Arg-tRNA synthetase links inflammatory metabolism to RNA splicing via nuclear condensates

SummaryCells respond to perturbations such as inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has long known connections to the inflammatory response. Here we show that depletion of arginine during inflammation decreased levels of arginyl-tRNA synthetase (ArgRS) in the nucleus. We found that nuclear ArgRS interacted with serine/arginine repetitive matrix protein 2 (SRRM2) in membrane-less, condensate-like, SRRM2-dependent nuclear speckles. This interaction impeded SRRM2 speckle trafficking and resulted in changes in alternative mRNA splicing. Splice site usage was regulated in opposite directions by ArgRS and SRRM2. These ArgRS- and SRRM2-dependent splicing changes cumulated in synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings delineate a novel mechanism whereby a tRNA synthetase responds to a metabolic change and modulates the splicing machinery via condensate trafficking for cellular responses to inflammatory injury.

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Abstract 235: Global RNA Splicing and Regulation in Cardiac Maturation and Diseases

Circulation Research ◽

10.1161/res.113.suppl_1.a235 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

CHEN GAO ◽

Vincent Ren ◽

Jae-Hyung Lee ◽

Xinshu (Grace) Xiao ◽

Jau-nian Chen ◽

...

Keyword(s):

Heart Failure ◽

Alternative Splicing ◽

Cardiac Function ◽

Rna Splicing ◽

Cardiac Development ◽

Mrna Splicing ◽

Pcr Analysis ◽

Mouse Heart ◽

Global Pattern ◽

Alternative Mrna Splicing

Background: The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is also implicated in many human diseases including cancer. However, the global pattern of alternative mRNA splicing during cardiac development and diseases is unknown, and the regulatory mechanisms remain unexplored. Methods and Results: Using deep RNA-Sequencing, we have identified global alternative splicing changes associated with both cardiac development and pathological remodeling in mouse heart following pressure-overload induced heart failure. The alternative RNA splicing events observed in failing hearts mimics the profile in fetal hearts, suggesting a fetal-like RNA splicing program induced in diseased hearts. Using RNA-Seq database and real-time PCR analysis, we examined the expression profile of a large number of known alternative splicing regulators. Among them, we identified Fox1 as a significantly induced regulator during cardiac development in zebrafish, mouse and human, and down-regulated in both mouse and human failing hearts. Morpholino mediated Fox1 knockdown in zebrafish embryos led to lethal phenotype associated with reduced cardiac function and defects in chamber specificity. This phenotype could be rescued by re-expressing both zebrafish and mouse Fox1 gene, suggesting a highly conserved cardiac function of Fox1 for normal cardiac development and function in vertebrates. Conclusion: Our study provided the first comprehensive analysis of mRNA splicing regulation in heart during post-natal development and heart failure, and identified Fox1 as a key regulator for alternative RNA splicing in heart. This study expands our current understanding to the complexity of cardiac transcriptome, and reveals the functional importance of RNA-splicing in cardiac development and diseases.

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Human isoleucyl-tRNA synthetase: Sequence of the cDNA, alternative mRNA splicing, and the characteristics of an unusually long C-terminal extension

Gene ◽

10.1016/0378-1119(94)00634-5 ◽

1995 ◽

Vol 155 (2) ◽

pp. 299-304 ◽

Cited By ~ 13

Author(s):

Ralph C. Nichols ◽

Nina Raben ◽

Cornelius F. Boerkoel ◽

Paul H. Plotz

Keyword(s):

Trna Synthetase ◽

Mrna Splicing ◽

Alternative Mrna Splicing

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Extracellular matrix protein-1 secretory isoform promotes ovarian cancer through increasing alternative mRNA splicing and stemness

Nature Communications ◽

10.1038/s41467-021-24315-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Huijing Yin ◽

Jingshu Wang ◽

Hui Li ◽

Yinjue Yu ◽

Xiaoling Wang ◽

...

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Mrna Splicing ◽

Extracellular Matrix Protein ◽

Good Survival ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Multiple Organs ◽

Extracellular Matrix Protein 1 ◽

Alternative Mrna Splicing ◽

Nuclear Ribonucleoprotein

AbstractExtracellular matrix protein-1 (ECM1) promotes tumorigenesis in multiple organs but the mechanisms associated to ECM1 isoform subtypes have yet to be clarified. We report in this study that the secretory ECM1a isoform induces tumorigenesis through the GPR motif binding to integrin αXβ2 and the activation of AKT/FAK/Rho/cytoskeleton signaling. The ATP binding cassette subfamily G member 1 (ABCG1) transduces the ECM1a-integrin αXβ2 interactive signaling to facilitate the phosphorylation of AKT/FAK/Rho/cytoskeletal molecules and to confer cancer cell cisplatin resistance through up-regulation of the CD326-mediated cell stemness. On the contrary, the non-secretory ECM1b isoform binds myosin and blocks its phosphorylation, impairing cytoskeleton-mediated signaling and tumorigenesis. Moreover, ECM1a induces the expression of the heterogeneous nuclear ribonucleoprotein L like (hnRNPLL) protein to favor the alternative mRNA splicing generating ECM1a. ECM1a, αXβ2, ABCG1 and hnRNPLL higher expression associates with poor survival, while ECM1b higher expression associates with good survival. These results highlight ECM1a, integrin αXβ2, hnRNPLL and ABCG1 as potential targets for treating cancers associated with ECM1-activated signaling.

