Journal: Molecular & Cellular Proteomics : MCP

Article Title: Characterization of R-Loop–Interacting Proteins in Embryonic Stem Cells Reveals Roles in rRNA Processing and Gene Expression

doi: 10.1016/j.mcpro.2021.100142

Figure Lengend Snippet: Many R-loop–binding proteins are enriched within the nucleolus. A , immunofluorescence staining of three R-loop–associated DEAD-box proteins, DDX18, DDX24, and DDX27, costained with FBRL to mark the nucleolus. DNA was stained by DAPI. The scale bar represents 10 μm. B , comparison of rRNA-related and rRNA-unrelated proteins enriched by S9.6. R-loop–associated proteins were classified as rRNA related or unrelated by GO term analysis, using the biological process term “ribosomebiogensis,” “rRNAmetabolicprocess,” and “rRNAprocessing.” Boxes of the box plot represent the first and third quartiles, the band represents the median, and the whiskers depict 1.5 times the interquartile range. C , Venn diagram of the overlap between the R-loop interactomes (with and without filtering) and the nucleolar proteome from Kar et al. . The upper diagram includes the 335 proteins enriched in S9.6 co-IP samples with the 76 most enriched (corresponding to the orange dots in Fig. 1 B ) depicted in the lower diagram . p Values were calculated using hypergeometric tests. DAPI, 4′,6-diamidino-2-phenylindole; FBRL, fibrillarin; GO, Gene Ontology; IP, immunoprecipitation.

Article Snippet: Antibodies used included DDX18 (Bethyl Laboratories; A300-535A), DDX24 (Abcam; ab70463), DDX27 (Bethyl Laboratories; A302-216A), DDX54 (MilliporeSigma; AV36498-100UL), DHX9 (Abcam; ab26271), RPB1 (Santa Cruz Biotechnology; sc-899x), fibrillarin (FBRL; Novus Biologicals; NB300-269), CEBPZ (Proteintech; 25612-1-AP), SFPQ (Abcam; ab38148), CTCF (MilliporeSigma; 07-729), and mouse IgG2a (Abcam; ab18413).

Techniques: Binding Assay, Immunofluorescence, Staining, Comparison, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: PLoS Genetics

Article Title: RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes

doi: 10.1371/journal.pgen.1007226

Figure Lengend Snippet: (A) Microscopic visualization of control and mutant larval zebrafish ( osoi ) at 5 days post fertilization (dpf). Mutant fish display leaner muscles (left panel) and exhibit highly reduced birefringence in comparison to control (right panel). Mutant fish also exhibit pericardial edema (arrow) (B) Genetic mapping of osoi mutant by initial bulk segregant analysis identified linkage on chromosome 6. Fine mapping of chromosome 6 resolved flanking markers z41548 and z14467, with a candidate genome region containing six candidate genes that were sequenced by Sanger sequencing (C) Overexpression of human DDX27 mRNA results in a significant decrease in mutant zebrafish phenotype (D) Whole-mount Immunofluorescence was performed on control and ddx27 mutant larvae (Z-stack confocal image, 4dpf) (scale bar: 50μm) (E) Immunofluorescence on newly isolated (Day 0) and cultured (Day1 and 3) EDL myofibers from wild-type mice (scale bar: 10μm). (F) Western blot showing relative expression of Ddx27 and myogenic markers (MyoD, MyoG and MF20) in proliferating C2C12 myoblasts in growth media (50% confluence) or in differentiation media for 3 days (D0-3). GAPDH was used as the control. (G) Schematic diagram of nucleolus depicting nucleolar domains. Eukaryotic nucleolus has tripartite architecture: Fibrillar center (FC); Dense fibrillar component (DFC) and granular compartment (GC). Immunofluorescence of human myoblasts with DDX27 and nucleolar markers labeling each compartment of nucleolus (scale bar: 2μm).

Article Snippet: Primary antibodies used in this study were: α-actinin (1:100; Sigma, A7811), RYR1 (1:100; Sigma, R-129), Fibrillarin (1:50; Santa Cruz, sc-25397), B23 (1:50; Santa Cruz, sc-5564), UBF (Sigma, 1:50, HPA006385), DDX27 (1:50, Santa Cruz, sc-81074), Pax7 (1:20; Developmental Studies Hybridoma Bank), Mef2 (1:20, Santa Cruz, sc-17785).

