title: Post-transcriptional regulation of Rbm5 expression in undifferentiated H9c2 myoblasts.
abstract: We previously examined the expression of Rbm5 during myoblast differentiation and found significantly more protein in the early stages of skeletal myoblast differentiation than during the later stages. We decided to determine if this elevated level was necessary for differentiation. Our hypothesis was that if high levels of Rbm5 protein expression were necessary for the initiation of skeletal myoblast differentiation, then inhibition of expression would prevent differentiation. Our long-term objective is to inhibit Rbm5 expression and examine the effect on H9c2 differentiation. Towards this end, stable knockdown clones and transient knockdown populations were generated. Expression analyses in H9c2 myoblasts demonstrated significant Rbm5 messenger RNA (mRNA) inhibition but, surprisingly, no effect on RBM5 protein levels. Expression of the Rbm5 paralogue Rbm10 was examined in order to (a) ensure no off-target knockdown effect, and (b) investigate any possible compensatory effects. RBM10 protein levels were found to be elevated, in both the clonal and transiently transfected populations. These results suggest that myoblast RBM5 expression is regulated by a process that includes RNA sequestration and/or controlled translation, and that (a) RBM5 function is compensated for by RBM10, and/or (b) RBM5 regulates RBM10 expression. We have developed a model to describe our findings, and suggest further experiments for testing its validity. Since upregulation of Rbm10 might compensate for downregulated Rbm5, and consequently might mask any potential knockdown effect, it could lead to incorrect conclusions regarding the importance of Rbm5 for differentiation. It is therefore imperative to determine how both RBM5 and RBM10 protein expression is regulated.
authors.affiliation: Biomolecular Sciences Program, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, P3E 2C6, Canada. jloiselle@amric.ca.|AMRIC, Health Sciences North, 41 Ramsey Lake Road, Sudbury, ON, P3E 5J1, Canada. jloiselle@amric.ca.
authors.forename: Julie J
authors.initials: JJ
authors.lastname: Loiselle
authors.affiliation: Department of Biology, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, P3E 2C6, Canada.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Tessier
authors.affiliation: Biomolecular Sciences Program, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, P3E 2C6, Canada.|AMRIC, Health Sciences North, 41 Ramsey Lake Road, Sudbury, ON, P3E 5J1, Canada.|Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, P3E 2C6, Canada.|Division of Medical Sciences, Northern Ontario School of Medicine, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, P3E 2C6, Canada.|Department of Medicine, Division of Medical Oncology, University of Ottawa, Ottawa, ON, Canada.
authors.forename: Leslie C
authors.initials: LC
authors.lastname: Sutherland
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000235:genetics*; D032446:Myoblasts / Q000166:cytology*; D009687:Nuclear Proteins / Q000096:biosynthesis*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D015533:Transcriptional Activation / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C000593492:RBM5 protein, rat; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C101166:Rbm10 protein, rat; D025521:Tumor Suppressor Proteins
keywords: Differentiation; H9c2; Rbm10; Rbm5; Regulation