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Effects of siRNA depletion on gene expression during C2C12 skeletal muscle differentiation initiation

ABSTRACT: In humans, skeletal muscles comprise nearly 40% of total body mass, which is maintained throughout adulthood by a balance of muscle protein synthesis and breakdown. Cellular amino acid (AA) levels are critical for these processes, and mammalian cells contain transporter proteins that import AAs to maintain homeostasis. Until recently, the control of transporter regulation has largely been studied at the transcriptional and post-translational levels; however, our recent work demonstrates that the RNA-binding protein YBX3 is critical to intracellular sustain AA in human cells. Here we report that YBX3 post-transcriptionally controls the levels of specific AA transporter messenger (m)RNAs in skeletal muscle cells, which impacts the intracellular levels transported by these proteins. Further, we find that reduction of YBX3 protein reduces proliferation during skeletal muscle differentiation, and that YBX3 expression increases substantially during skeletal muscle differentiation, which is independent to changes in itsmRNA levels. Taken together, our findings suggest that YBX3 regulates AA transport in skeletal muscle cells and that its expression is critical to maintain skeletal muscle cell proliferation during differentiation.

ORGANISM(S): Mus musculus

PROVIDER: GSE240445 | GEO | 2023/12/20

REPOSITORIES: GEO

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YBX3 controls amino acid import via post-transcriptional regulation of Solute Carrier mRNAs

Project description:Amino acids (AA) support protein synthesis, and thus cell growth and proliferation. Specialized transporters mediate AA exchange across membranes and their regulation is critical for AA homeostasis. Here, we report that the DNA- and RNA-binding protein YBX3 regulates the expression of AA transporters. To investigate the functions of YBX3, we used a multiomics approach including proteomics, transcriptomics and metabolomics. We identified a subset of mRNAs that require YBX3 for de novo translation and are also bound to YBX3, including multiple solute carrier (SLC) AA transporters (SLC7A5, SLC3A2 and SLC1A3). We show that YBX3 stabilizes these SLC mRNAs and that the 3’UTR of SLC7A5 is necessary and sufficient for YBX3-mediated regulation. Further, YBX3 depletion results in diminished intracellular levels of SLC7A5/SLC3A2 substrate AA. Importantly, expression of an exogenous SLC7A5 not susceptible to YBX3 regulation restores AA levels. Thus, YBX3 is a key post-transcriptional regulator of large neutral AA import.

2022-03-01 | PXD007136 | Pride

YBX3 is a versatile RNA-binding protein that controls amino acid import by regulating SLC mRNA abundance

Project description:Summary: Amino acids (AA) support protein synthesis, and thus cell growth and proliferation. Specialized transporters mediate AA exchange across membranes and their regulation is critical for AA homeostasis. Here, we report that the DNA- and RNA-binding protein YBX3 regulates the expression of AA transporters. To investigate the functions of YBX3, we integrated proteomics and transcriptomics data with YBX3-RNA complex data to identify RNAs directly regulated by YBX3. The data revealed YBX3 as a versatile RNA-binding protein (RBP) that regulates as both a translational repressor and mRNA stabilizer. Among the direct YBX3 targets, we We identified two solute carrier (SLC) AA transporters (SLC7A5 and SLC3A2). We show that YBX3 stabilizes these SLC mRNAs and that the 3 UTR of SLC7A5 is necessary and sufficient for YBX3-mediated regulation. Further, YBX3 depletion results in diminished intracellular levels of SLC7A5/SLC3A2 substrates, large neutral AA. Importantly, expression of an exogenous SLC7A5 not susceptible to YBX3 regulation restores AA levels. Thus, YBX3 is a key post-transcriptional regulator of large neutral AA import.

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Culture induced changes in Blood-Brain Barrier transcriptome: implications for amino acid transporters in vivo

Project description:Tight homeostatic control of brain amino acids (AA) depends on transport via solute family carrier proteins expressed by the Blood-Brain Barrier (BBB) microvascular endothelial cells (BMEC). To characterize the mouse BMEC transcriptome and probe culture-induced changes microarray analyses of PECAM-1+ endothelial cells (ppMBMECs) were compared with primary MBMECs (pMBMEC) cultured in the presence or absence of glial cells, and with b.End5 endothelioma cell-line. Selected cell marker and AA transporter mRNA levels were further verified by real-time RT PCR. Regardless of glial co-culture expression of a large subset of genes was strongly altered by a brief culture step. This is consistent with the known dependence of BMECs on in vivo interactions to maintain physiological functions, e.g. tight barrier formation, and their consequent de-differentiation in culture. Seven (4F2hc, Lat1, Taut, Snat3, Snat5, Xpct, Cat1) of nine highly in vivo expressed AA transporter mRNAs were strongly down-regulated for all cultures and two (Snat2, Eaat3) were variably regulated. In contrast, five AA transporter mRNAs with low in vivo expression, including y+Lat2, xCT, and Snat1, were strongly up-regulated by culture. We hypothesized that the AA transporters highly expressed in ppMBMECs and strongly down-regulated in culture play a major in vivo role for BBB transendothelial transport. Keywords: culture vs non-cultured RNA was prepared from 4 sources of endothelial cells: 1) isolated mouse brain microvascular endothelial cells that were cultured in transwells with and 2) without non-contact co-culture with glial cultures or 3) further purified using anti-PECAM1 magnetic bead sorting and 4) the endothelioma b.End5 cell-line.

2010-05-18 | E-GEOD-14375 | biostudies-arrayexpress

Culture induced changes in Blood-Brain Barrier transcriptome: implications for amino acid transporters in vivo

2009-06-01 | GSE14375 | GEO

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2020-10-02 | GSE133505 | GEO

Myf6/MRF4 Mediated Myokine Signaling is Required for the Maintenance of the Adult Skeletal Muscle Stem Cell Pool

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