RBM20-Mediated Pre-mRNA Splicing Has Muscle-Specificity and Differential Hormonal Responses between Muscles and in Muscle Cell Cultures

Pre-mRNA splicing plays an important role in muscle function and diseases. The RNA binding motif 20 (RBM20) is a splicing factor that is predominantly expressed in muscle tissues and primarily regulates pre-mRNA splicing of Ttn, encoding a giant muscle protein titin that is responsible for muscle function and diseases. RBM20-mediated Ttn splicing has been mostly studied in heart muscle, but not in skeletal muscle. In this study, we investigated splicing specificity in different muscle types in Rbm20 knockout rats and hormonal effects on RBM20-mediated splicing both in cellulo and in vivo studies. The results revealed that RBM20 is differentially expressed across muscles and RBM20-mediated splicing is muscle-type specific. In the presence of RBM20, Ttn splicing responds to hormones in a muscle-type dependent manner, while in the absence of RBM20, Ttn splicing is not affected by hormones. In differentiated and undifferentiated C2C12 cells, RBM20-mediated splicing in response to hormonal effects is mainly through genomic signaling pathway. The knowledge gained from this study may help further understand muscle-specific gene splicing in response to hormone stimuli in different muscle types.

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Role of HuR in Skeletal Myogenesis through Coordinate Regulation of Muscle Differentiation Genes

Molecular and Cellular Biology ◽

10.1128/mcb.23.14.4991-5004.2003 ◽

2003 ◽

Vol 23 (14) ◽

pp. 4991-5004 ◽

Cited By ~ 123

Author(s):

Angélica Figueroa ◽

Ana Cuadrado ◽

Jinshui Fan ◽

Ulus Atasoy ◽

George E. Muscat ◽

...

Keyword(s):

Rna Binding ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Dependent Manner ◽

Skeletal Myogenesis ◽

Critical Function ◽

Mouse Muscle ◽

Coordinate Regulation

ABSTRACT In this report, we investigate the role of the RNA-binding protein HuR during skeletal myogenesis. At the onset of myogenesis in differentiating C2C12 myocytes and in vivo in regenerating mouse muscle, HuR cytoplasmic abundance increased dramatically, returning to a predominantly nuclear presence upon completion of myogenesis. mRNAs encoding key regulators of myogenesis-specific transcription (myogenin and MyoD) and cell cycle withdrawal (p21), bearing AU-rich regions, were found to be targets of HuR in a differentiation-dependent manner. Accordingly, mRNA half-lives were highest during differentiation, declining when differentiation was completed. Importantly, HuR-overexpressing C2C12 cells displayed increased target mRNA expression and half-life and underwent precocious differentiation. Our findings underscore a critical function for HuR during skeletal myogenesis linked to HuR's coordinate regulation of muscle differentiation genes.

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miRNA-Based Regulation of Alternative RNA Splicing in Metazoans

International Journal of Molecular Sciences ◽

10.3390/ijms222111618 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11618

Author(s):

Anna L. Schorr ◽

Marco Mangone

Keyword(s):

Neurological Disorders ◽

Rna Splicing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Splicing ◽

Splicing Factors ◽

Transcriptional Level ◽

Dependent Manner ◽

Non Coding Rnas ◽

Specific Mrna

Alternative RNA splicing is an important regulatory process used by genes to increase their diversity. This process is mainly executed by specific classes of RNA binding proteins that act in a dosage-dependent manner to include or exclude selected exons in the final transcripts. While these processes are tightly regulated in cells and tissues, little is known on how the dosage of these factors is achieved and maintained. Several recent studies have suggested that alternative RNA splicing may be in part modulated by microRNAs (miRNAs), which are short, non-coding RNAs (~22 nt in length) that inhibit translation of specific mRNA transcripts. As evidenced in tissues and in diseases, such as cancer and neurological disorders, the dysregulation of miRNA pathways disrupts downstream alternative RNA splicing events by altering the dosage of splicing factors involved in RNA splicing. This attractive model suggests that miRNAs can not only influence the dosage of gene expression at the post-transcriptional level but also indirectly interfere in pre-mRNA splicing at the co-transcriptional level. The purpose of this review is to compile and analyze recent studies on miRNAs modulating alternative RNA splicing factors, and how these events contribute to transcript rearrangements in tissue development and disease.

