The Muscleblind-like protein MBL-1 regulates microRNA expression in Caenorhabditis elegans through an evolutionarily conserved autoregulatory mechanism.

The Muscleblind-like (MBNL) family is a highly conserved set of RNA-binding proteins (RBPs) that regulate RNA metabolism during the differentiation of various animal tissues. Functional insufficiency of MBNL affects muscle and central nervous system development, and contributes to the myotonic dystrophies (DM), a set of incurable multisystemic disorders. Studies on the regulation of MBNL genes are essential to provide insight into the gene regulatory networks controlled by MBNL proteins and to understand how dysregulation within these networks causes disease. In this study, we demonstrate the evolutionary conservation of an autoregulatory mechanism that governs the function of MBNL proteins by generating two distinct protein isoform types through alternative splicing. Our aim was to further our understanding of the regulatory principles that underlie this conserved feedback loop in a whole-organismal context, and to address the biological significance of the respective isoforms. Using an alternative splicing reporter, our studies show that, during development of the Caenorhabditis elegans central nervous system, the orthologous mbl-1 gene shifts production from long protein isoforms that localize to the nucleus to short isoforms that also localize to the cytoplasm. Using isoform-specific CRISPR/Cas9-generated strains, we showed that expression of short MBL-1 protein isoforms is required for healthy neuromuscular function and neurodevelopment, while expression of long MBL-1 protein isoforms is dispensable, emphasizing a key role for cytoplasmic functionalities of the MBL-1 protein. Furthermore, RNA-seq and lifespan analyses indicated that short MBL-1 isoforms are crucial regulators of miRNA expression and, in consequence, required for normal lifespan. In conclusion, this study provides support for the disruption of cytoplasmic RNA metabolism as a contributor in myotonic dystrophy and paves the way for further exploration of miRNA regulation through MBNL proteins during development and in disease models.

Asymmetric inheritance of RNA toxicity in C. elegans expressing CTG repeats.

Nucleotide repeat expansions are a hallmark of over 40 neurodegenerative diseases and cause RNA toxicity and multisystemic symptoms that worsen with age. Through an unclear mechanism, RNA toxicity can trigger severe disease manifestation in infants if the repeats are inherited from their mother. Here we use Caenorhabditis elegans bearing expanded CUG repeats to show that this asymmetric intergenerational inheritance of toxicity contributes to disease pathogenesis. In addition, we show that this mechanism is dependent on small RNA pathways with maternal repeat-derived small RNAs causing transcriptomic changes in the offspring, reduced motility, and shortened lifespan. We rescued the toxicity phenotypes in the offspring by perturbing the RNAi machinery in the affected hermaphrodites. This points to a novel mechanism linking maternal bias and the RNAi machinery and suggests that toxic RNA is transmitted to offspring, causing disease phenotypes through intergenerational epigenetic inheritance.

Loss of muscleblind splicing factor shortens Caenorhabditis elegans lifespan by reducing the activity of p38 MAPK/PMK-1 and transcription factors ATF-7 and Nrf/SKN-1.

Muscleblind-like splicing regulators (MBNLs) are RNA-binding factors that have an important role in developmental processes. Dysfunction of these factors is a key contributor of different neuromuscular degenerative disorders, including Myotonic Dystrophy type 1 (DM1). Since DM1 is a multisystemic disease characterized by symptoms resembling accelerated aging, we asked which cellular processes do MBNLs regulate that make them necessary for normal lifespan. By utilizing the model organism Caenorhabditis elegans, we found that loss of MBL-1 (the sole ortholog of mammalian MBNLs), which is known to be required for normal lifespan, shortens lifespan by decreasing the activity of p38 MAPK/PMK-1 as well as the function of transcription factors ATF-7 and SKN-1. Furthermore, we show that mitochondrial stress caused by the knockdown of mitochondrial electron transport chain components promotes the longevity of mbl-1 mutants in a partially PMK-1-dependent manner. Together, the data establish a mechanism of how DM1-associated loss of muscleblind affects lifespan. Furthermore, this study suggests that mitochondrial stress could alleviate symptoms caused by the dysfunction of muscleblind splicing factor, creating a potential approach to investigate for therapy.

