scholarly journals A Candidate RNAi Screen Reveals Diverse RNA-Binding Protein Phenotypes in Drosophila Flight Muscle

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2505
Author(s):  
Shao-Yen Kao ◽  
Elena Nikonova ◽  
Sabrina Chaabane ◽  
Albiona Sabani ◽  
Alexandra Martitz ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Processing ◽  
Muscle Development ◽  
Rna Binding ◽  
Flight Muscle ◽  
Rna Binding Proteins ◽  
Fiber Type ◽  
Expression Patterns ◽  
Fine Tuning ◽  
Fiber Types

The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability among muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing, including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently, only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. Here, we demonstrate the utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. We find that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. We performed knockdown with 105 RNAi hairpins targeting 35 RBPs and report associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa, Rm62, mub, mbl, sbr, and clu. Knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. We further show that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of kettin to the Z-disc. Our results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a powerful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.

scholarly journals A Candidate RNAi Screen Reveals Diverse RNA-Binding Protein Phenotypes in Drosophila Flight Muscle

2021 ◽  
Author(s):  
Shao-Yen Kao ◽  
Elena Nikonova ◽  
Sabrina Chaabane ◽  
Albiona Sabani ◽  
Alexandra Martitz ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Processing ◽  
Muscle Development ◽  
Rna Binding ◽  
Flight Muscle ◽  
Rna Binding Proteins ◽  
Fiber Type ◽  
Expression Patterns ◽  
Fine Tuning ◽  
Fiber Types

The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability between muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy, and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. Here we demonstrate the utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. We find that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. We performed knockdown with 105 RNAi hairpins targeting 35 RBPs and report associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa, Rm62, mub, mbl, sbr, and clu. Interestingly, knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. We further show that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of Kettin to the Z-disc. Our results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a useful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.

scholarly journals Rbfox1 is required for myofibril development and maintaining fiber type–specific isoform expression in Drosophila muscles

2022 ◽  
Vol 5 (4) ◽  
pp. e202101342
Author(s):  
Elena Nikonova ◽  
Amartya Mukherjee ◽  
Ketaki Kamble ◽  
Christiane Barz ◽  
Upendra Nongthomba ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Muscle Development ◽  
Rna Binding ◽  
Troponin I ◽  
Rna Binding Proteins ◽  
Fiber Type ◽  
Structural Defects ◽  
Specific Gene ◽  
Fiber Types ◽  
Isoform Expression

Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. In this study, we report that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila. Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type–specific gene and splice isoform expression, notably loss of an indirect flight muscle–specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. We further show that Rbfox1 directly binds the 3′-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type–specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type–specific splicing and expression dynamics of identity genes and structural proteins.

scholarly journals Rbfox1 is required for myofibril development and maintaining fiber-type specific isoform expression in Drosophila muscles

2021 ◽  
Author(s):  
Elena Nikonova ◽  
Ketaki Kamble ◽  
Amartya Mukherjee ◽  
Christiane Barz ◽  
Upendra Nongthomba ◽  
...  
Keyword(s):  
Muscle Development ◽  
Rna Binding ◽  
Troponin I ◽  
Rna Binding Proteins ◽  
Fiber Type ◽  
Specific Gene ◽  
Fiber Types ◽  
Transcript Levels ◽  
Splice Isoform ◽  
Isoform Expression

Protein isoform transitions confer distinct properties on muscle fibers and are regulated predominantly by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to control skeletal muscle function. However, the detailed mechanisms by which Rbfox1 contributes to normal muscle development and physiology remain obscure. In this study, we report that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila. Rbfox1 is differentially expressed in tubular and fibrillar muscle fiber types. RNAi knockdown of Rbfox1 leads to a loss of flight, climbing and jumping ability, as well as eclosion defects. Myofibers in knockdown muscle are frequently torn, and sarcomeres are hypercontracted. These defects arise from mis-regulation of fiber-type specific gene and splice isoform expression, notably loss of an IFM-specific isoform of Troponin-I that is critical for regulating myosin activity. We find that Rbfox1 influences mRNA transcript levels through 1) direct binding of 3'-UTRs of target transcripts as well as 2) through regulation of myogenic transcription factors, including Mef2, Exd and Salm. Moreover, Rbfox1 modulates splice isoform expression through 1) direct regulation of target splice events in structural genes and 2) regulation of the CELF-family RNA-binding protein Bruno1. Our data indicate that cross-regulatory interactions observed between FOX and CELF family RNA-binding proteins in vertebrates are conserved between their counterparts, Rbfox1 and Bruno1 in flies. Rbfox1 thus affects muscle development by regulation of both fiber-type specific gene and gene isoform expression dynamics of identity genes and structural proteins.