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Robust RBM3 and β-klotho expression in developing neurons in the human brain

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x19878889 ◽

2019 ◽

Vol 39 (12) ◽

pp. 2355-2367 ◽

Cited By ~ 3

Author(s):

Travis C Jackson ◽

Keri Janesko-Feldman ◽

Shaun W Carlson ◽

Shawn E Kotermanski ◽

Patrick M Kochanek

Keyword(s):

Cortical Neurons ◽

Rna Binding ◽

Transmembrane Protein ◽

Adult Brain ◽

Human Infant ◽

Fibroblast Growth Factor 21 ◽

Binding Motif ◽

Dependent Manner ◽

Neuroprotective Effects

RNA binding motif 3 (RBM3) is a powerful neuroprotectant that inhibits neurodegenerative cell death in vivo and is a promising therapeutic target in brain ischemia. RBM3 is increased by the hormone fibroblast growth factor 21 (FGF21) in an age- and temperature-dependent manner in rat cortical neurons. FGF21 receptor binding is controlled by the transmembrane protein β-klotho, which is mostly absent in the adult brain. We discovered that RBM3/β-klotho is unexpectedly high in the human infant vs. adult brain (hippocampus/prefrontal cortex). The use of tissue homogenates in that study precluded a comparison of RBM3/β-klotho expression among different CNS cell-types, thus, omitted key evidence (i.e. confirmation of neuronal expression) that would otherwise provide a critical link to support their possible direct neuroprotective effects in humans. This report addresses that knowledge gap. High-quality fixed human hippocampus, cortex, and hypothalamic tissues were acquired from the NIH Neurobiobank (<1 yr (premature born) infants, 1 yr, 4 yr, and 34 yr). Dual labeling of cell-type markers vs. RBM3/β-klotho revealed enriched staining of targets in neurons in the developing brain. Identifying that RBM3/β-klotho is abundant in neurons in the immature brain is fundamentally important to guide protocol design and conceptual frameworks germane to future testing of these neuroprotective pathways in humans.

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Effect of cold acclimation on troponin I isoform expression in striated muscle of rainbow trout

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00127.2012 ◽

2012 ◽

Vol 303 (2) ◽

pp. R168-R176 ◽

Cited By ~ 13

Author(s):

Sarah L. Alderman ◽

Jordan M. Klaiman ◽

Courtney A. Deck ◽

Todd E. Gillis

Keyword(s):

Rainbow Trout ◽

Cold Acclimation ◽

Troponin I ◽

Striated Muscle ◽

Protein Isoforms ◽

Specific Gene ◽

Muscle Type ◽

Gene Transcripts ◽

Muscle Types ◽

Isoform Expression

In vertebrates each of the three striated muscle types (fast skeletal, slow skeletal, and cardiac) contain distinct isoforms of a number of different contractile proteins including troponin I (TnI). The functional characteristics of these proteins have a significant influence on muscle function and contractility. The purpose of this study was to characterize which TnI gene and protein isoforms are expressed in the different muscle types of rainbow trout ( Oncorhynchus mykiss) and to determine whether isoform expression changes in response to cold acclimation (4°C). Semiquantitative real-time PCR was used to characterize the expression of seven different TnI genes. The sequence of these genes, cloned from Atlantic salmon ( Salmo salar) and rainbow trout, were obtained from the National Center for Biotechnology Information databases. One-dimensional gel electrophoresis and tandem mass spectrometry were used to identify the TnI protein isoforms expressed in each muscle type. Interestingly, the results indicate that each muscle type expresses the gene transcripts of up to seven TnI isoforms. There are significant differences, however, in the expression pattern of these genes between muscle types. In addition, cold acclimation was found to increase the expression of specific gene transcripts in each muscle type. The proteomics analysis demonstrates that fast skeletal and cardiac muscle contain three TnI isoforms, whereas slow skeletal muscle contains four. No other vertebrate muscle to date has been found to express as many TnI protein isoforms. Overall this study underscores the complex molecular composition of teleost striated muscle and suggests there is an adaptive value to the unique TnI profiles of each muscle type.