Expanded CUG Repeats Trigger Disease Phenotype and Expression Changes through the RNAi Machinery in C. elegans.

Myotonic dystrophy type 1 is an autosomal-dominant inherited disorder caused by the expansion of CTG repeats in the 3' untranslated region of the DMPK gene. The RNAs bearing these expanded repeats have a range of toxic effects. Here we provide evidence from a Caenorhabditis elegans myotonic dystrophy type 1 model that the RNA interference (RNAi) machinery plays a key role in causing RNA toxicity and disease phenotypes. We show that the expanded repeats systematically affect a range of endogenous genes bearing short non-pathogenic repeats and that this mechanism is dependent on the small RNA pathway. Conversely, by perturbating the RNA interference machinery, we reversed the RNA toxicity effect and reduced the disease pathogenesis. Our results unveil a role for RNA repeats as templates (based on sequence homology) for moderate but constant gene silencing. Such a silencing effect affects the cell steady state over time, with diverse impacts depending on tissue, developmental stage, and the type of repeat. Importantly, such a mechanism may be common among repeats and similar in human cells with different expanded repeat diseases.

The chromatin remodeling factor ISW-1 integrates organismal responses against nuclear and mitochondrial stress.

Age-associated changes in chromatin structure have a major impact on organismal longevity. Despite being a central part of the ageing process, the organismal responses to the changes in chromatin organization remain unclear. Here we show that moderate disturbance of histone balance during C. elegans development alters histone levels and triggers a stress response associated with increased expression of cytosolic small heat-shock proteins. This stress response is dependent on the transcription factor, HSF-1, and the chromatin remodeling factor, ISW-1. In addition, we show that mitochondrial stress during developmental stages also modulates histone levels, thereby activating a cytosolic stress response similar to that caused by changes in histone balance. These data indicate that histone and mitochondrial perturbations are both monitored through chromatin remodeling and involve the activation of a cytosolic response that affects organismal longevity. HSF-1 and ISW-1 hence emerge as a central mediator of this multi-compartment proteostatic response regulating longevity.

Identification of genes in toxicity pathways of trinucleotide-repeat RNA in C. elegans.

Myotonic dystrophy disorders are caused by expanded CUG repeats in noncoding regions. Here we used Caenorhabditis elegans expressing CUG repeats to identify genes that modulate the toxicity of such repeats. We identified 15 conserved genes that function as suppressors or enhancers of CUG repeat-induced toxicity and that modulate formation of nuclear foci by CUG-repeat RNA. These genes regulate CUG repeat-induced toxicity through distinct mechanisms including RNA export and clearance, thus suggesting that CUG-repeat toxicity is mediated by multiple pathways. A subset of the genes are also involved in other degenerative disorders. The nonsense-mediated mRNA decay (NMD) pathway has a conserved role in regulating CUG-repeat-RNA transcript levels and toxicity, and NMD recognition of toxic RNAs depends on 3'-untranslated-region GC-nucleotide content. Our studies suggest a broader surveillance role for NMD in which variations in this pathway influence multiple degenerative diseases.

The Caenorhabditis elegans RDE-10/RDE-11 complex regulates RNAi by promoting secondary siRNA amplification.

BACKGROUND: In nematodes, plants, and fungi, RNAi is remarkably potent and persistent due to the amplification of initial silencing signals by RNA-dependent RNA polymerases (RdRPs). In Caenorhabditis elegans (C. elegans), the interaction between the RNA-induced silencing complex (RISC) loaded with primary small interfering RNAs (siRNAs) and the target messenger RNA (mRNA) leads to the recruitment of RdRPs and synthesis of secondary siRNAs using the target mRNA as the template. The mechanism and genetic requirements for secondary siRNA accumulation are not well understood. RESULTS: From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10, and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, and other candidate RNAi factors. The RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10, rde-11, and other dosage-sensitive RNAi-defective genes rsd-2, rsd-6, and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways. CONCLUSIONS: The RDE-10/RDE-11 complex is essential for the amplification of RNAi in C. elegans by promoting secondary siRNA accumulation.