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.

RBM10 promotes transformation-associated processes in small cell lung cancer and is directly regulated by RBM5

“…In the results presented herein, however, using an aggressive RBM5 -null SCLC model, we show an opposite effect for RBM10, correlating its expression with promotion of various transformation-associated processes. These results suggested to us that RBM5 may be a regulator of RBM10 function, a relationship previously supported by our work in a rat myoblast system [ 20 ]. To investigate if and how RBM5 might influence RBM10, we examined RBM10 mRNA and protein expression in our stable RBM5 -expressing GLC20 sublines T2 and C4.…”

“…This is an extension of a previous model we developed in a non-transformed rat myoblast system, which described how RBM10 might influence RBM5 [20]. We hypothesize that RBM10’s binding to RBM10v2 mRNA results in a downregulation of RBM10v2 protein expression levels.…”

“…Based on our results and those of previous RBM10-related studies, we propose a working model describing RBM5’s influence on RBM10 and yet to be confirmed functional interactions between RBM5 and RBM10 ( S2 Fig ). This is an extension of a previous model we developed in a non-transformed rat myoblast system, which described how RBM10 might influence RBM5 [ 20 ]. We hypothesize that RBM10’s binding to RBM10v2 mRNA results in a downregulation of RBM10v2 protein expression levels.…”

“…Furthermore, RBM10 is mutated in pancreatic intraductal papillary mucinous neoplasms [ 56 ], 7% of lung adenocarcinomas [ 57 ], and 21% of invasive lung adenocarcinomas [ 21 ]. RBM10 has also been suggested to be important to rat myoblast differentiation [ 20 ]. Therefore, understanding the regulation of RBM10, as well as the range of its downstream effects, is of interest to various areas of cell, developmental and cancer biology.…”

“…Considered as a whole, these results suggest that the regulation of cell growth is an important aspect of RBM10 function, but that the mechanism regulating this function requires elucidation. Limited evidence suggests that this regulation depends, at least in part, on the related RNA binding protein RBM5 [ 20 ].…”