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

Gene expression in a tissue-specific context depends on the combined efforts of epigenetic, transcriptional and post-transcriptional processes that lead to the production of specific proteins that are important determinants of cellular identity. Ribosomes are a central component of the protein biosynthesis machinery in cells; however, their regulatory roles in the translational control of gene expression in skeletal muscle remain to be defined. In a genetic screen to identify critical regulators of myogenesis, we identified a DEAD-Box RNA helicase, DDX27, that is required for skeletal muscle growth and regeneration. We demonstrate that DDX27 regulates ribosomal RNA (rRNA) maturation, and thereby the ribosome biogenesis and the translation of specific transcripts during myogenesis. These findings provide insight into the translational regulation of gene expression in myogenesis and suggest novel functions for ribosomes in regulating gene expression in skeletal muscles

Skeletal muscle abnormalities in <i>ddx27</i> mutant zebrafish.

<p>(A) Microscopic visualization of control and mutant larval zebrafish (<i>osoi</i>) 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 <i>osoi</i> 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 <i>DDX27</i> mRNA results in a significant decrease in mutant zebrafish phenotype (D) Whole-mount Immunofluorescence was performed on control and <i>ddx27</i> 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).</p

Skeletal muscle hypotrophy and precocious differentiation in Ddx27 deficiency.

<p>(A-B) Histology of longitudinal skeletal muscle sections in control and <i>ddx27</i> 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 <i>ddx27</i> mutant (5 dpf). Arrows indicating disorganized sarcomere (H) Quantification of myofiber size in control and <i>ddx27</i> mutant fish (5 dpf) (n = 10) (I) qRT-PCR of control and <i>ddx27</i> mutant fish showed a reduction in the expression of muscle stem cell markers (<i>pax3</i>, <i>pax7</i>) and an increase in expression of myogenic commitment genes (<i>myod1</i> and <i>myf5</i>). The expression of late differentiation markers was reduced in <i>ddx27</i> fish suggesting that pre-mature expression of early myogenic genes results in abnormal disorganization of skeletal muscles.</p

Disruption in nucleolar architecture, rRNA synthesis and ribosomes in Ddx27 deficiency.

<p>(A) Immunofluorescence of control and <i>ddx27</i> 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 <i>ddx27</i> 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 <i>ddx27</i> mutant larvae (5 dpf).</p

Decrease in muscle precursor cells (MPC) proliferation and skeletal muscle regeneration in Ddx27 deficiency.

<p>(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 <i>ddx27</i> mutant during post-embryonic skeletal muscle growth (4 dpf) (scale bar: 50μm) (B) Control and <i>ddx27</i> 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 <i>ddx27</i> 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 <i>ddx27</i> muscles (arrow) (scale bar: 50μm).</p

Decreased contractile force and prolonged muscle relaxation in Ddx27-deficient skeletal muscle.

<p>(A) Representative twitch (left) and tetanic force (right) records from control and <i>ddx27</i> zebrafish (5 dpf) preparation (B) Peak tetanic force (C) Peak tetanic force normalized to preparation cross-sectional area. Tetanic force is significantly reduced in <i>ddx27</i> mutant fish (D) Peak twitch force (E) Peak twitch force normalized to preparation cross-sectional area. Twitch force is significantly reduced in <i>ddx27</i> 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 <i>ddx27</i> (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.</p

Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation

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