The role of MBNL1 and MBNL2 in nuclear foci formation and repeat expansion transcript retention in Myotonic Dystrophy 1
Year: 2023
Type: dissertation
Abstract: Myotonic dystrophy (dystrophia myotonica, DM) is a multisystemic disorder that is dominantly inherited. There are two types of DM: DM1, which is caused by a CTG repeat expansion in the 3’ UTR of the DMPK gene, and DM2, which is caused by a CCTG repeat expansion in intron 1 of the CNBP gene. In vitro experiments have shown that once transcribed, expanded C(C)UG repeats fold into very stable and long RNA hairpin structures that sequester MBNLs and other nuclear factors to form ribonuclear foci, in both types of DM. It is important to note that MBNLs are not just passively trapped within [C(C)UG]exp, but also actively participate in foci formation and cause nuclear retention of [C(C)UG]exp transcripts. The sequestration of MBNLs directly impairs the splicing of several key regulatory pre-mRNAs in muscles and neural cells. Missplicing of muscle chloride channel (CLCN1), calcium channel voltage-dependent L type alpha 1S subunit (CACNA1S), bridging integrator 1 (BIN1) and insulin receptor (INSR), results in myotonia, muscle weakness and insulin resistance, respectively, which are key hallmarks of DM. This study aimed to investigate the dynamics of MBNL1 and MBNL2 co-localisation with (CUG)exp DMPK transcripts to shed light on the mechanisms underlying the aggregation, nuclear retention and degradation of mRNA containing CUG-triplet repeats. To achieve this, I conducted the double knock-out of MBNL1 and MBNL2 in DM1 fibroblast cells using the CRISPR/Cas9 system, which led to the loss of full-length MBNL1 and 2 proteins. The effects of MBNL1 and 2 double knock-out on DM1 are complex and multi-faceted. It exacerbated many mis-splicing events and the mis-regulation of expression of many genes implicated in the disease. Additionally, it led to the emergence of novel aberrant splicing events and dysregulated gene expression. Furthermore, the double-inactivation of MBNL1 and 2 caused the instability of RNA foci formed by the mutant DMPK transcript, facilitating the dissipation of RNA foci and the degradation or nuclear export of the mutant transcript. To gain a better understanding of the dynamics of (CUG)exp RNA foci, I utilised super-resolution single-molecule localisation microscopy (SMLM) to detect DMPK transcripts. I designed probes from three intervals of the DMPK mRNA which allowed me to determine signals from the regions 5’ and 3’ to the repeat expansion and from the repeat itself. My results uncovered several key findings. First, I discovered large (CUG)exp foci are primarily formed by multiple DMPK transcripts, while micro (CUG)exp foci are mainly formed by single DMPK transcripts, suggesting that micro (CUG)exp foci may be the intermediate degradation products of large (CUG)exp foci. Second, I found that in the absence of MBNL, mutant DMPK transcripts can only form micro (CUG)exp foci, which rapidly dissipate or export to the cytoplasm. Third, I observed that micro (CUG)exp foci with or without 5' or 3' flanking regions are present in the DM1 cytoplasm, potentially leading to the source of RAN translation in DM1. I also examined the participation of various RNA decay factors in DM1 cells and their derivatives through shRNA lentiviral transduction. The successful knock-down of XRN2, EXOSC10, UPF1 and STAU1 produced a slight but significant increase in the foci area and the abundance of mutant DMPK mRNA in DM1 cells. Notably, a more pronounced increase was observed in MBNL-deficient DM1 cells. These findings highlight the critical role played by various RNA decay pathways in reducing the accumulation of mutant DMPK RNA. Furthermore, the sequestration of MBNLs to the mutant RNA may impede its degradation process, as the absence of MBNLs appears to enhance the efficiency of these pathways. In summary, my investigations uncovered valuable data on RNA foci dynamics in DM1, revealing the intricate mechanisms that underlie their formation, stability, and turnover. In addition, I contributed to delineate the complex pathways involved in the transportation and degradation of the mutant mRNA and provided insights into the critical role played by MBNL proteins in these processes.
Author: Xing, Xiaomeng
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7218155176