mRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation

Nainita Roy; Swetha Sundar; Malini Pillai; Farah Patell-Socha; Sravya Ganesh; Ajoy Aloysius; Mohammed Rumman; Hardik Gala; Simon M Hughes; Peter S Zammit; Jyotsna Dhawan

doi:10.1186/s13395-021-00270-9

mRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation

Nainita Roy, Swetha Sundar, Malini Pillai, Farah Patell-Socha, Sravya Ganesh, Ajoy Aloysius, Mohammed Rumman, Hardik Gala, Simon M Hughes, Peter S Zammit, Jyotsna Dhawan

Research output: Contribution to journal › Article › peer-review

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Abstract

BACKGROUND: During skeletal muscle regeneration, satellite stem cells use distinct pathways to repair damaged myofibers or to self-renew by returning to quiescence. Cellular/mitotic quiescence employs mechanisms that promote a poised or primed state, including altered RNA turnover and translational repression. Here, we investigate the role of mRNP granule proteins Fragile X Mental Retardation Protein (Fmrp) and Decapping protein 1a (Dcp1a) in muscle stem cell quiescence and differentiation.

METHODS: Using isolated single muscle fibers from adult mice, we established differential enrichment of mRNP granule proteins including Fmrp and Dcp1a in muscle stem cells vs. myofibers. We investigated muscle tissue homeostasis in adult Fmr1-/- mice, analyzing myofiber cross-sectional area in vivo and satellite cell proliferation ex vivo. We explored the molecular mechanisms of Dcp1a and Fmrp function in quiescence, proliferation and differentiation in a C2C12 culture model. Here, we used polysome profiling, imaging and RNA/protein expression analysis to establish the abundance and assembly status of mRNP granule proteins in different cellular states, and the phenotype of knockdown cells.

RESULTS: Quiescent muscle satellite cells are enriched for puncta containing the translational repressor Fmrp, but not the mRNA decay factor Dcp1a. MuSC isolated from Fmr1-/- mice exhibit defective proliferation, and mature myofibers show reduced cross-sectional area, suggesting a role for Fmrp in muscle homeostasis. Expression and organization of Fmrp and Dcp1a varies during primary MuSC activation on myofibers, with Fmrp puncta prominent in quiescence, but Dcp1a puncta appearing during activation/proliferation. This reciprocal expression of Fmrp and Dcp1a puncta is recapitulated in a C2C12 culture model of quiescence and activation: consistent with its role as a translational repressor, Fmrp is enriched in non-translating mRNP complexes abundant in quiescent myoblasts; Dcp1a puncta are lost in quiescence, suggesting stabilized and repressed transcripts. The function of each protein differs during proliferation; whereas Fmrp knockdown led to decreased proliferation and lower cyclin expression, Dcp1a knockdown led to increased cell proliferation and higher cyclin expression. However, knockdown of either Fmrp or Dcp1a led to compromised differentiation. We also observed cross-regulation of decay versus storage mRNP granules; knockdown of Fmrp enhances accumulation of Dcp1a puncta, whereas knockdown of Dcp1a leads to increased Fmrp in puncta.

CONCLUSIONS: Taken together, our results provide evidence that the balance of mRNA turnover versus utilization is specific for distinct cellular states.

Original language	English
Article number	18
Pages (from-to)	18
Journal	Skeletal Muscle
Volume	11
Issue number	1
DOIs	https://doi.org/10.1186/s13395-021-00270-9
Publication status	Published - 8 Jul 2021

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10.1186/s13395-021-00270-9

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Roy, N., Sundar, S., Pillai, M., Patell-Socha, F., Ganesh, S., Aloysius, A., Rumman, M., Gala, H., Hughes, S. M., Zammit, P. S., & Dhawan, J. (2021). mRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation. Skeletal Muscle, 11(1), 18. Article 18. https://doi.org/10.1186/s13395-021-00270-9

