Son S et al. (2023), Role of the proline-rich disordered domain of D...

XB-ART-59840

Genes Dev 2023 May 01;379-10:383-397. doi: 10.1101/gad.350275.122.

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Role of the proline-rich disordered domain of DROSHA in intronic microRNA processing.

Son S , Kim B , Yang J , Kim VN .

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DROSHA serves as a gatekeeper of the microRNA (miRNA) pathway by processing primary transcripts (pri-miRNAs). While the functions of structured domains of DROSHA have been well documented, the contribution of N-terminal proline-rich disordered domain (PRD) remains elusive. Here we show that the PRD promotes the processing of miRNA hairpins located within introns. We identified a DROSHA isoform (p140) lacking the PRD, which is produced by proteolytic cleavage. Small RNA sequencing revealed that p140 is significantly impaired in the maturation of intronic miRNAs. Consistently, our minigene constructs demonstrated that PRD enhances the processing of intronic hairpins, but not those in exons. Splice site mutations did not affect the PRD's enhancing effect on intronic constructs, suggesting that the PRD acts independently of splicing reaction by interacting with sequences residing within introns. The N-terminal regions from zebrafish and Xenopus DROSHA can replace the human counterpart, indicating functional conservation despite poor sequence alignment. Moreover, we found that rapidly evolving intronic miRNAs are generally more dependent on PRD than conserved ones, suggesting a role of PRD in miRNA evolution. Our study reveals a new layer of miRNA regulation mediated by a low-complexity disordered domain that senses the genomic contexts of miRNA loci.

