Nuclear localization of MBNL1: splicing-mediated autoregulation and repression of repeat-derived aberrant proteins

Abstract

In some neurological diseases caused by repeat expansions such as myotonic dystrophy, the RNA-binding protein muscleblind-like 1 (MBNL1) accumulates in intranuclear inclusions containing mutant repeat RNA. The interaction between MBNL1 and mutant RNA in the nucleus is a key event leading to loss of MBNL function, yet the details of this effect have been elusive. Here, we investigated the mechanism and significance of MBNL1 nuclear localization. We found that MBNL1 contains two classes of nuclear localization signal (NLS), a classical bipartite NLS and a novel conformational NLS. Alternative splicing of exon 7 acts as a switch between these NLS types and couples MBNL1 activity and intracellular localization. Depending on its nuclear localization, MBNL1 promoted nuclear accumulation of mutant RNA containing a CUG or CAG repeat, some of which produced proteins containing homopolymeric tracts such as polyglutamine. Furthermore, MBNL1 repressed the expression of these homopolymeric proteins including those presumably produced through repeat-associated non-ATG (RAN) translation. These results suggest that nuclear retention of expanded RNA reflects a novel role of MBNL proteins in repressing aberrant protein expression and may provide pathological and therapeutic implications for a wide range of repeat expansion diseases associated with nuclear RNA retention and/or RAN translation.

Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila

“…Of note, binding sites of dme-miR-277 and dme-miR-304 overlap in the mblD 3′ UTR, which could explain the difference observed between in vivo and luciferase assays for mblD regulation by dme-miR-277 . Interestingly, miR-304SP and miR-277SP were able to downregulate the expression of mblB and mblC , respectively, instead of increasing expression, which could also be a consequence of inter-isoform regulation as it has been previously shown for MBNL proteins5354.…”

Mechanistic determinants of MBNL activity

“…The variance in amino acid sequences of different isoforms of MBNL paralogs depends on the presence or absence of three highly conserved alternative exons which consist of 54, 36 and 95 nucleotides, herein called ex.54nt, ex.36nt and ex.95nt (previously numbered ex.5, ex.7 and ex.8, respectively) (2) (Figure 1A and Supplementary Figure S1A). The protein sequences encoded by MBNL1 and MBNL2 ex.54nt, which contains a nuclear localization signals, are 72% identical whereas no such sequence exists in MBNL3 (Supplementary Figure S1A) (16,17,19). The amino acid sequences encoded by ex.36nt and ex.95nt are 60-82% and 81-87% identical between the three paralogs, respectively, and form a C-terminus of the protein.…”

“…All MBNL paralogs contain two N-terminal tandem zinc finger (ZnF) domains which bind preferentially to specific RNA sequences and/or structures containing two or more clustered GC steps flanked by pyrimidines (YGCY) (12–15). Each MBNL paralog may contain variable amino acid sequences encoded by alternative exons that modulate its cellular localization, the number of ZnF domains and the distance between them, multimerization capacity, affinity to RNA sequence motifs and AS activity (16–19). A comparison of MBNL activities might be reflected by splicing activities.…”

Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy

“…Inclusion of MBNL exon 5 controls localization of the protein, and appears to be tightly regulated by MBNL and other proteins [29,40,41]. This region is an “ultra-conserved region” [31], and previous work has shown that in mouse myoblasts, MBNL1 protein interacts with the region mutated in these experiments [29].…”