Nuclear retention of full-length HTT RNA is mediated by splicing factors MBNL1 and U2AF65

Abstract

Huntington’s disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. Recent evidence suggests that HD is a consequence of multimodal, non-mutually exclusive mechanisms of pathogenesis that involve both HTT protein- and HTT RNA-triggered mechanisms. Here we provide further evidence for the role of expanded HTT (expHTT) RNA in HD by demonstrating that a fragment of expHTT is cytotoxic in the absence of any translation and that the extent of cytotoxicity is similar to the cytotoxicity of an expHTT protein fragment encoded by a transcript of similar length and with a similar repeat size. In addition, full-length (FL) expHTT is retained in the nucleus. Overexpression of the splicing factor muscleblind-like 1 (MBNL1) increases nuclear retention of expHTT and decreases the expression of expHTT protein in the cytosol. The splicing and nuclear export factor U2AF65 has the opposite effect, decreasing expHTT nuclear retention and increasing expression of expHTT protein. This suggests that MBNL1 and U2AF65 play a role in nuclear export of expHTT RNA.

RAN Translation Regulated by Muscleblind Proteins in Myotonic Dystrophy Type 2

“…Additionally, MBNL1 also decreases RAN protein levels expressed across CUG, and similar to a previous report (Sun et al, 2015) CAG expansion RNAs (Figures S7). These results are consistent with our CCUG findings because muscleblind proteins have been shown to bind to CAG and CUG expansion RNAs (Ho et al, 2005; Kino et al, 2004; Mykowska et al, 2011; Yuan et al, 2007).…”

ATXN2‐AS, a gene antisense to ATXN2, is associated with spinocerebellar ataxia type 2 and amyotrophic lateral sclerosis

“…30 Fluorescent in situ hybridization (FISH) with DNaseor RNase treatment was performed as previously described. 31 …”

“…33 The green fluorescent protein (GFP)-tagged MBNL1 (GFP-MBNL1) plasmid was a kind gift from Dr Charles A. Thornton. 31,34 …”

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

“…Since then, proteins that are trapped not only by CUG repeats but also by CAG, CGG, CCUG, AUUCU and GGGGCC repeats have been the subject of many studies, the results of which have broadened our understanding of the pathomechanisms of these disorders (Figure 4) (Table 1 and Supplementary Table S2). These abnormal RNA–protein interactions affect the alternative splicing of specific pre-mRNAs (144), alter the use of alternative polyadenylation sites of a number of mRNAs (145), change nuclear transport and export (146,147), affect translation (148), induce nucleolar stress (94), and dysregulate miRNA processing (149). What makes these expanded repeats so attractive for cellular proteins?…”