PABPN1-Dependent mRNA Processing Induces Muscle Wasting

Riaz, Muhammad Bilal; Raz, Yotam; Putten, Maaike van; Soriano, Guillem Paniagua; Krom, Yvonne D.; Florea, Bogdan I.; Raz, Vered

doi:10.1371/journal.pgen.1006031

Cited by 43 publications

(115 citation statements)

References 44 publications

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“…In HeLa cells, all three β‐subunits show similar signal intensity,17 but in the muscle cell culture, we found that the β5 signal is two‐fold to four‐fold higher than β2 and β1, respectively. We recently reported that in the C2C12 cell culture, β5 protein levels are lower than β2;14 this indicates that β5 activity in C2C12 is higher than β2 activity. Also, in mouse thymus, the β5 signal is higher compared with the other subunits 18.…”

Section: Resultsmentioning

confidence: 92%

“…shUsp14 cell line was generated by transfection of shRNA clone TRCN0000007428 (Sigma‐Aldrich, MO, USA) into C2C12 using Lipofectamin 2000 (Invitrogen) according to the manufacturer manual. Scramble shRNA line was previously described 14. Stable culture was generated by puromycin selection.…”

Section: Methodsmentioning

confidence: 99%

“…High‐throughput imaging was carried out with ArrayScan VTI HCA, Cellomics (Thermo Scientific), in cells seeds in a 96‐well plate, images were taken directly after washings. Image analysis was carried out with the Cellomics (Thermofisher) compartmental bio‐application as described in Riaz et al 14. In brief, the nucleus was defined as a reference, as detected by Hoechst staining.…”

Section: Methodsmentioning

confidence: 99%

“…The immunofluorescence procedure was carried out as detailed in Riaz et al 14, 25. Images were captured with the DM5500 microscope (Leica) using the LAS AF software version 2.3.6.…”

Section: Methodsmentioning

confidence: 99%

“…Decreased proteasome activity in human muscles is age‐associated,12 whereas in rats, proteasome activity in muscles increases with age 13. We reported that proteasome activity is reduced in a mouse model for muscle ageing 14. Despite the prominent role of the proteasome in maintaining protein homeostasis in muscles, the changes in proteasomal activity with age are not fully understood.…”

Section: Introductionmentioning

confidence: 99%

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Proteasomal activity‐based probes mark protein homeostasis in muscles

Raz¹,

Raz²,

Soriano

et al. 2017

J cachexia sarcopenia muscle

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BackgroundProtein homeostasis, primarily regulated by the ubiquitin–proteasome system is crucial for proper function of cells. In tissues of post‐mitotic cells, the impaired ubiquitin–proteasome system is found in a wide range of neuromuscular disorders. Activity‐based probes (ABPs) measure proteasomal proteolytic subunits and can be used to report protein homeostasis. Despite the crucial role of the proteasome in neuromuscular pathologies, ABPs were not employed in muscle cells and tissues, and measurement of proteasomal activity was carried out in vitro using low‐throughput procedures.MethodsWe screened six ABPs for specific application in muscle cell culture using high throughput call‐based imaging procedures. We then determined an in situ proteasomal activity in myofibers of muscle cryosections.ResultsWe demonstrate that LWA300, a pan‐reactive proteasomal probe, is most suitable to report proteasomal activity in muscle cells using cell‐based bio‐imaging. We found that proteasomal activity is two‐fold and three‐fold enhanced in fused muscle cell culture compared with non‐fused cells. Moreover, we found that proteasomal activity can discriminate between muscles. Across muscles, a relative higher proteasomal activity was found in hybrid myofibers whereas fast‐twitch myofibers displayed lower activity.ConclusionsOur study demonstrates that proteasomal activity differ between muscles and between myofiber types. We suggest that ABPs can be used to report disease progression and treatment efficacy.

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Section: Resultsmentioning

confidence: 92%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The immunofluorescence procedure was carried out as detailed in Riaz et al 14, 25. Images were captured with the DM5500 microscope (Leica) using the LAS AF software version 2.3.6.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Proteasomal activity‐based probes mark protein homeostasis in muscles

Raz¹,

Raz²,

Soriano

et al. 2017

J cachexia sarcopenia muscle

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Discovering fiber type architecture over the entire muscle using data‐driven analysis

et al. 2021

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Skeletal muscle function is inferred from the spatial arrangement of muscle fiber architecture, which corresponds to myofiber molecular and metabolic features.Myofiber features are often determined using immunofluorescence on a local sampling, typically obtained from a median region. This median region is assumed to represent the entire muscle. However, it remains largely unknown to what extent this local sampling represents the entire muscle. We present a pipeline to study the architecture of muscle fiber features over the entire muscle, including sectioning, staining, imaging to image quantification and data-driven analysis with Myofiber type were identified by the expression of myosin heavy chain (MyHC) isoforms, representing contraction properties. We reconstructed muscle architecture from consecutive cross-sections stained for laminin and MyHC isoforms. Examining the entire muscle using consecutive cross-sections is extremely laborious, we provide consideration to reduce the dataset without loosing spatial information. Data-driven analysis with over 150,000 myofibers showed spatial variations in myofiber geometric features, myofiber type, and the distribution of neuromuscular junctions over the entire muscle. We present a workflow to study histological changes over the entire muscle using high-throughput imaging, image quantification, and data-driven analysis. Our results suggest that asymmetric spatial distribution of these features over the entire muscle could impact muscle function. Therefore, instead of a single sampling from a median region, representative regions covering the entire muscle should be investigated in future studies. K E Y W O R D S data-driven analysis, muscle architecture, myofiber type, quantitative image analysis 1 | INTRODUCTION Skeletal muscles facilitate the mobilization and stability of the skeleton, which is greatly determined by muscle fiber architecture. Muscle function changes in physiological, pathological conditions, and during development [1, 2]. Muscle fiber spatial arrangement is broadly described by their orientation relative to the axis of force generation, which can be classified into three main classes: 1) fibers arrangement

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Alternative polyadenylation: An enigma of transcript length variation in health and disease

et al. 2021

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Alternative polyadenylation (APA) is a molecular mechanism during a pre‐mRNA processing that involves usage of more than one polyadenylation site (PA‐site) generating transcripts of varying length from a single gene. The location of a PA‐site affects transcript length and coding potential of an mRNA contributing to both mRNA and protein diversification. This variation in the transcript length affects mRNA stability and translation, mRNA subcellular and tissue localization, and protein function. APA is now considered as an important regulatory mechanism in the pathophysiology of human diseases. An important consequence of the changes in the length of 3′‐untranslated region (UTR) from disease‐induced APA is altered protein expression. Yet, the relationship between 3′‐UTR length and protein expression remains a paradox in a majority of diseases. Here, we review occurrence of APA, mechanism of PA‐site selection, and consequences of transcript length variation in different diseases. Emerging evidence reveals coordinated involvement of core RNA processing factors including poly(A) polymerases in the PA‐site selection in diseases‐associated APAs. Targeting such APA regulators will be therapeutically significant in combating drug resistance in cancer and other complex diseases. This article is categorized under: RNA Processing > 3′ End Processing RNA in Disease and Development > RNA in Disease Translation > Regulation

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PABPN1-Dependent mRNA Processing Induces Muscle Wasting

Cited by 43 publications

References 44 publications

Proteasomal activity‐based probes mark protein homeostasis in muscles

Proteasomal activity‐based probes mark protein homeostasis in muscles

Discovering fiber type architecture over the entire muscle using data‐driven analysis

Alternative polyadenylation: An enigma of transcript length variation in health and disease

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