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Genes referenced: drosha

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	Figure 1. Proteolytic cleavage yields a DROSHA isoform (p140) lacking the proline-rich domain. (A) Domain structure of human DROSHA. The lower horizontal lines indicate the epitopes of three antibodies against DROSHA used in this study. SPOT-disorder-single and Valdar01 methods were used for disorderliness prediction and amino acid conservation scoring, respectively (see the Supplemental Material for details). (P-rich) Proline-rich, (RS-rich) arginine/serine-rich, (RIIID) RNase III domain, (dsRBD) double-stranded RNA-binding domain. (B) Identification of an N-terminal-truncated form of DROSHA (p140). HEK293T cells were transfected with control (siCtrl) or DROSHA (siDro) siRNAs for 2 d and then subjected to subcellular fractionation followed by Western blotting. (C) Localization of DROSHA (red) visualized using antibodies against the N terminus (left) or C terminus (right) in HEK293T cells. Costaining was done with an antibody against GM130 (green), a Golgi apparatus marker. The insets are enlarged views from the white rectangles. Scale bars, 10 µm. (D) Detection of the N-terminal fragment of ∼25 kDa (p25) in the membrane fraction. These blots are from the lower parts of the same blots in B. The asterisks indicate cross-reacting bands. (E) Fragments generated from ectopically expressed DROSHA. A plasmid with DROSHA coding sequence linked to the HA and FLAG tags was transfected to HEK293T cells. After 48 h, subcellular fractionation was performed, and the membrane fractions were used for Western blotting. (F) Schematics of the observed DROSHA fragments.
	Figure 2. Identification of the residues critical for proteolytic cleavage of DROSHA. (A) Schematics of the DROSHA constructs harboring deleted or substituted regions. (B–D) Western blot analyses of DROSHA mutants with 20 amino acid deletions (B), 10 amino acid deletions (C), or three amino acid substitutions (D) collectively indicate that the region around the 200th residue is crucial for the proteolytic cleavage. Subcellular fractionation was performed 48 h post-transfection, and the membrane fractions were used for Western blotting. The asterisk indicates a nonspecific band.
	Figure 3. The PRD is necessary for the processing of a subset of pri-miRNAs in cells. (A) Experimental design to compare miRNA expression levels in cells expressing full-length DROSHA or p140. The Δ195–204 or ΔN200 DROSHA construct was transiently transfected to HCT116 or HEK293E DROSHA KO cells. Total RNAs and cell lysates were extracted for small RNA sequencing and Western blotting, respectively. (B) Differentially expressed miRNAs from HCT116 (left) or HEK293E (right). Vertical dotted lines were set based on the maximum fold changes of spike-in RNAs and indicate 2.9-fold and 2.1-fold enrichment in HCT116 and HEK293E, respectively. Horizontal dotted lines denote an adjusted P-value of 0.05 (n = 3). Only miRNAs that passed the cutoffs are colored. Numbers at the top corners indicate the numbers of significantly down-regulated or up-regulated miRNAs in each group. Fold changes and P-values for each miRNA are listed in Supplemental Table S2. (C) Comparison between log2 fold changes measured in two cell lines. In the case of canonical miRNAs, two values are highly correlated. (r) Pearson correlation coefficient. (D) Relative expression levels of mature and pri-miRNAs for selected miRNAs. Validating the sequencing results (gray bars), mature miRNAs decreased when measured by qRT-PCR (purple bars). Pri-miRNA levels determined by RT-qPCR increased (dark blue), suggesting a defect in pri-miRNA processing. U6 snRNA and GAPDH were used as internal controls for mature miRNAs and pri-miRNAs, respectively. The abundance of pri-mir-92a-1 was measured because of the low expression of pri-mir-92a-2. The error bars indicate mean ± standard deviations (n = 3). Two-tailed unpaired Student's t-test: () P < 0.05, (*) P < 0.005.
	Figure 4. The PRD dependency cannot be recapitulated in vitro with the purified Microprocessor. (A,B, top) Representative gel images from in vitro processing assays with Microprocessor composed of either recombinant p140 or full-length DROSHA mimic proteins (8–120 fmol). The log2(p140m/FLm) value of miR-186 is −2.541, and that of miR-125a is 0.165 according to the rescue and sequencing data shown in Figure 3. F2 corresponds to pre-miRNA, while F1 and F3 are the flanking fragments. (Bottom) Quantification of pre-miRNA (F2) band intensities from three independent replicates. The bars indicate mean ± standard deviations (n = 3) after minimum–maximum normalization.
	Figure 5. Searching for pri-miRNA features related to PRD dependency. (A) Schematic diagram of local features of a pri-miRNA hairpin. (B–D) PRD dependency measured in HCT116 as quantified by log2 miRNA expression fold changes (p140m/FLm). miRNAs were grouped based on each sequence motif (B), clustered genomic arrangement (C), or intronic/exonic locations (D). The box indicates the upper and lower quartiles. The horizontal line within the box plot denotes the median. Whiskers represent 1.5× interquartile range. The values from the two groups were compared using the two-tailed Mann–Whitney U-test: (n.s.) not significant (P = 0.05). (E) The PRD dependency of intronic and exonic miRNAs in HCT116 and HEK293E cells. (F) Analysis of DROSHA and miRNA expressions on HCT116 wild-type (WT) cells exposed to serum-starved conditions for 72 h. (G) Serum deprivation induces accumulation of p140 and p25. (H,I) Log2 fold changes of miRNA expression levels under normal or serum-starved conditions. miRNAs were grouped based on the intronic/exonic locations (H) or PRD dependency (I). Two-tailed Mann–Whitney U-test (n = 3).
	Figure 6. The PRD is required for intronic miRNA maturation. (A) Schematics of intronic and exonic constructs. The intronic constructs include exon 3, intron 3, and exon 4 of the BCL2L2 gene. A 125-nt pri-miRNA was inserted into the middle of the intron. In the exonic constructs, pri-miRNAs were inserted into multiple cloning sites downstream from the PGK promoter. Each construct was designed to express the Renilla luciferase (luc) from an independent promoter downstream from the miRNA cassette to normalize transfection efficiency. (B,C) Relative expression levels of miRNAs transcribed from endogenous genomic loci (as determined by small RNA sequencing), intronic constructs, or exonic constructs (as determined by miRNA TaqMan qPCR). Endogenous genomic locations of miRNA genes in B and C are intronic and exonic, respectively. All miRNAs become more dependent on the PRD in the intronic environment, regardless of the original genomic location. miRNA levels from exonic or intronic constructs were normalized to U6 snRNA and Renilla luciferase transcript. The miRNA levels in the p140m-expressing condition were set to 1. The error bars indicate mean ± standard deviations (n = 3). Two-tailed unpaired Student's t-test: () P < 0.05, () P < 0.005, (n.s.) not significant. (D) The relative expression levels of miR-627 transcribed from different intronic or exonic backbones. The number at the right of the schematic indicates the length of the corresponding intron or exon. The dependency is not affected by the length of the transcript. Normalization and statistical tests were performed in the same way as in B and C. (E) Point and deletion mutants of the BCL2L2 intronic construct. Mutations were introduced into the 5′/3′splice site (ss), polypyrimidine tract (PPT), or branch point (BP). Δ1–Δ10 have a 120-nt length deletion. Δ4 includes the 5′ splice site, while Δ9 includes the 3′ splice site. (F) PRD dependency of intronic constructs with mutations in the core splicing elements. miRNA levels from exonic or intronic constructs were normalized to U6 snRNA and Renilla luciferase transcript. The error bars indicate mean ± standard deviations (n = 3). Two-tailed unpaired Student's t-test: (n.s.) not significant (P = 0.05). (G) PRD dependency of deletion mutants Δ1–Δ10. Normalization and statistical tests were applied in the same way as in F. Two-tailed unpaired Student's t-test: () P < 0.05, (**) P < 0.005. The bars without an asterisk do not show statistically significant differences. (H) “PRD-RE” (spanning Δ8 and a part of Δ9 regions) was inserted upstream (US) of or downstream (DS) from the exonic construct. (I) Measured PRD dependency of insertion mutants. Normalization and statistical tests were done in the same way as in G.
	Figure 7. Evolutionary implications of the DROSHA PRD. (A) Comparison of the N-terminal part of Drosha homologs across species. The N-terminal part was defined by taking the upstream 391st amino acid of human DROSHA and the corresponding N-terminal parts from the homologs in other species. The disorderliness was predicted by SPOT-disorder-single (Hanson et al. 2018). UniProt protein IDs are listed in Supplemental Table S4. (NTD) N-terminal domain. (B, top) Hybrid DROSHA proteins with the N-terminal part from other species and the C-terminal part from human DROSHA. (Bottom) The exonic or intronic construct containing pri-mir-627 was transfected into HCT116 DROSHA KO cells together with one of the hybrid proteins. FLm was used for the H. sapiens NTD. miRNA levels were normalized to U6 snRNA and the Renilla luciferase transcript. The miRNA levels in the p140m-expressing condition were set to 1. The error bars indicate mean ± standard deviations (n = 3). Two-tailed unpaired Student's t-test: () P < 0.005, (n.s.) not significant (P = 0.05). (C, top) The P > A mutant in which all prolines within the first 1–200 amino acids were replaced with alanines was cloned. miRNA expression tests were done as in B. (D) A model for the function of the DROSHA PRD in pri-miRNA processing. The PRD may interact with the intronic sequences directly or indirectly via an intron-binding protein(s), thereby assisting the Microprocessor to access the intronic miRNA hairpins. (E) Conservation analysis of DROSHA-dependent canonical miRNAs. Conservation was quantified as the average of phyloP scores using the pre-miRNA sequence. Different miRNA groups were compared using the two-tailed Mann–Whitney U test: (*) P < 0.0005, (n.s.) not significant.

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