Intragenic alternative splicing coordination is essential for Caenorhabditis elegans slo-1 gene function

The extent of and the oncogenic role played by alternative splicing (AS) in cancer are well documented. Nonetheless, only few studies have attempted to dissect individual gene function at an isoform level. Here, we focus on the AS of splicing factors during prostate cancer progression, as these factors are known to undergo extensive AS and have the potential to affect hundreds of downstream genes. We identified exon 7 (ex7) in the MBNL1 (Muscleblind-like 1) transcript as being the most differentially included exon in cancer, both in cell lines and in patients' samples. In contrast, MBNL1 overall expression was down-regulated, consistently with its described role as a tumor suppressor. This observation holds true in the majority of cancer types analyzed. We first identified components associated to the U2 splicing complex (SF3B1, SF3A1, and PHF5A) as required for efficient ex7 inclusion and we confirmed that this exon is fundamental for MBNL1 protein homodimerization. We next used splice-switching antisense oligonucleotides (AONs) or siRNAs to compare the effect of MBNL1 splicing isoform switching with knockdown. We report that whereas the absence of MBNL1 is tolerated in cancer cells, the expression of isoforms lacking ex7 (MBNL1 Δex7) induces DNA damage and inhibits cell viability and migration, acting as dominant negative proteins. Our data demonstrate the importance of studying gene function at the level of alternative spliced isoforms and support our conclusion that MBNL1 Δex7 proteins are antisurvival factors with a defined tumor suppressive role that cancer cells tend to down-regulate in favor of MBNL +ex7 isoforms.

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Global Genetic Robustness of the Alternative Splicing Machinery in Caenorhabditis elegans

Genetics ◽

10.1534/genetics.110.119677 ◽

2010 ◽

Vol 186 (1) ◽

pp. 405-410 ◽

Cited By ~ 37

Author(s):

Yang Li ◽

Rainer Breitling ◽

L. Basten Snoek ◽

K. Joeri van der Velde ◽

Morris A. Swertz ◽

...

Keyword(s):

Alternative Splicing ◽

Caenorhabditis Elegans ◽

Genetic Robustness ◽

Splicing Machinery

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A new view of transcriptome complexity and regulation through the lens of local splicing variations

eLife ◽

10.7554/elife.11752 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 140

Author(s):

Jorge Vaquero-Garcia ◽

Alejandro Barrera ◽

Matthew R Gazzara ◽

Juan González-Vallinas ◽

Nicholas F Lahens ◽

...

Keyword(s):

Genetic Variation ◽

Alternative Splicing ◽

Gene Function ◽

Specific Protein ◽

Mrna Splicing ◽

New Approach ◽

Genome Wide ◽

Mouse Tissues ◽

Splice Variation ◽

Novel Isoforms

Alternative splicing (AS) can critically affect gene function and disease, yet mapping splicing variations remains a challenge. Here, we propose a new approach to define and quantify mRNA splicing in units of local splicing variations (LSVs). LSVs capture previously defined types of alternative splicing as well as more complex transcript variations. Building the first genome wide map of LSVs from twelve mouse tissues, we find complex LSVs constitute over 30% of tissue dependent transcript variations and affect specific protein families. We show the prevalence of complex LSVs is conserved in humans and identify hundreds of LSVs that are specific to brain subregions or altered in Alzheimer's patients. Amongst those are novel isoforms in the Camk2 family and a novel poison exon in Ptbp1, a key splice factor in neurogenesis. We anticipate the approach presented here will advance the ability to relate tissue-specific splice variation to genetic variation, phenotype, and disease.

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High Qualitative and Quantitative Conservation of Alternative Splicing in Caenorhabditis elegans and Caenorhabditis briggsae

Molecular Biology and Evolution ◽

10.1093/molbev/msm023 ◽

2007 ◽

Vol 24 (4) ◽

pp. 909-917 ◽

Cited By ~ 22

Author(s):

Jakob Lewin Rukov ◽

Manuel Irimia ◽

Søren Mørk ◽

Viktor Karlovich Lund ◽

Jeppe Vinther ◽

...

Keyword(s):

Alternative Splicing ◽

Caenorhabditis Elegans ◽

Caenorhabditis Briggsae ◽

Qualitative And Quantitative

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New strains for tissue-specific RNAi studies in Caenorhabditis elegans

10.1101/283325 ◽

2018 ◽

Author(s):

Jason S. Watts ◽

Henry F. Harrison ◽

Shizue Omi ◽

Quentin Guenthers ◽

James Dalelio ◽

...

Keyword(s):

Fatty Acid ◽

Caenorhabditis Elegans ◽

Gene Function ◽

Target Genes ◽

Resistant Mutant ◽

Knockout Mutant ◽

Tissue Specific ◽

Double Stranded Rna ◽

C Elegans ◽

Insight Into

AbstractRNA interference is a powerful tool for dissecting gene function. In Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. We attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1 (ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, we found that the before mentioned strain, rde-1 (ne219) and the reported germline only RNAi strain, rrf-1 (pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments. However, the knockout mutant rde-1 (ne300) was strongly resistant to dsRNA induced RNAi, and thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.