Techniques: Control, Mutagenesis, Muscles, Comparison, Sequencing, Over Expression, Immunofluorescence, Isolation, Cell Culture, Western Blot, Expressing, Labeling

Journal: PLoS Genetics

Article Title: RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes

doi: 10.1371/journal.pgen.1007226

Figure Lengend Snippet: (A-B) Histology of longitudinal skeletal muscle sections in control and ddx27 mutant stained with toluidine blue exhibiting enlarged nucleoli (arrowhead) and areas lacking sarcomeres (arrow) at 5 dpf. High magnification view (boxed area) (C-G) Transmission electron micrographs of skeletal muscles (longitudinal view: C-E, and cross-section view: F-G) in control and ddx27 mutant (5 dpf). Arrows indicating disorganized sarcomere (H) Quantification of myofiber size in control and ddx27 mutant fish (5 dpf) (n = 10) (I) qRT-PCR of control and ddx27 mutant fish showed a reduction in the expression of muscle stem cell markers ( pax3 , pax7 ) and an increase in expression of myogenic commitment genes ( myod1 and myf5 ). The expression of late differentiation markers was reduced in ddx27 fish suggesting that pre-mature expression of early myogenic genes results in abnormal disorganization of skeletal muscles.

Article Snippet: Primary antibodies used in this study were: α-actinin (1:100; Sigma, A7811), RYR1 (1:100; Sigma, R-129), Fibrillarin (1:50; Santa Cruz, sc-25397), B23 (1:50; Santa Cruz, sc-5564), UBF (Sigma, 1:50, HPA006385), DDX27 (1:50, Santa Cruz, sc-81074), Pax7 (1:20; Developmental Studies Hybridoma Bank), Mef2 (1:20, Santa Cruz, sc-17785).

Techniques: Control, Mutagenesis, Staining, Transmission Assay, Muscles, Quantitative RT-PCR, Expressing

Journal: PLoS Genetics

Article Title: RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes

doi: 10.1371/journal.pgen.1007226

Figure Lengend Snippet: (A) Representative twitch (left) and tetanic force (right) records from control and ddx27 zebrafish (5 dpf) preparation (B) Peak tetanic force (C) Peak tetanic force normalized to preparation cross-sectional area. Tetanic force is significantly reduced in ddx27 mutant fish (D) Peak twitch force (E) Peak twitch force normalized to preparation cross-sectional area. Twitch force is significantly reduced in ddx27 mutant fish (F) Maximal rate of twitch tension development. (G) Maximum rate of twitch force relaxation. (H) Twitch to tetanic force ratio. Each symbol represents an individual control (n = 10) or ddx27 (n = 12) preparation with the group mean indicated by a horizontal line. t-tests indicated significant differences between control and mutant means for all 7 variables (P < 0.001 to P < 0.0001). Abbreviations: CSA, cross-sectional area; +dP/dt, maximal rate of tension development; -dP/dt, maximal rate of tension relaxation; Pt, peak twitch force; Po, peak tetanic force.

Article Snippet: Primary antibodies used in this study were: α-actinin (1:100; Sigma, A7811), RYR1 (1:100; Sigma, R-129), Fibrillarin (1:50; Santa Cruz, sc-25397), B23 (1:50; Santa Cruz, sc-5564), UBF (Sigma, 1:50, HPA006385), DDX27 (1:50, Santa Cruz, sc-81074), Pax7 (1:20; Developmental Studies Hybridoma Bank), Mef2 (1:20, Santa Cruz, sc-17785).

Techniques: Control, Mutagenesis

Journal: PLoS Genetics

Article Title: RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes

doi: 10.1371/journal.pgen.1007226

Figure Lengend Snippet: (A) Whole mount immunofluorescence of zebrafish at different time intervals (2 dpf and 4 dpf) with MPC marker (Pax7) and late differentiation marker (Mef2) demonstrating a decrease in of MPC in ddx27 mutant during post-embryonic skeletal muscle growth (4 dpf) (scale bar: 50μm) (B) Control and ddx27 mutant zebrafish were pulse-labeled with EdU for 2hr and immunostained with Pax7. Fish were analyzed for EdU and Pax7 labeling (4 dpf) by whole mount immunofluorescence. The proportion of proliferative Pax7 population was estimated by quantifying Pax7 + /Edu + double-positive nuclei out of total Pax7 + nuclei in control and mutant fish (scale bar: 50μm) (C) Trunk muscles in control and ddx27 zebrafish were injected with cardiotoxin (3 dpf). Skeletal muscles were analyzed at 5 dpf by whole mount immunofluorescence with Pax7 and phalloidin. Control muscles show an accumulation of Pax7 expressing cells at the site of injury (arrow) that was lacking in ddx27 muscles (arrow) (scale bar: 50μm).

