Transcriptomic Analysis of Single Isolated Myofibers Identifies miR-27a-3p and miR-142-3p as Regulators of Metabolism in Skeletal Muscle

Chemello, Francesco; Grespi, Francesca; Zulian, Alessandra; Cancellara, Pasqua; Hébert-Chatelain, Étienne; Martini, Paolo; Bean, Camilla; Alessio, Enrico; Buson, Lisa; Bazzega, Martina; Armani, Andrea; Sandri, Marco; Ferrazza, Ruggero; Laveder, Paolo; Guella, Graziano; Reggiani, Carlo; Romualdi, Chiara; Bernardi, Paolo; Scorrano, Luca; Cagnin, Stefano; Lanfranchi, Gerolamo

doi:10.1016/j.celrep.2019.02.105

Cited by 60 publications

(66 citation statements)

References 71 publications

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“…We used the SMART-seq based method to identify mature miRNAs expressed in skeletal muscle single fibers evidencing 137 miRNAs. We confirmed the expression of miRNAs detected with single-cell sequencing by using qRT-PCR showing an average correlation of almost 0.8 among the two techniques for 55 tested miRNAs [113]. As we profiled miRNAs and mRNAs from the same myofibers [113], also Xiao and colleagues [218] proposed the Holo-seq method to profile mRNAs and miRNAs from the same sample.…”

Section: Discussionsupporting

confidence: 65%

“…The analysis of lncRNAs requires a deeper mechanistic exploration of their cellular expression, subcellular localization, interacting partners to interpret their function in cell reprogramming and several other biological contexts. We and other researchers demonstrated the importance of using a single-cell approach to evidence the function of specific ncRNAs [113,114,212]. This result is related to the cell-specific expression of ncRNAs not detectable analyzing RNA expression using whole tissues or a heterogeneous cell population.…”

Section: Discussionmentioning

confidence: 91%

“…Several methods have been established to isolate single cells, each with particular benefits and drawbacks. The micropipette isolation method consists in diluting the cells to a very low concentration and seeding them into a tube [113,114] or 96 well plate [115] ( Figure 3A). This technique does not require sophisticated platforms but is laborious and not very efficient.…”

Section: Single-cell Isolationmentioning

confidence: 99%

“…Oligo-dT primers can be used to initiate the cDNA reaction, but they will only anneal on polyadenylated RNAs, such as mRNAs and some lncRNAs. Thus, the analysis of other classes of non-coding RNAs requires specific protocols (e.g., for circularRNAs [129], for total RNA [130,131], or miRNAs [113,132]). There are two main strategies to run the cDNA amplification: The template switching and the in vitro transcription.…”

Section: Library Preparationmentioning

confidence: 99%

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A Single Cell but Many Different Transcripts: A Journey into the World of Long Non-Coding RNAs

Alessio

Bonadio

Buson

et al. 2020

IJMS

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In late 2012 it was evidenced that most of the human genome is transcribed but only a small percentage of the transcripts are translated. This observation supported the importance of non-coding RNAs and it was confirmed in several organisms. The most abundant non-translated transcripts are long non-coding RNAs (lncRNAs). In contrast to protein-coding RNAs, they show a more cell-specific expression. To understand the function of lncRNAs, it is fundamental to investigate in which cells they are preferentially expressed and to detect their subcellular localization. Recent improvements of techniques that localize single RNA molecules in tissues like single-cell RNA sequencing and fluorescence amplification methods have given a considerable boost in the knowledge of the lncRNA functions. In recent years, single-cell transcription variability was associated with non-coding RNA expression, revealing this class of RNAs as important transcripts in the cell lineage specification. The purpose of this review is to collect updated information about lncRNA classification and new findings on their function derived from single-cell analysis. We also retained useful for all researchers to describe the methods available for single-cell analysis and the databases collecting single-cell and lncRNA data. Tables are included to schematize, describe, and compare exposed concepts.Int. J. Mol. Sci. 2020, 21, 302 2 of 32 not achieved by transcription factors (TFs) alone because of their low number (approximately 1500 in mammals [4]) compared to genes to regulate (90% of the genome is transcribed in human [5]). A single TF is estimated to regulate only ten thousand genes according to Chromatin immunoprecipitation and DNA sequencing experiments (ChIp-seq) [6]. Moreover, the involvement of non-coding RNAs in gene expression regulation was evident [7,8]. The evidence that the non-protein coding component of the human genome is actively transcribed and carries out crucial functions limits the importance of coding genes in genome regulation. Non-coding transcripts become one of the stars of modern biology, especially because of their involvement in a wide range of regulatory processes and changed the association of non-coding regions to junk DNA [3].The genome of multicellular eukaryotes is mostly comprised of non-coding DNA (less than 2% of the human genome codes for proteins [9] while 90% of the human genome is transcribed). Non-coding DNA is transcribed into different classes of non-coding RNAs (ncRNAs) that include structural RNAs (rRNAs and tRNAs) and regulatory RNAs [10]. rRNAs and tRNAs are involved in mRNA translation and can be long or short in size. Regulatory RNAs are further divided into three classes: Small, medium, and long non-coding RNAs. Small non-coding RNAs are between 20 and 50 nucleotides long and include microRNAs (miRNAs) that participate in post-transcriptional regulation [11], small interfering RNAs (siRNAs) that are double-stranded RNAs involved in gene silencing through RNA interfering pathway [12], piwi interacting R...