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An efficient genome editing strategy to generate putative null mutants in Caenorhabditis elegans using CRISPR/Cas9

10.1101/391243 ◽

2018 ◽

Cited By ~ 1

Author(s):

Han Wang ◽

Heenam Park ◽

Jonathan Liu ◽

Paul W. Sternberg

Keyword(s):

Dna Repair ◽

Caenorhabditis Elegans ◽

Genome Editing ◽

Gene Function ◽

Unique Sequence ◽

Target Site ◽

Null Alleles ◽

Wild Type ◽

C Elegans ◽

Null Mutants

AbstractNull mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long stop knock-in (STOP-IN) cassette into the early exons of target genes. This cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette right at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two 35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2. We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for additional 20 genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.SummaryWe report a simple and efficient CRISPR/Cas9 genome editing strategy to generate putative null C. elegans mutants by inserting a small universal stop knock-in (STOP-IN) cassette with stop codons in three frames and frameshifts. The strategy is cloning-free, with the mixture consisting of preassembled Cas9 ribonucleoprotein and single stranded repair DNA oligos directly injected into gonads of adult C. elegans. The universal STOP-IN cassette also contains a unique sequence that simplifies detection of successful knock-in events via PCR and an exogenous Cas9 target sequence that allows further genome editing.

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Translational suppression via IFG-1/eIF4G confers resistance to stress-induced RNA alternative splicing in Caenorhabditis elegans

10.1101/2021.11.29.470369 ◽

2021 ◽

Author(s):

Samantha C Chomyshen ◽

Cheng-Wei Wu

Keyword(s):

Alternative Splicing ◽

Caenorhabditis Elegans ◽

Rna Splicing ◽

Molecular Mechanisms ◽

Protein Translation ◽

Initiation Factor ◽

A Genome ◽

Dividing Cells ◽

Extended Lifespan

Splicing of pre-mRNA is an essential process for dividing cells and splicing defects have been linked to aging and various chronic diseases. Environmental stress has recently been shown to alter splicing fidelity and molecular mechanisms that protect against splicing disruption remains unclear. Using an in vivo RNA splicing reporter, we performed a genome-wide RNAi screen in Caenorhabditis elegans and found that protein translation suppression via silencing of the conserved initiation factor 4G (IFG-1/eIF4G) protects against cadmium-induced splicing disruption. Transcriptome analysis of an ifg-1 deficient mutant revealed an overall increase in splicing fidelity and resistance towards cadmium-induced alternative splicing compared to the wild-type. We found that the ifg-1 mutant up-regulates >80 RNA splicing regulatory genes that are controlled by the TGF-β transcription factor SMA-2. The extended lifespan of the ifg-1 mutant is partially reduced upon sma-2 depletion and completely nullified when core spliceosome genes including snr-1, snr-2, and uaf-2 are knocked down. Together, these data describe a molecular mechanism that provides resistance towards stress-induced alternative splicing and demonstrate an essential role for RNA homeostasis in promoting longevity in a translation-compromised mutant.

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MUTATIONS WITH DOMINANT EFFECTS ON THE BEHAVIOR AND MORPHOLOGY OF THE NEMATODE CAENORHABDITIS ELEGANS

Genetics ◽

10.1093/genetics/113.4.821 ◽

1986 ◽

Vol 113 (4) ◽

pp. 821-852

Author(s):

Eun-Chung Park ◽

H Robert Horvitz

Keyword(s):

Caenorhabditis Elegans ◽

Random Sample ◽

Gene Function ◽

The Novel ◽

Wild Type ◽

Nematode Caenorhabditis Elegans ◽

Type Function ◽

C Elegans ◽

Wild Type Phenotype ◽

Gene Functions

ABSTRACT We have analyzed 31 mutations that have dominant effects on the behavior or morphology of the nematode Caenorhabditis elegans. These mutations appear to define 15 genes. We have studied ten of these genes in some detail and have been led to two notable conclusions. First, loss of gene function for four of these ten genes results in a wild-type phenotype; if these genes represent a random sample from the genome, then we would estimate that null mutations in about half of the genes in C. elegans would result in a nonmutant phenotype. Second, the dominant effects of mutations in nine of these ten genes are caused by novel gene functions, and in all nine cases the novel function is antagonized by the wild-type function.

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Mosaic analysis of the let-23 gene function in vulval induction of Caenorhabditis elegans

Development ◽

10.1242/dev.121.8.2655 ◽

1995 ◽

Vol 121 (8) ◽

pp. 2655-2666 ◽

Cited By ~ 18

Author(s):

M. Koga ◽

Y. Ohshima

Keyword(s):

Caenorhabditis Elegans ◽

Gene Function ◽

Larval Survival ◽

Precursor Cell ◽

Kinase Gene ◽

Genetic Mosaic ◽

Tyrosine Kinase Gene ◽

Receptor Tyrosine Kinase Gene ◽

Almost All ◽

Vulval Precursor Cell

The let-23 receptor tyrosine kinase gene is required for vulval induction and larval survival in the nematode Caenorhabditis elegans. We carried out genetic mosaic analyses of the let-23 gene function by using the cloned let-23 and ncl-1 genes. The wild-type let-23 gene was required in a vulval precursor cell to adopt the 1 degree vulval fate in animals carrying a let-23 vulvaless or lethal chromosomal mutation. In almost all the animals, vulval precursor cells adjacent to a 1 degree fate cell were induced to the 2 degrees vulval fate regardless of the let-23 genotypes. These findings indicate that the vulval induction signal from an anchor cell induces a vulval precursor cell to adopt the 1 degree fate through LET-23, and then a 1 degree fate cell induces adjacent cells to adopt the 2 degrees fate, for which LET-23 is not required. Foci of lethality of the let-23 (mn23) mutation were found in ABal and ABplp lineages.

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