@article{1f4078c1029e4e0190e9ae2464f0d6a2,

title = "mRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation",

abstract = "BACKGROUND: During skeletal muscle regeneration, satellite stem cells use distinct pathways to repair damaged myofibers or to self-renew by returning to quiescence. Cellular/mitotic quiescence employs mechanisms that promote a poised or primed state, including altered RNA turnover and translational repression. Here, we investigate the role of mRNP granule proteins Fragile X Mental Retardation Protein (Fmrp) and Decapping protein 1a (Dcp1a) in muscle stem cell quiescence and differentiation.METHODS: Using isolated single muscle fibers from adult mice, we established differential enrichment of mRNP granule proteins including Fmrp and Dcp1a in muscle stem cells vs. myofibers. We investigated muscle tissue homeostasis in adult Fmr1-/- mice, analyzing myofiber cross-sectional area in vivo and satellite cell proliferation ex vivo. We explored the molecular mechanisms of Dcp1a and Fmrp function in quiescence, proliferation and differentiation in a C2C12 culture model. Here, we used polysome profiling, imaging and RNA/protein expression analysis to establish the abundance and assembly status of mRNP granule proteins in different cellular states, and the phenotype of knockdown cells.RESULTS: Quiescent muscle satellite cells are enriched for puncta containing the translational repressor Fmrp, but not the mRNA decay factor Dcp1a. MuSC isolated from Fmr1-/- mice exhibit defective proliferation, and mature myofibers show reduced cross-sectional area, suggesting a role for Fmrp in muscle homeostasis. Expression and organization of Fmrp and Dcp1a varies during primary MuSC activation on myofibers, with Fmrp puncta prominent in quiescence, but Dcp1a puncta appearing during activation/proliferation. This reciprocal expression of Fmrp and Dcp1a puncta is recapitulated in a C2C12 culture model of quiescence and activation: consistent with its role as a translational repressor, Fmrp is enriched in non-translating mRNP complexes abundant in quiescent myoblasts; Dcp1a puncta are lost in quiescence, suggesting stabilized and repressed transcripts. The function of each protein differs during proliferation; whereas Fmrp knockdown led to decreased proliferation and lower cyclin expression, Dcp1a knockdown led to increased cell proliferation and higher cyclin expression. However, knockdown of either Fmrp or Dcp1a led to compromised differentiation. We also observed cross-regulation of decay versus storage mRNP granules; knockdown of Fmrp enhances accumulation of Dcp1a puncta, whereas knockdown of Dcp1a leads to increased Fmrp in puncta.CONCLUSIONS: Taken together, our results provide evidence that the balance of mRNA turnover versus utilization is specific for distinct cellular states.",

author = "Nainita Roy and Swetha Sundar and Malini Pillai and Farah Patell-Socha and Sravya Ganesh and Ajoy Aloysius and Mohammed Rumman and Hardik Gala and Hughes, {Simon M} and Zammit, {Peter S} and Jyotsna Dhawan",

note = "Funding Information: We thank Debarya Saha (CCMB) for help with global transcript analysis and Louise Moyle (KCL) for help with single myofibers. We gratefully acknowledge Colin Crist (McGill Univ.) for detailed discussions and sharing data before publication, U Varshney (IISc), R Muddashetty and Vishal Tiwari (InStem) for help with polysome profiling and S Chattarji (NCBS) for access to Fmr1 knockout mouse tissue used in this study. We thank J Joseph (NCCS) for Dcp1a antibody. We are grateful for access to flow cytometry and imaging facilities (CIFF at NCBS-InStem and AIF at CCMB), as well as use of the National Mouse Resource animal facility at NCBS-InStem and the Laboratory Animal Facility at CCMB. Funding Information: This work was supported by postdoctoral fellowships from the Govt. of India Department of Biotechnology (DBT) to NR, FPS and MP, graduate fellowships from the Council of Scientific and Industrial Research to MR, HG, and SS, and from the Tata Institute of Fundamental Research to AA, and grants from the Indo-Australia Biotechnology Fund (DBT) and the Indo-Danish Strategic Research Fund (DBT) to JD. Animal experiments were carried out at the National Mouse Resource in NCBS-InStem (partially funded by DBT grant BT/PR5981/MED/31/181/2012). SMH is a Medical Research Council Scientist with Programme Grant (G1001029 and MR/N021231/1) support. The lab of P.S.Z. is supported in this work by grants from the Medical Research Council (MR/P023215/1 and MR/S002472/1). The collaborative work was supported by a BBSRC Partnership Award to King{\textquoteright}s College London and InStem. Publisher Copyright: {\textcopyright} 2021, The Author(s). Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",