scholarly journals Posttranscriptional Regulation of Splicing Factor SRSF1 and Its Role in Cancer Cell Biology

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Vânia Gonçalves ◽  
Peter Jordan
Keyword(s):  
Alternative Splicing ◽  
Cancer Cell ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Current Knowledge ◽  
Expression Patterns ◽  
Regulatory Protein ◽  
Gene Transcript

Over the past decade, alternative splicing has been progressively recognized as a major mechanism regulating gene expression patterns in different tissues and disease states through the generation of multiple mRNAs from the same gene transcript. This process requires the joining of selected exons or usage of different pairs of splice sites and is regulated by gene-specific combinations of RNA-binding proteins. One archetypical splicing regulator is SRSF1, for which we review the molecular mechanisms and posttranscriptional modifications involved in its life cycle. These include alternative splicing of SRSF1 itself, regulatory protein phosphorylation events, and the role of nuclear versus cytoplasmic SRSF1 localization. In addition, we resume current knowledge on deregulated SRSF1 expression in tumors and describe SRSF1-regulated alternative transcripts with functional consequences for cancer cell biology at different stages of tumor development.

scholarly journals Rapidly Growing Protein-Centric Technologies to Extensively Identify Protein–RNA Interactions: Application to the Analysis of Co-Transcriptional RNA Processing

2021 ◽  
Vol 22 (10) ◽  
pp. 5312
Author(s):  
Akio Masuda ◽  
Toshihiko Kawachi ◽  
Kinji Ohno
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Detection Efficiency ◽  
Fine Tuning ◽  
Interaction Sites ◽  
Rnap Ii ◽  
Rna Interaction ◽  
In Cells

During mRNA transcription, diverse RNA-binding proteins (RBPs) are recruited to RNA polymerase II (RNAP II) transcription machinery. These RBPs bind to distinct sites of nascent RNA to co-transcriptionally operate mRNA processing. Recent studies have revealed a close relationship between transcription and co-transcriptional RNA processing, where one affects the other’s activity, indicating an essential role of protein–RNA interactions for the fine-tuning of mRNA production. Owing to their limited amount in cells, the detection of protein–RNA interactions specifically assembled on the transcribing RNAP II machinery still remains challenging. Currently, cross-linking and immunoprecipitation (CLIP) has become a standard method to detect in vivo protein–RNA interactions, although it requires a large amount of input materials. Several improved methods, such as infrared-CLIP (irCLIP), enhanced CLIP (eCLIP), and target RNA immunoprecipitation (tRIP), have shown remarkable enhancements in the detection efficiency. Furthermore, the utilization of an RNA editing mechanism or proximity labeling strategy has achieved the detection of faint protein–RNA interactions in cells without depending on crosslinking. This review aims to explore various methods being developed to detect endogenous protein–RNA interaction sites and discusses how they may be applied to the analysis of co-transcriptional RNA processing.

scholarly journals Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks

2015 ◽  
Author(s):  
Endre Sebestyén ◽  
Babita Singh ◽  
Belén Miñana ◽  
Amadís Pagès ◽  
Francesca Mateo ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Copy Number ◽  
Large Scale ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Copy Number Variations ◽  
Eukaryotic Genes ◽  
Cancer Drivers ◽  
Tumor Types

AbstractAlternative splicing is regulated by multiple RNA-binding proteins and influences the expression of most eukaryotic genes. However, the role of this process in human disease, and particularly in cancer, is only starting to be unveiled. We systematically analyzed mutation, copy number and gene expression patterns of 1348 RNA-binding protein (RBP) genes in 11 solid tumor types, together with alternative splicing changes in these tumors and the enrichment of binding motifs in the alternatively spliced sequences. Our comprehensive study reveals widespread alterations in the expression of RBP genes, as well as novel mutations and copy number variations in association with multiple alternative splicing changes in cancer drivers and oncogenic pathways. Remarkably, the altered splicing patterns in several tumor types recapitulate those of undifferentiated cells. These patterns are predicted to be mainly controlled by MBNL1 and involve multiple cancer drivers, including the mitotic gene NUMA1. We show that NUMA1 alternative splicing induces enhanced cell proliferation and centrosome amplification in non-tumorigenic mammary epithelial cells. Our study uncovers novel splicing networks that potentially contribute to cancer development and progression.

