Conservation of context-dependent splicing activity in distant Muscleblind homologs

Oddo, Julia C.; Saxena, Tanvi; McConnell, Ona; Berglund, J. Andrew; Wang, Eric T.

doi:10.1093/nar/gkw735

Cited by 14 publications

(20 citation statements)

References 40 publications

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“…Immunofluorescence detection in transfected HeLa cells showed predominant nuclear localization with a modest signal in the cytoplasm for MBNL-AB and both synthetic proteins ( Supplemental Figure S2A ). This distribution is comparable to past results, including those using full-length MBNL1 ( 31 , 39 ). The only noticeable difference in the subcellular distribution of the synthetic proteins was a lack of nucleolar definition in cells expressing MBNL-BB.…”

Section: Resultssupporting

confidence: 90%

“…Next, we tested the AS activity of the synthetic MBNL proteins for 15 endogenous splicing events across a range of protein expression generated via doxycycline gradient. These 15 endogenous events (9 inclusion, 6 exclusion) were selected from RNAseq data sets previously published from the mbnl 1/2 knockout MEFs ( 39 ). RT-PCR was then performed to determine ψ of each individual point along the protein gradient (representative images to calculate ψ are shown in Supplemental Figure S10 ) and plotted against log [MBNL] levels to generate dose–response curves (Figure 4A – C , Supplemental Figure S11 ).…”

Section: Resultsmentioning

confidence: 99%

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An engineered RNA binding protein with improved splicing regulation

Hale

Richardson

Day

et al. 2018

Nucleic Acids Research

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The muscleblind-like (MBNL) family of proteins are key developmental regulators of alternative splicing. Sequestration of MBNL proteins by expanded CUG/CCUG repeat RNA transcripts is a major pathogenic mechanism in the neuromuscular disorder myotonic dystrophy (DM). MBNL1 contains four zinc finger (ZF) motifs that form two tandem RNA binding domains (ZF1–2 and ZF3–4) which each bind YGCY RNA motifs. In an effort to determine the differences in function between these domains, we designed and characterized synthetic MBNL proteins with duplicate ZF1–2 or ZF3–4 domains, referred to as MBNL-AA and MBNL-BB, respectively. Analysis of splicing regulation revealed that MBNL-AA had up to 5-fold increased splicing activity while MBNL-BB had 4-fold decreased activity compared to a MBNL protein with the canonical arrangement of zinc finger domains. RNA binding analysis revealed that the variations in splicing activity are due to differences in RNA binding specificities between the two ZF domains rather than binding affinity. Our findings indicate that ZF1–2 drives splicing regulation via recognition of YGCY RNA motifs while ZF3–4 acts as a general RNA binding domain. Our studies suggest that synthetic MBNL proteins with improved or altered splicing activity have the potential to be used as both tools for investigating splicing regulation and protein therapeutics for DM and other microsatellite diseases.

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Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

An engineered RNA binding protein with improved splicing regulation

Hale

Richardson

Day

et al. 2018

Nucleic Acids Research

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show abstract

“…The muscleblind (MBNL) family of RNA binding proteins is highly conserved in 46 structure and function across multicellular species, and vertebrates have three family 47 members: MBNL1, MBNL2, and MBNL3 (Oddo et al, 2016). MBNL proteins use zinc 48 finger motifs (two pairs in the case of vertebrates) to recognize and bind to consensus 49 YGCY sequences (where Y is a pyrimidine) in target RNAs and regulate multiple 50 aspects of RNA metabolism, the most well-characterized of which is alternative splicing 51 Fig.…”

Section: Introduction 45mentioning

confidence: 99%

Zebrafish mbnl mutants model physical and molecular phenotypes of myotonic dystrophy

Hinman

Richardson

Sockol

et al. 2019

Preprint

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The muscleblind RNA binding proteins (MBNL1, MBNL2, and MBNL3) are highly conserved across vertebrates and are important regulators of RNA alternative splicing. Loss of MBNL protein function through sequestration by CUG or CCUG RNA repeats is largely responsible for the phenotypes of the human genetic disorder myotonic dystrophy (DM). We generated the first stable zebrafish (Danio rerio) models of DM-associated MBNL loss of function through mutation of the three zebrafish mbnl genes. In contrast to mouse models, zebrafish double and triple homozygous mbnl mutants were viable to adulthood. Zebrafish mbnl mutants displayed disease-relevant physical phenotypes including decreased body size and impaired movement. They also exhibited widespread alternative splicing changes, including the misregulation of many DM-relevant exons. Physical and molecular phenotypes were more severe in compound mbnl mutants than in single mbnl mutants, suggesting partially redundant functions of Mbnl proteins. The high fecundity and larval optical transparency of this complete series of zebrafish mbnl mutants will make them useful for studying DM-related phenotypes and how individual Mbnl proteins contribute to them, and for testing potential therapeutics.

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“…Similar functions are carried out by the Drosophila MBL protein, which is expressed in the fly brain and muscle. Drosophila contains only one muscleblind homolog, MBL (34)(35)(36).…”

Section: Introductionmentioning

confidence: 99%

Anin vivoknockdown strategy reveals multiple functions for circMbl

Krishnamoorthy

et al. 2018

Preprint

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Circular RNAs (circRNAs) are highly abundant and evolutionary conserved RNAs of mostly unknown functions. circRNAs are enriched in the brain and accumulate with age in flies, worms and mice. Despite their abundance, little is known about their functions, especially in the context of whole organisms. Here we report the development and use of shRNAs to knock down and study the function of circMbl, the most abundant circRNA in Drosophila. This circRNA is highly conserved through evolution and is generated from the locus of the essential splicing factor muscleblind (mbl). Briefly, we generated flies in which circMbl is reduced more than 90% without measurable off-target effects in the hosting gene as well as in other RNAs. These flies display specific defects that suggest roles of circMbl in muscle and neural tissues during development and in adult flies. More specifically, whole organism downregulation of circMbl leads to male developmental lethality, altered gene expression, behavioral defects, wing posture-and flight defects. Moreover, these phenotypes are recapitulated by a second shRNA targeting circMbl.Importantly, knockdown and overexpression of circMbl affect mostly the same genes but in the opposite direction. Last but not least, downregulation of circMbl in the fly central nervous system caused abnormal synaptic function. Together, our results demonstrate the functionality of circMbl at the organismal level likely by acting in multiple tissues. Moreover, here we provide the first proof of functionality of circRNAs in Drosophila as well as a methodological approach that enables the comprehensive study of circRNAs in vivo. SIGNIFICANCE STATEMENTCircular RNAs (circRNAs) are highly abundant and evolutionary conserved RNAs of mostly unknown functions. Here we report the development and use of a shRNA-based system to knockdown specific circRNAs in vivo. We generated flies in which circMbl, the most abundant circRNA is reduced more than 90% without measurable off-target effects. These flies display male developmental lethality, altered gene expression, behavioral defects, wing posture-and flight defects. These phenotypes are recapitulated by a second shRNA targeting circMbl. Moreover, downregulation of circMbl in the fly central nervous system caused abnormal synaptic function.Together, our results demonstrate the functionality of circMbl at the organismal level and provide a methodological approach that enables the comprehensive study of circRNAs in vivo.

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Conservation of context-dependent splicing activity in distant Muscleblind homologs

Cited by 14 publications

References 40 publications

An engineered RNA binding protein with improved splicing regulation

An engineered RNA binding protein with improved splicing regulation

Zebrafish mbnl mutants model physical and molecular phenotypes of myotonic dystrophy

Anin vivoknockdown strategy reveals multiple functions for circMbl

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