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Section: Discussionsupporting

confidence: 65%

Section: Discussionmentioning

confidence: 91%

Section: Single-cell Isolationmentioning

confidence: 99%

Section: Library Preparationmentioning

confidence: 99%

See 2 more Smart Citations

A Single Cell but Many Different Transcripts: A Journey into the World of Long Non-Coding RNAs

Alessio

Bonadio

Buson

et al. 2020

IJMS

Self Cite

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“…Fast oxidative fibers express type IIa, IIx, or a mixture of those two faster MyHCs. They also are more oxidative than type I fibers, presumably to accommodate the greater ATP demands of types IIa and IIx MyHCs compared to type I (Chemello et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

miR-206 Enforces a Slow Muscle Phenotype

Bjorkman

Guess

Harrison

et al. 2019

Preprint

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11Striated muscle is a highly specialized collection of tissues with contractile properties varying according to 12 functional needs. Although muscle fiber types are established postnatally, lifelong plasticity facilitates stimulus-13 dependent adaptation. Functional adaptation requires molecular adaptation, partially provided by miRNA-14 mediated post-transcriptional regulation. miR-206 is a muscle-specific miRNA enriched in slow muscles. We 15 investigated whether miR-206 drives the slow muscle phenotype or is merely an outcome. We found that miR-16 206 expression increases in both physiologic (including female sex and endurance exercise) and pathologic 17 conditions that promote a slow phenotype. Consistent with that observation, the slow soleus muscle of male miR-18 206 knockout mice displays a faster phenotype than wild-type mice. Moreover, their left ventricles have a faster 19 myosin profile accompanied by male-specific dilation and systolic dysfunction. Thus, miR-206 appears necessary 20 to enforce a slow skeletal and cardiac muscle phenotype and to play a key role in muscle sexual dimorphisms. 21 22 2014). Notably, despite a general sex difference in muscle fiber types, it is not known whether biological sex 72 influences miR-206 expression. Thus, we sought to determine in both sexes whether the miR-206 slow muscle 73 enrichment is an outcome or a driver of the oxidative phenotype. 74 Materials and Methods 75Cloning and mutagenesis: The minimal promoter (minTATA) and MyoG enhancer firefly luciferase reporter gene 76 consructs were previously described (Cheung et al., 2007). We cloned the miR-206 enhancer (GRCm38/mm10 77 chr1: 20,678,678,259) in the same manner as described for MyoG. We generated E-box point mutations 78 (CANNTG à CANNTA) with the QuikChange II site-directed mutagenesis kit (Agilent, 200523, Santa Clara, 79 CA) per the manufacturer's instructions. We verified all clones by Sanger sequencing. MRF expression constructs 80 were a kind gift from Dr. Xuedong Liu (University of Colorado Boulder). Primer sequences are listed in 81 Supplementary Table 1. 82 Cell culture and transfection: We grew C2C12 myoblasts in Growth Medium (GM): high glucose DMEM 83 (Invitrogen, 11960069, Waltham, MA) supplemented with 20% fetal bovine serum, 2 mM L-glutamine, 100 84 U/mL penicillin and 100 μg/mL streptomycin, and 1 mM sodium pyruvate. We differentiated them to myotubes 85 by changing media to Differentiation Medium (DM): high glucose DMEM supplemented with 5% adult horse 86serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, and 1 mM sodium pyruvate. When 87 differentiating, we refreshed DM every day to prevent media acidification. We grew 10T ½ cells in GM but with 88 10% FBS. For luciferase assays, we plated cells in triplicate in 6-well dishes at a density of 50,000 cells/well 89 (C2C12) or 100,000 cells/well (10T ½) 24 hours before transfection. We repeated each experiment at least twice 90 with independent thaws of cells. We transfected with TransIT-LT1 (Mirus Bio, MIR 2305, Madiso...

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MiRNAs as biomarkers of phenotype in neutral lipid storage disease with myopathy

et al. 2019

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Background: Neutral lipid storage disease with myopathy (NLSDM) is a rare lipid metabolism disorder. In this study, we evaluated some circulating miRNAs levels in serum samples and the MRI of three affected siblings.Methods: Three members of one NLSDM family were identified: two brothers and one sister. Muscles of lower and right upper extremities were studied by MRI.Expression profile of miRNAs, obtained from serum samples, was detected using qRT-PCR.Results: Two brothers presented with progressive skeletal myopathy, while the sister had severe hepatosteatosis and diabetes. NLSDM patients showed a significant increase of muscle-specific miRNAs expression compared with healthy subjects. We found a correlation between hepatic damage and elevation of miRNAs expression profile of liver origin. Conclusions:The dysregulation of miRNAs might represent an indicator of skeletal and hepatic damage and it might be useful to monitor the progression of NLSDM. K E Y W O R D Slipid metabolism, miRNAs, myomiRs, neutral lipid storage disease with myopathy, PNPLA2

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Transcriptomic Analysis of Single Isolated Myofibers Identifies miR-27a-3p and miR-142-3p as Regulators of Metabolism in Skeletal Muscle

Abstract: Highlights d Transcriptional networking distinguishes myofibers as glycolytic or oxidative d miR-27a-3p and miR-142-3p influence mitochondrial morphology d miR-27a-3p improves lipid use and increases glycogen storage d miR-142-3p reduces lipid use

Cited by 60 publications

References 71 publications

A Single Cell but Many Different Transcripts: A Journey into the World of Long Non-Coding RNAs

A Single Cell but Many Different Transcripts: A Journey into the World of Long Non-Coding RNAs

miR-206 Enforces a Slow Muscle Phenotype

MiRNAs as biomarkers of phenotype in neutral lipid storage disease with myopathy

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