scholarly journals CELF Family Proteins in Cancer: Highlights on the RNA-Binding Protein/Noncoding RNA Regulatory Axis

2021 ◽  
Vol 22 (20) ◽  
pp. 11056
Author(s):  
Maryam Nasiri-Aghdam ◽  
Texali C. Garcia-Garduño ◽  
Luis Felipe Jave-Suárez
Keyword(s):  
Alternative Splicing ◽  
Rna Processing ◽  
Noncoding Rna ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Cancer Proliferation ◽  
Functional Consequences ◽  
Rna Biogenesis ◽  
Non Coding Rnas ◽  
Proliferation And Invasion

Post-transcriptional modifications to coding and non-coding RNAs are unquestionably a pivotal way in which human mRNA and protein diversity can influence the different phases of a transcript’s life cycle. CELF (CUGBP Elav-like family) proteins are RBPs (RNA-binding proteins) with pleiotropic capabilities in RNA processing. Their responsibilities extend from alternative splicing and transcript editing in the nucleus to mRNA stability, and translation into the cytoplasm. In this way, CELF family members have been connected to global alterations in cancer proliferation and invasion, leading to their identification as potential tumor suppressors or even oncogenes. Notably, genetic variants, alternative splicing, phosphorylation, acetylation, subcellular distribution, competition with other RBPs, and ultimately lncRNAs, miRNAs, and circRNAs all impact CELF regulation. Discoveries have emerged about the control of CELF functions, particularly via noncoding RNAs, and CELF proteins have been identified as competing, antagonizing, and regulating agents of noncoding RNA biogenesis. On the other hand, CELFs are an intriguing example through which to broaden our understanding of the RBP/noncoding RNA regulatory axis. Balancing these complex pathways in cancer is undeniably pivotal and deserves further research. This review outlines some mechanisms of CELF protein regulation and their functional consequences in cancer physiology.

scholarly journals Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

2021 ◽  
Vol 4 (1) ◽  
pp. 22
Author(s):  
Mrinmoyee Majumder ◽  
Viswanathan Palanisamy
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Control Of Gene Expression ◽  
Regulatory Pathways ◽  
Advantages And Disadvantages ◽  
Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

scholarly journals Targeting Poison Exons to Treat Developmental and Epileptic Encephalopathy

2021 ◽  
pp. 1-6
Author(s):  
Miriam C. Aziz ◽  
Patricia N. Schneider ◽  
Gemma L. Carvill
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Autism Spectrum ◽  
Epileptic Encephalopathy ◽  
Nonsense Mediated Decay ◽  
Subsequent Degradation ◽  
Alternative Splicing Events ◽  
Genetic Epilepsies

Developmental and epileptic encephalopathies (DEEs) describe a subset of neurodevelopmental disorders categorized by refractory epilepsy that is often associated with intellectual disability and autism spectrum disorder. The majority of DEEs are now known to have a genetic basis with de novo coding variants accounting for the majority of cases. More recently, a small number of individuals have been identified with intronic <i>SCN1A</i> variants that result in alternative splicing events that lead to ectopic inclusion of poison exons (PEs). PEs are short highly conserved exons that contain a premature truncation codon, and when spliced into the transcript, lead to premature truncation and subsequent degradation by nonsense-mediated decay. The reason for the inclusion/exclusion of these PEs is not entirely clear, but research suggests an autoregulatory role in gene expression and protein abundance. This is seen in proteins such as RNA-binding proteins and serine/arginine-rich proteins. Recent studies have focused on targeting these PEs as a method for therapeutic intervention. Targeting PEs using antisense oligonucleotides (ASOs) has shown to be effective in modulating alternative splicing events by decreasing the amount of transcripts harboring PEs, thus increasing the abundance of full-length transcripts and thereby the amount of protein in haploinsufficient genes implicated in DEE. In the age of personalized medicine, cellular and animal models of the genetic epilepsies have become essential in developing and testing novel precision therapeutics, including PE-targeting ASOs in a subset of DEEs.

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