scholarly journals Secondary motifs enable concentration-dependent regulation by Rbfox family proteins

2019 ◽  
Author(s):  
Bridget E. Begg ◽  
Marvin Jens ◽  
Peter Y. Wang ◽  
Christopher B. Burge
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Neuronal Development ◽  
Splicing Factors ◽  
Reporter Assay ◽  
Animal Development ◽  
High Affinity ◽  
Second Wave ◽  
Relevant Range

AbstractThe Rbfox family of splicing factors regulate alternative splicing during animal development and in disease, impacting thousands of exons in the maturing brain, heart, and muscle. Rbfox proteins have long been known to bind to the RNA sequence GCAUG with high affinity, but just half of Rbfox CLIP peaks contain a GCAUG motif. We incubated recombinant RBFOX2 with over 60,000 transcriptomic sequences to reveal significant binding to several moderate-affinity, non-GCAYG sites at a physiologically relevant range of RBFOX concentrations. We find that many of these “secondary motifs” bind Rbfox robustly in vivo and that several together can exert regulation comparable to a GCAUG in a trichromatic splicing reporter assay. Furthermore, secondary motifs regulate RNA splicing in neuronal development and in neuronal subtypes where cellular Rbfox concentrations are highest, enabling a second wave of splicing changes as Rbfox levels increase.

scholarly journals ALG-1 Influences Accurate mRNA Splicing Patterns in the Caenorhabditis elegans Intestine and Body Muscle Tissues by Modulating Splicing Factor Activities

Genetics ◽  
2019 ◽  
Vol 212 (3) ◽  
pp. 931-951 ◽  
Author(s):  
Kasuen Kotagama ◽  
Anna L. Schorr ◽  
Hannah S. Steber ◽  
Marco Mangone
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Rna Splicing ◽  
Specific Gene ◽  
Splicing Factors ◽  
Tissue Specific ◽  
Link Type ◽  
Body Muscle ◽  
Mirna Targets ◽  
Mirna Pathway

MicroRNAs (miRNAs) are known to modulate gene expression, but their activity at the tissue-specific level remains largely uncharacterized. To study their contribution to tissue-specific gene expression, we developed novel tools to profile putative miRNA targets in the Caenorhabditis elegans intestine and body muscle. We validated many previously described interactions and identified ∼3500 novel targets. Many of the candidate miRNA targets curated are known to modulate the functions of their respective tissues. Within our data sets we observed a disparity in the use of miRNA-based gene regulation between the intestine and body muscle. The intestine contained significantly more putative miRNA targets than the body muscle highlighting its transcriptional complexity. We detected an unexpected enrichment of RNA-binding proteins targeted by miRNA in both tissues, with a notable abundance of RNA splicing factors. We developed in vivo genetic tools to validate and further study three RNA splicing factors identified as putative miRNA targets in our study (asd-2, hrp-2, and smu-2), and show that these factors indeed contain functional miRNA regulatory elements in their 3′UTRs that are able to repress their expression in the intestine. In addition, the alternative splicing pattern of their respective downstream targets (unc-60, unc-52, lin-10, and ret-1) is dysregulated when the miRNA pathway is disrupted. A reannotation of the transcriptome data in C. elegans strains that are deficient in the miRNA pathway from past studies supports and expands on our results. This study highlights an unexpected role for miRNAs in modulating tissue-specific gene isoforms, where post-transcriptional regulation of RNA splicing factors associates with tissue-specific alternative splicing.

scholarly journals Poison-Exon Inclusion in DHX9 Reduces Its Expression and Sensitizes Ewing Sarcoma Cells to Chemotherapeutic Treatment

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 328 ◽  
Author(s):  
Ramona Palombo ◽  
Veronica Verdile ◽  
Maria Paola Paronetto
Keyword(s):  
Alternative Splicing ◽  
Ewing Sarcoma ◽  
Splicing Factors ◽  
Mrna Variants ◽  
Aggressive Tumor ◽  
Chemotherapeutic Treatment ◽  
Sarcoma Cells ◽  
Combinatorial Mechanism ◽  
Worse Prognosis

Alternative splicing is a combinatorial mechanism by which exons are joined to produce multiple mRNA variants, thus expanding the coding potential and plasticity of eukaryotic genomes. Defects in alternative splicing regulation are associated with several human diseases, including cancer. Ewing sarcoma is an aggressive tumor of bone and soft tissue, mainly affecting adolescents and young adults. DHX9 is a key player in Ewing sarcoma malignancy, and its expression correlates with worse prognosis in patients. In this study, by screening a library of siRNAs, we have identified splicing factors that regulate the alternative inclusion of a poison exon in DHX9 mRNA, leading to its downregulation. In particular, we found that hnRNPM and SRSF3 bind in vivo to this poison exon and suppress its inclusion. Notably, DHX9 expression correlates with that of SRSF3 and hnRNPM in Ewing sarcoma patients. Furthermore, downregulation of SRSF3 or hnRNPM inhibited DHX9 expression and Ewing sarcoma cell proliferation, while sensitizing cells to chemotherapeutic treatment. Hence, our study suggests that inhibition of hnRNPM and SRSF3 expression or activity could be exploited as a therapeutic tool to enhance the efficacy of chemotherapy in Ewing sarcoma.

Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors

Science ◽  
1994 ◽  
Vol 265 (5179) ◽  
pp. 1706-1709 ◽  
Author(s):  
J. Caceres ◽  
S Stamm ◽  
D. Helfman ◽  
A. Krainer
Keyword(s):  
Alternative Splicing ◽  
Splicing Factors

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

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.

scholarly journals Efficacy of longevity interventions in C. elegans is determined by early life activity of RNA splicing factors

2021 ◽  
Author(s):  
Sneha Dutta ◽  
Caroline Heintz ◽  
Maria C. Perez-Matos ◽  
Ayse Sena Mutlu ◽  
Mary E Piper ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Early Life ◽  
Late Onset ◽  
Natural Aging ◽  
Splicing Factors ◽  
Life Activity ◽  
C Elegans ◽  
Lipid Utilization ◽  
Early Inhibition

Geroscience aims to target the aging process to extend healthspan. However, even isogenic individuals show heterogeneity in natural aging rate and responsiveness to pro-longevity interventions, limiting translational potential. Using in vivo mini gene reporters in isogenic C. elegans, we show that alternative splicing of mRNAs related to lipid metabolism in young animals is coupled to subsequent life expectancy. Further, activity of RNA splicing factors REPO-1 and SFA-1 early in life modulates effectiveness of specific longevity interventions via POD-2/ACC1 and regulation of lipid utilization. In addition, early inhibition of REPO-1 renders animals refractory to late onset suppression of the TORC1 pathway. Together these data suggest that activity of RNA splicing factors and the metabolic landscape early in life can modulate responsiveness to longevity interventions and may explain variance in efficacy between individuals.

Alternative splicing of the mouse profilin II gene generates functionally different profilin isoforms

2000 ◽  
Vol 113 (21) ◽  
pp. 3795-3803 ◽  
Author(s):  
A. Di Nardo ◽  
R. Gareus ◽  
D. Kwiatkowski ◽  
W. Witke
Keyword(s):  
Alternative Splicing ◽  
Cell Motility ◽  
Hela Cells ◽  
The Other ◽  
Actin Dynamics ◽  
High Affinity ◽  
Other Hand ◽  
The Brain

Profilins are a conserved family of proteins participating in actin dynamics and cell motility. In the mouse, two profilin genes are known. Profilin I is expressed universally at high levels, while profilin II is expressed mainly in the brain. Here we describe the occurrence of two mouse profilin II isoforms, A and B, which are derived by alternative splicing. They are identical through residue 107 of the protein, but then have distinct C-terminal sequences. Profilin IIA binds to poly-L-proline and actin with high affinity similar to profilin I. Profilin IIB on the other hand does not bind to actin and the affinity for poly-L-proline is greatly diminished. However, tubulin was found to bind to GST-profilin IIB, and in vivo GFP-profilin IIB was recruited to spindles and asters during mitosis in HeLa cells. Our results indicate unexpected diversity in the functions of the profilin family of proteins, and suggest that in mouse profilin IIA is intimately involved in actin dynamics, while profilin IIB associates with other cytoskeletal components.

scholarly journals miRNA activity contributes to accurate RNA splicing inC. elegansintestine and body muscle tissues

2018 ◽  
Author(s):  
Kasuen Kotagama ◽  
Anna L Schorr ◽  
Hannah S Steber ◽  
Marco Mangone
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Rna Splicing ◽  
Specific Gene ◽  
Splicing Factors ◽  
Tissue Specific ◽  
C Elegans ◽  
Body Muscle ◽  
Mirna Targets ◽  
Mirna Pathway

ABSTRACTMicroRNAs (miRNAs) are known to modulate gene expression, but their activity at the tissue-specific level remains largely uncharacterized. In order to study their contribution to tissue-specific gene expression, we developed novel tools to profile miRNA targets in theC. elegansintestine and body muscle.We validated many previously described interactions, and identified ~3,500 novel targets. Many of the miRNA targets curated are known to modulate the functions of their respective tissues. Within our datasets we observed a disparity in the use of miRNA-based gene regulation between the intestine and body muscle. The intestine contained significantly more miRNA targets than the body muscle highlighting its transcriptional complexity. We detected an unexpected enrichment of RNA binding proteins targeted by miRNA in both tissues, with a notable abundance of RNA splicing factors.We developedin vivogenetic tools to validate and further study three RNA splicing factors identified as miRNA targets in our study (asd-2,hrp-2andsmu-2), and show that these factors indeed contain functional miRNA regulatory elements in their 3’UTRs that are able to repress their expression in the intestine. In addition, the alternative splicing pattern of their respective downstream targets (unc-60,unc-52,lin-10andret-1) is dysregulated when the miRNA pathway is disrupted.A re-annotation of the transcriptome data inC. elegansstrains that are deficient in the miRNA pathway from past studies supports and expands on our results. This study highlights an unexpected role for miRNAs in modulating tissue-specific gene isoforms, where post-transcriptional regulation of RNA splicing factors associates with tissue-specific alternative splicing.