Article Snippet: Primary antibodies used in this study were: α-actinin (1:100; Sigma, A7811), RYR1 (1:100; Sigma, R-129), Fibrillarin (1:50; Santa Cruz, sc-25397), B23 (1:50; Santa Cruz, sc-5564), UBF (Sigma, 1:50, HPA006385), DDX27 (1:50, Santa Cruz, sc-81074), Pax7 (1:20; Developmental Studies Hybridoma Bank), Mef2 (1:20, Santa Cruz, sc-17785).

Techniques: Immunofluorescence, Marker, Mutagenesis, Control, Labeling, Muscles, Injection, Expressing

Journal: PLoS Genetics

Article Title: RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes

doi: 10.1371/journal.pgen.1007226

Figure Lengend Snippet: (A) Immunofluorescence of control and ddx27 mutant fish with antibodies labeling different nucleolar compartments at 5 dpf (scale bar: 10μm) (B) rRNA transcription was measured in MPCs (labeled with Pax7) in myotome (labeled with myosin) or myonuclei (labeled with Actn2/3) at 5 dpf by quantifying the incorporation of 5-ethynl uridine (5-EU). Zebrafish or myofibers were treated with Actinomycin D for two hours to block background transcription and subsequently, were incubated with or without Actinomycin D and freshly synthesized rRNA was quantified by incorporation of 5-EU by fluorescent detection. Representative single Z-section images are shown. (scale bar: 5μm) (C) Northern blot analysis of total RNAs extracted from skeletal muscles of control and mutant ddx27 zebrafish larvae (5 dpf). 5’ETS, 5’ITS1 and ITS2 probes were used to identify pre-rRNA and intermediate species targeted different steps of the processing pathways. The pre-rRNA intermediates are described in zebrafish. The corresponding human precursors are indicated into brackets. (D) Quantification of the pre-rRNA intermediates in zebrafish skeletal muscles. (E) Polysomal profiles of skeletal muscle in control and ddx27 mutant larvae (5 dpf).

Article Snippet: Primary antibodies used in this study were: α-actinin (1:100; Sigma, A7811), RYR1 (1:100; Sigma, R-129), Fibrillarin (1:50; Santa Cruz, sc-25397), B23 (1:50; Santa Cruz, sc-5564), UBF (Sigma, 1:50, HPA006385), DDX27 (1:50, Santa Cruz, sc-81074), Pax7 (1:20; Developmental Studies Hybridoma Bank), Mef2 (1:20, Santa Cruz, sc-17785).

Techniques: Immunofluorescence, Control, Mutagenesis, Labeling, Blocking Assay, Incubation, Synthesized, Northern Blot, Muscles

Journal: PLoS Genetics

Article Title: RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes

doi: 10.1371/journal.pgen.1007226

Figure Lengend Snippet: (A) Polysomal profiling and RNA sequencing of total cellular mRNA transcripts and polysomal fractions were performed in control and Ddx27 mutant C2C12 myoblasts. (B) mRNAs transcripts exhibiting expression values of Log 2 Fold change >+1 were considered upregulated and those displaying Log 2 Fold change <+1 were considered downregulated in the polysomal fraction in comparison to total cellular mRNA. Upregulated and downregulated mRNAs populations in control and Ddx27 myoblasts were compared (C) Expression of mRNA transcripts exhibiting maximum differences in control and Ddx27 mutant myoblasts (D) Immunofluorescence and Western blot quantification of HDDC2 expression in control and Ddx27 proliferating myoblasts (scale bar: 100μm).

Article Snippet: Primary antibodies used in this study were: α-actinin (1:100; Sigma, A7811), RYR1 (1:100; Sigma, R-129), Fibrillarin (1:50; Santa Cruz, sc-25397), B23 (1:50; Santa Cruz, sc-5564), UBF (Sigma, 1:50, HPA006385), DDX27 (1:50, Santa Cruz, sc-81074), Pax7 (1:20; Developmental Studies Hybridoma Bank), Mef2 (1:20, Santa Cruz, sc-17785).

Techniques: RNA Sequencing, Control, Mutagenesis, Expressing, Comparison, Immunofluorescence, Western Blot