year = "2021",

month = jul,

day = "8",

doi = "10.1186/s13395-021-00270-9",

language = "English",

volume = "11",

pages = "18",

journal = "Skeletal Muscle",

issn = "2044-5040",

publisher = "BioMed Central",

number = "1",

}

TY - JOUR

T1 - mRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation

AU - Roy, Nainita

AU - Sundar, Swetha

AU - Pillai, Malini

AU - Patell-Socha, Farah

AU - Ganesh, Sravya

AU - Aloysius, Ajoy

AU - Rumman, Mohammed

AU - Gala, Hardik

AU - Hughes, Simon M

AU - Zammit, Peter S

AU - Dhawan, Jyotsna

N1 - Funding Information: We thank Debarya Saha (CCMB) for help with global transcript analysis and Louise Moyle (KCL) for help with single myofibers. We gratefully acknowledge Colin Crist (McGill Univ.) for detailed discussions and sharing data before publication, U Varshney (IISc), R Muddashetty and Vishal Tiwari (InStem) for help with polysome profiling and S Chattarji (NCBS) for access to Fmr1 knockout mouse tissue used in this study. We thank J Joseph (NCCS) for Dcp1a antibody. We are grateful for access to flow cytometry and imaging facilities (CIFF at NCBS-InStem and AIF at CCMB), as well as use of the National Mouse Resource animal facility at NCBS-InStem and the Laboratory Animal Facility at CCMB. Funding Information: This work was supported by postdoctoral fellowships from the Govt. of India Department of Biotechnology (DBT) to NR, FPS and MP, graduate fellowships from the Council of Scientific and Industrial Research to MR, HG, and SS, and from the Tata Institute of Fundamental Research to AA, and grants from the Indo-Australia Biotechnology Fund (DBT) and the Indo-Danish Strategic Research Fund (DBT) to JD. Animal experiments were carried out at the National Mouse Resource in NCBS-InStem (partially funded by DBT grant BT/PR5981/MED/31/181/2012). SMH is a Medical Research Council Scientist with Programme Grant (G1001029 and MR/N021231/1) support. The lab of P.S.Z. is supported in this work by grants from the Medical Research Council (MR/P023215/1 and MR/S002472/1). The collaborative work was supported by a BBSRC Partnership Award to King’s College London and InStem. Publisher Copyright: © 2021, The Author(s). Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2021/7/8

Y1 - 2021/7/8

N2 - BACKGROUND: During skeletal muscle regeneration, satellite stem cells use distinct pathways to repair damaged myofibers or to self-renew by returning to quiescence. Cellular/mitotic quiescence employs mechanisms that promote a poised or primed state, including altered RNA turnover and translational repression. Here, we investigate the role of mRNP granule proteins Fragile X Mental Retardation Protein (Fmrp) and Decapping protein 1a (Dcp1a) in muscle stem cell quiescence and differentiation.METHODS: Using isolated single muscle fibers from adult mice, we established differential enrichment of mRNP granule proteins including Fmrp and Dcp1a in muscle stem cells vs. myofibers. We investigated muscle tissue homeostasis in adult Fmr1-/- mice, analyzing myofiber cross-sectional area in vivo and satellite cell proliferation ex vivo. We explored the molecular mechanisms of Dcp1a and Fmrp function in quiescence, proliferation and differentiation in a C2C12 culture model. Here, we used polysome profiling, imaging and RNA/protein expression analysis to establish the abundance and assembly status of mRNP granule proteins in different cellular states, and the phenotype of knockdown cells.RESULTS: Quiescent muscle satellite cells are enriched for puncta containing the translational repressor Fmrp, but not the mRNA decay factor Dcp1a. MuSC isolated from Fmr1-/- mice exhibit defective proliferation, and mature myofibers show reduced cross-sectional area, suggesting a role for Fmrp in muscle homeostasis. Expression and organization of Fmrp and Dcp1a varies during primary MuSC activation on myofibers, with Fmrp puncta prominent in quiescence, but Dcp1a puncta appearing during activation/proliferation. This reciprocal expression of Fmrp and Dcp1a puncta is recapitulated in a C2C12 culture model of quiescence and activation: consistent with its role as a translational repressor, Fmrp is enriched in non-translating mRNP complexes abundant in quiescent myoblasts; Dcp1a puncta are lost in quiescence, suggesting stabilized and repressed transcripts. The function of each protein differs during proliferation; whereas Fmrp knockdown led to decreased proliferation and lower cyclin expression, Dcp1a knockdown led to increased cell proliferation and higher cyclin expression. However, knockdown of either Fmrp or Dcp1a led to compromised differentiation. We also observed cross-regulation of decay versus storage mRNP granules; knockdown of Fmrp enhances accumulation of Dcp1a puncta, whereas knockdown of Dcp1a leads to increased Fmrp in puncta.CONCLUSIONS: Taken together, our results provide evidence that the balance of mRNA turnover versus utilization is specific for distinct cellular states.

