Muscleblind-Like 1 and Muscleblind-Like 3 Depletion Synergistically Enhances Myotonia by Altering Clc-1 RNA Translation

Abstract

Loss of Muscleblind-like 1 (Mbnl1) is known to alter Clc-1 splicing to result in myotonia. Mbnl1ΔE3/ΔE3/Mbnl3ΔE2 mice, depleted of Mbnl1 and Mbnl3, demonstrate a profound enhancement of myotonia and an increase in the number of muscle fibers with very low Clc-1 currents, where gClmax values approach ~ 1 mS/cm2, with the absence of a further enhancement in Clc-1 splice errors, alterations in polyA site selection or Clc-1 localization. Significantly, Mbnl1ΔE3/ΔE3/Mbnl3ΔE2 muscles demonstrate an aberrant accumulation of Clc-1 RNA on monosomes and on the first polysomes. Mbnl1 and Mbnl3 bind Clc-1 RNA and both proteins bind Hsp70 and eEF1A, with these associations being reduced in the presence of RNA. Thus binding of Mbnl1 and Mbnl3 to Clc-1 mRNA engaged with ribosomes can facilitate an increase in the local concentration of Hsp70 and eEF1A to assist Clc-1 translation. Dual depletion of Mbnl1 and Mbnl3 therefore initiates both Clc-1 splice errors and translation defects to synergistically enhance myotonia. As the HSALR model for myotonic dystrophy (DM1) shows similar Clc-1 defects, this study demonstrates that both splice errors and translation defects are required for DM1 pathology to manifest.Research in contextResearch in context: Myotonic Dystrophy type 1 (DM1) is a dominant disorder resulting from the expression of expanded CUG repeat RNA, which aberrantly sequesters and inactivates the muscleblind-like (MBNL) family of proteins. In mice, inactivation of Mbnl1 is known to alter Clc-1 splicing to result in myotonia. We demonstrate that concurrent depletion of Mbnl1 and Mbnl3 results in a synergistic enhancement of myotonia, with an increase in muscle fibers showing low chloride currents. The observed synergism results from the aberrant accumulation of Clc-1 mRNA on monosomes and the first polysomes. This translation error reflects the ability of Mbnl1 and Mbnl3 to act as adaptors that recruit Hsp70 and eEF1A to the Clc-1 mRNA engaged with ribosomes, to facilitate translation. Thus our study demonstrates that Clc-1 RNA translation defects work coordinately with Clc-1 splice errors to synergistically enhance myotonia in mice lacking Mbnl1 and Mbnl3.

Sarcolemmal excitability in the myotonic dystrophies

“…Quantification of chloride channel function via the parameter 5XRSN would also be of interest. 37 In conclusion, using MVRCs, we found evidence that the RMMP is relatively depolarized in persons at a later clinical stage of DM1 compared with those at earlier stages of disease, patients with DM2, and NCs. These results extend the findings of Tan et al 7 and most likely represent defects in the proper function of the sodium pump.…”

“…Through analysis in changes in parameters such as MRRP and ESN, MVRCs are well placed to track any fluctuations in the RMMP that may arise from ASO administration. Quantification of chloride channel function via the parameter 5XRSN would also be of interest …”

Muscleblind-like 3 deficit results in a spectrum of age-associated pathologies observed in myotonic dystrophy

“…Thus, although we cannot exclude the possibility of rare DM1-like splice errors underlying the observed age-associated pathologies, our data suggest that mechanisms distinct from the adult retention of embryonic splice isoforms may make important contributions to the onset of age-associated pathologies in DM1. As noted previously, it is possible that altered levels or translation of Mbnl3 38kD target RNAs could underlie the phenotypes observed in Mbnl3 ΔE2 mice 34 35 . Poulos and colleagues demonstrate inhibition of myogenic differentiation of muscle satellite cells leading to impaired muscle regeneration in Mbnl3 ΔE2 mice 35 .…”

“…To test the potential effects of MBNL3 deficiency in the development of DM1 pathophysiology we developed Mbnl3 ΔE2 mice on a 129sv background, in which exon 2 of the X-linked Mbnl3 gene was replaced by a Neomycin expression cassette 34 . As previously reported 12 34 we observe Mbnl3 expression in the adult mouse spleen and diminished but clearly detectable Mbnl3 mRNA expression in the adult soleus muscle and in the adult mouse heart, lens and brain 34 ( Supplementary Figure S1 ).…”

“…We deleted exon 2 of Mbnl3 , which is an X-linked gene, to develop Mbnl3 ΔE2 mice 34 . This mutation results in the absence of the full-length 38 kD Mbnl3 protein (Mbnl3 38kD ) and the retention of a truncated 27 kD Mbnl3 isoform (Mbnl3 27kD ) translated from an ATG codon present in Mbnl3 exon 3, as previously described by us and Poulos and colleagues 34 35 . Mbnl3 ΔE2 mice demonstrate an accelerated onset of a subset of age-associated phenotypes observed in DM1, over a range of 4–13 months of age.…”