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Structural Protein 4.1 in the Nucleus of Human Cells: Dynamic Rearrangements during Cell Division

The Journal of Cell Biology ◽

10.1083/jcb.137.2.275 ◽

1997 ◽

Vol 137 (2) ◽

pp. 275-289 ◽

Cited By ~ 75

Author(s):

Sharon Wald Krauss ◽

Carolyn A. Larabell ◽

Stephen Lockett ◽

Philippe Gascard ◽

Sheldon Penman ◽

...

Keyword(s):

Cell Division ◽

Nuclear Matrix ◽

Mammalian Cells ◽

Matrix Protein ◽

Protein Isoforms ◽

Red Cell ◽

Structural Protein ◽

Red Cell Membrane ◽

Protein 4.1 ◽

Alternative Mrna Splicing

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.

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Abstract 15: Global RNA Splicing Regulation in Cardiac Maturation

Circulation Research ◽

10.1161/res.117.suppl_1.15 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Chen Gao ◽

Shuxun Ren ◽

Jae-Hyung Lee ◽

Yun-Hua Esther Hsiao ◽

Xinshu (Grace) Xiao ◽

...

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Rna Sequencing ◽

Rna Splicing ◽

Mrna Splicing ◽

Splicing Regulation ◽

Sequencing Analysis ◽

Cell Based Therapy ◽

Alternative Mrna Splicing ◽

Cardiac Maturation

Background: The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is implicated in both development and disease. However, the change of alternative mRNA splicing during cardiomyocytes maturation is unknown, and the regulatory mechanisms remain unexplored. Methods and Results: Using deep RNA-Sequencing, we identified global alternative splicing changes associated with both cardiac development and pathological remodeling in mouse heart. Further, we identified a highly conserved splicing regulator-RBFox1 to be significantly induced during zebrafish, mouse and human cardiac maturation. RBFox1 expression was also detected in cardiomyocytes derived from both mouse and human embryonic stem cells but at much lower levels comparing to adult heart. In zebrafish embryos, inactivation of RBFox1 caused cardiomyocyte maturation defects. Expression of RBFox1 in cultured neonatal cardiomyocytes was sufficient to promote maturation by reducing fetal marker gene expression while increasing calcium handling gene expression including RyR and promoting sarcomere organization. Deep RNA-Sequencing analysis showed that RBFox1 expression promoted alternative splicing in genes involved in calcium cycling, blood vessel development and muscle contraction. Finally, we identified a highly conserved mutually exclusive alternative splicing event of transcription factor MEF2 to be a direct downstream target of RBFox1. Expression of individual MEF2 splicing variants led to different cardiac developmental phenotypes in zebrafish, indicating their different transcriptional activities. Conclusion: Our study provided the first comprehensive analysis of mRNA splicing regulation in heart during post-natal development and heart failure, and identified RBFox1 as a key regulator for alternative RNA splicing during cardiomyocytes maturation. Further exploration of RBFox1 mediated RNA splicing regulation in heart may yield novel insight to the underlying mechanisms of cardiac maturation and new approach to improve cell based therapy for heart diseases.

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A Cytoplasmic RNA Virus Alters the Function of the Cell Splicing Protein SRSF2

Journal of Virology ◽

10.1128/jvi.02488-16 ◽

2017 ◽

Vol 91 (7) ◽

Cited By ~ 9

Author(s):

Efraín E. Rivera-Serrano ◽

Ethan J. Fritch ◽

Elizabeth H. Scholl ◽

Barbara Sherry

Keyword(s):

