asterisks), with a stronger effect on let-7g. Biogenesis of non-CNNC pri-miRNA constructs was not affected by Drosha mutations. (F) In-vitro M²P² of pri-let-7c with SRSF3-mutants at the Drosha-SRSF3 interface. (G) SRSF3 pull-down assay with MP^he and pri-let-7f1. SRSF3-WT co-elutes with the MP^he-pri-let-7f1 as analyzed on SDS-PAGE (upper panel) and urea-PAGE (lower panel) gels.