AB - BACKGROUND: During skeletal muscle regeneration, satellite stem cells use distinct pathways to repair damaged myofibers or to self-renew by returning to quiescence. Cellular/mitotic quiescence employs mechanisms that promote a poised or primed state, including altered RNA turnover and translational repression. Here, we investigate the role of mRNP granule proteins Fragile X Mental Retardation Protein (Fmrp) and Decapping protein 1a (Dcp1a) in muscle stem cell quiescence and differentiation.METHODS: Using isolated single muscle fibers from adult mice, we established differential enrichment of mRNP granule proteins including Fmrp and Dcp1a in muscle stem cells vs. myofibers. We investigated muscle tissue homeostasis in adult Fmr1-/- mice, analyzing myofiber cross-sectional area in vivo and satellite cell proliferation ex vivo. We explored the molecular mechanisms of Dcp1a and Fmrp function in quiescence, proliferation and differentiation in a C2C12 culture model. Here, we used polysome profiling, imaging and RNA/protein expression analysis to establish the abundance and assembly status of mRNP granule proteins in different cellular states, and the phenotype of knockdown cells.RESULTS: Quiescent muscle satellite cells are enriched for puncta containing the translational repressor Fmrp, but not the mRNA decay factor Dcp1a. MuSC isolated from Fmr1-/- mice exhibit defective proliferation, and mature myofibers show reduced cross-sectional area, suggesting a role for Fmrp in muscle homeostasis. Expression and organization of Fmrp and Dcp1a varies during primary MuSC activation on myofibers, with Fmrp puncta prominent in quiescence, but Dcp1a puncta appearing during activation/proliferation. This reciprocal expression of Fmrp and Dcp1a puncta is recapitulated in a C2C12 culture model of quiescence and activation: consistent with its role as a translational repressor, Fmrp is enriched in non-translating mRNP complexes abundant in quiescent myoblasts; Dcp1a puncta are lost in quiescence, suggesting stabilized and repressed transcripts. The function of each protein differs during proliferation; whereas Fmrp knockdown led to decreased proliferation and lower cyclin expression, Dcp1a knockdown led to increased cell proliferation and higher cyclin expression. However, knockdown of either Fmrp or Dcp1a led to compromised differentiation. We also observed cross-regulation of decay versus storage mRNP granules; knockdown of Fmrp enhances accumulation of Dcp1a puncta, whereas knockdown of Dcp1a leads to increased Fmrp in puncta.CONCLUSIONS: Taken together, our results provide evidence that the balance of mRNA turnover versus utilization is specific for distinct cellular states.

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U2 - 10.1186/s13395-021-00270-9

DO - 10.1186/s13395-021-00270-9

M3 - Article

C2 - 34238354

SN - 2044-5040

VL - 11

SP - 18

JO - Skeletal Muscle

JF - Skeletal Muscle

IS - 1

M1 - 18

ER -

mRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation

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