scholarly journals Cell-specific exon methylation and CTCF binding in neurons regulate calcium ion channel splicing and function

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Eduardo Javier López Soto ◽  
Diane Lipscombe
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Molecular Mechanisms ◽  
Ctcf Binding ◽  
Dna Hypomethylation ◽  
Cell Functions ◽  
Specific Alternative ◽  
Opioid Sensitivity ◽  
And Function

Cell-specific alternative splicing modulates myriad cell functions and is disrupted in disease. The mechanisms governing alternative splicing are known for relatively few genes and typically focus on RNA splicing factors. In sensory neurons, cell-specific alternative splicing of the presynaptic CaV channel Cacna1b gene modulates opioid sensitivity. How this splicing is regulated is unknown. We find that cell and exon-specific DNA hypomethylation permits CTCF binding, the master regulator of mammalian chromatin structure, which, in turn, controls splicing in a DRG-derived cell line. In vivo, hypomethylation of an alternative exon specifically in nociceptors, likely permits CTCF binding and expression of CaV2.2 channel isoforms with increased opioid sensitivity in mice. Following nerve injury, exon methylation is increased, and splicing is disrupted. Our studies define the molecular mechanisms of cell-specific alternative splicing of a functionally validated exon in normal and disease states – and reveal a potential target for the treatment of chronic pain.

scholarly journals Comprehensive network analysis reveals alternative splicing-related lncRNAs in hepatocellular carcinoma

2019 ◽  
Author(s):  
Junqing Wang ◽  
Yixin Chen ◽  
Keli Xu ◽  
Yin-yuan Mo ◽  
Yunyun Zhou
Keyword(s):  
Hepatocellular Carcinoma ◽  
Alternative Splicing ◽  
Rna Splicing ◽  
Normal Liver ◽  
Splicing Factors ◽  
Primary Concern ◽  
Rna Seq ◽  
Genome Wide ◽  
Tumorigenesis And Progression ◽  
Extensive Collection

AbstractA number of recent studies have highlighted the findings that certain lncRNAs are associated with alternative splicing (AS) in tumorigenesis and progression. Although existing work showed the importance of linking certain misregulations of RNA splicing with lncRNAs, a primary concern is the lack of genome-wide comprehensive analysis for their associations.We analyzed an extensive collection of RNA-seq data, quantified 198,619 isoform expressions, and found systematic isoform usage changes between hepatocellular carcinoma (HCC) and normal liver tissue. We identified a total of 1375 splicing switched isoforms and further analyzed their biological functions.To predict which lncRNAs are associated with these AS genes, we integrated the co-expression networks and epigenetic interaction networks collected from text mining and database searching, linking lncRNA modulators such as splicing factors, transcript factors, and miRNAs with their targeted AS genes in HCC. To model the heterogeneous networks in a single framework, we developed a multi-graphic random walk (RWMG) network method to prioritize the lncRNAs associated with AS in HCC. RWMG showed a good performace evaluated by ROC curve based on cross-validation and bootstrapping strategy.As a summary, we identified 31 AS-related lncRNAs including MALAT1 and HOXA11-AS, which have been reported before, as well as some novel lncRNAs such as DNM1P35, HAND2-AS1, and DLX6-AS1. Survival analysis further confirmed the clinical significance of identified lncRNAs.

scholarly journals Alternative RNA splicing in the endothelium mediated in part by Rbfox2 regulates the arterial response to low flow

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Patrick A Murphy ◽  
Vincent L Butty ◽  
Paul L Boutz ◽  
Shahinoor Begum ◽  
Amy L Kimble ◽  
...  
Keyword(s):  
Blood Flow ◽  
Alternative Splicing ◽  
Rna Splicing ◽  
Vascular Inflammation ◽  
Low Flow ◽  
Transcriptional Responses ◽  
Binding Motifs ◽  
Endothelial Response ◽  
Arterial Endothelium

Low and disturbed blood flow drives the progression of arterial diseases including atherosclerosis and aneurysms. The endothelial response to flow and its interactions with recruited platelets and leukocytes determine disease progression. Here, we report widespread changes in alternative splicing of pre-mRNA in the flow-activated murine arterial endothelium in vivo. Alternative splicing was suppressed by depletion of platelets and macrophages recruited to the arterial endothelium under low and disturbed flow. Binding motifs for the Rbfox-family are enriched adjacent to many of the regulated exons. Endothelial deletion of Rbfox2, the only family member expressed in arterial endothelium, suppresses a subset of the changes in transcription and RNA splicing induced by low flow. Our data reveal an alternative splicing program activated by Rbfox2 in the endothelium on recruitment of platelets and macrophages and demonstrate its relevance in transcriptional responses during flow-driven vascular inflammation.

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