Rna Splicing ◽

Cytopathic Effect ◽

Rna Virus ◽

Mrna Splicing ◽

Splicing Factor ◽

Messenger Rnas ◽

Nuclear Speckles ◽

Protein Variant ◽

Antiviral Responses ◽

Reovirus Replication

ABSTRACT To replicate efficiently, viruses must create favorable cell conditions and overcome cell antiviral responses. We previously reported that the reovirus protein μ2 from strain T1L, but not strain T3D, represses one antiviral response: alpha/beta interferon signaling. We report here that T1L, but not T3D, μ2 localizes to nuclear speckles, where it forms a complex with the mRNA splicing factor SRSF2 and alters its subnuclear localization. Reovirus replicates in cytoplasmic viral factories, and there is no evidence that reovirus genomic or messenger RNAs are spliced, suggesting that T1L μ2 might target splicing of cell RNAs. Indeed, RNA sequencing revealed that reovirus T1L, but not T3D, infection alters the splicing of transcripts for host genes involved in mRNA posttranscriptional modifications. Moreover, depletion of SRSF2 enhanced reovirus replication and cytopathic effect, suggesting that T1L μ2 modulation of splicing benefits the virus. This provides the first report of viral antagonism of the splicing factor SRSF2 and identifies the viral protein that determines strain-specific differences in cell RNA splicing. IMPORTANCE Efficient viral replication requires that the virus create favorable cell conditions. Many viruses accomplish this by repressing specific antiviral responses. We demonstrate here that some mammalian reoviruses, RNA viruses that replicate strictly in the cytoplasm, express a protein variant that localizes to nuclear speckles, where it targets a cell mRNA splicing factor. Infection with a reovirus strain that targets this splicing factor alters splicing of cell mRNAs involved in the maturation of many other cell mRNAs. Depletion of this cell splicing factor enhances reovirus replication and cytopathic effect. Our results provide the first evidence of viral antagonism of this splicing factor and suggest that downstream consequences to the cell are global and benefit the virus.

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Asian Zika Virus Isolate Significantly Changes the Transcriptional Profile and Alternative RNA Splicing Events in a Neuroblastoma Cell Line

Viruses ◽

10.3390/v12050510 ◽

2020 ◽

Vol 12 (5) ◽

pp. 510 ◽

Cited By ~ 3

Author(s):

Gaston Bonenfant ◽

Ryan Meng ◽

Carl Shotwell ◽

Pheonah Badu ◽

Anne F. Payne ◽

...

Keyword(s):

Alternative Splicing ◽

Zika Virus ◽

Rna Splicing ◽

Virus Isolate ◽

Neuroblastoma Cell ◽

Neuroblastoma Cell Line ◽

Mrna Splicing ◽

Transcriptional Profile ◽

Protein Isoforms ◽

Functionally Diverse

The alternative splicing of pre-mRNAs expands a single genetic blueprint to encode multiple, functionally diverse protein isoforms. Viruses have previously been shown to interact with, depend on, and alter host splicing machinery. The consequences, however, incited by viral infection on the global alternative slicing (AS) landscape are under-appreciated. Here, we investigated the transcriptional and alternative splicing profile of neuronal cells infected with a contemporary Puerto Rican Zika virus (ZIKVPR) isolate, an isolate of the prototypical Ugandan ZIKV (ZIKVMR), and dengue virus 2 (DENV2). Our analyses revealed that ZIKVPR induced significantly more differential changes in expressed genes compared to ZIKVMR or DENV2, despite all three viruses showing equivalent infectivity and viral RNA levels. Consistent with the transcriptional profile, ZIKVPR induced a higher number of alternative splicing events compared to ZIKVMR or DENV2, and gene ontology analyses highlighted alternative splicing changes in genes associated with mRNA splicing. In summary, we show that ZIKV affects cellular RNA homeostasis not only at the transcriptional levels but also through the alternative splicing of cellular transcripts. These findings could provide new molecular insights into the neuropathologies associated with this virus.

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C3G dynamically associates with Nuclear speckles and regulates mRNA splicing

10.1101/159269 ◽

2017 ◽

Author(s):

Dhruv Kumar Shakyawar ◽

Bhattiprolu Muralikrishna ◽

Vegesna Radha

Keyword(s):

Rna Splicing ◽

Mrna Splicing ◽

C2c12 Cells ◽

Splicing Factors ◽

Nuclear Speckles ◽

Protein Levels ◽

Ras Family ◽

Reversible Association ◽

And Function ◽

Mammalian Embryonic Development

AbstractC3G (RapGEF1), essential for mammalian embryonic development, is ubiquitously expressed and undergoes regulated nucleo-cytoplasmic exchange. Here we show that C3G localizes to SC35 positive nuclear speckles, and regulates splicing activity. Reversible association of C3G with speckles was seen upon inhibition of transcription and splicing. C3G shows partial colocalization with SC35, and is recruited to a chromatin and RNase sensitive fraction of speckles. Its presence in speckles is dependent on intact cellular actin cytoskeleton, and is lost upon expression of the kinase, Clk1. Rap1, a substrate of C3G, is also present in nuclear speckles and inactivation of Rap signalling by expression of GFP- Rap1GAP, alters speckle morphology and number. Enhanced association of C3G with speckles is seen upon GSK3β inhibition, or differentiation of C2C12 cells to myotubes. CRISPR/Cas9 mediated knockdown of C3G resulted in decreased splicing activity and reduced staining for SC35 in speckles. C3G knockout clones of C2C12 as well as MDA-MB- 231 showed reduced protein levels of several splicing factors compared to control cells. Our results identify C3G and Rap1 as novel components of nuclear speckles and a role for C3G in regulating cellular RNA splicing activity.SummaryNuclear speckles are sites for pre-mRNA splicing. We provide evidence for localization and function of a Ras family GTPase, Rap1 and its exchange factor C3G in nuclear speckles.

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