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Alternative Splicing Mediated by RNA-Binding Protein RBM24 Facilitates Cardiac Myofibrillogenesis in a Differentiation Stage-Specific Manner

Circulation Research ◽

10.1161/circresaha.121.320080 ◽

2021 ◽

Author(s):

Serena Huei-An Lu ◽

Kang-Zheng Lee ◽

Paul Wei-Che Hsu ◽

Liang-Yu Su ◽

Yu-Chen Yeh ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Binding ◽

Temporal Dynamics ◽

Building Blocks ◽

Cardiac Differentiation ◽

Actin Binding ◽

Binding Motif ◽

Dependent Manner ◽

Sarcomeric Proteins ◽

Sarcomere Assembly

Background: Mutations in genes encoding sarcomeric proteins lead to failures in sarcomere assembly, the building blocks of contracting muscles, resulting in cardiomyopathies that are a leading cause of morbidity and mortality worldwide. Splicing variants of sarcomeric proteins are crucial at different stages of myofibrillogenesis, accounting for sarcomeric structural integrity. RNA-binding motif protein 24 (RBM24) is known as a tissue-specific splicing regulator that plays an essential role in cardiogenesis. However, it had been unclear if the developmental stage-specific alternative splicing facilitated by RBM24 contributes to sarcomere assembly and cardiogenesis. Our aim isto study the molecular mechanism by which RBM24 regulates cardiogenesis and sarcomere assembly in a temporal-dependent manner. Methods: We ablated RBM24 from human embryonic stem cells (hESCs) using CRISPR/Cas9 techniques. Results: Although RBM24 -/- hESCs still differentiated into sarcomere-hosting cardiomyocytes, they exhibited disrupted sarcomeric structures with punctate Z-lines due to impaired myosin replacement during early myofibrillogenesis. Transcriptomics revealed >4000 genes regulated by RBM24. Among them, core myofibrillogenesis proteins (e.g. ACTN2, TTN, and MYH10) were misspliced. Consequently, MYH6 cannot replace non-muscle myosin MYH10, leading to myofibrillogenesis arrest at the early premyofibril stage and causing disrupted sarcomeres. Intriguingly, we found that the actin-binding domain (ABD; encoded by exon 6) of the Z-line anchor protein ACTN2 is predominantly excluded from early cardiac differentiation, whereas it is consistently included in human adult heart. CRISPR/Cas9-mediated deletion of exon 6 from ACTN2 in hESCs, as well as forced expression of full-length ACTN2 in RBM24 -/- hESCs, further corroborated that inclusion of exon 6 is critical for sarcomere assembly. Overall, we have demonstrated that RBM24-facilitated inclusion of exon 6 in ACTN2 at distinct stages of cardiac differentiation is evolutionarily conserved and crucial to sarcomere assembly and integrity. Conclusions: RBM24 acts as a master regulator to modulate the temporal dynamics of core myofibrillogenesis genes and thereby orchestrates sarcomere organization.

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Synovial Sarcoma Translocation (SYT) Encodes a Nuclear Receptor Coactivator

Endocrinology ◽

10.1210/en.2004-1513 ◽

2005 ◽

Vol 146 (9) ◽

pp. 3892-3899 ◽

Cited By ~ 14

Author(s):

Toshiharu Iwasaki ◽

Noriyuki Koibuchi ◽

William W. Chin

Keyword(s):

Synovial Sarcoma ◽

Hormone Receptors ◽

Rna Binding ◽

Thyroid Hormone Receptor ◽

Single Gene ◽

Binding Motif ◽

Dependent Manner ◽

Interacting Protein ◽

C Terminus ◽

The Brain

Abstract We previously cloned and characterized a novel RNA-binding motif-containing coactivator, named coactivator activator (CoAA), as a thyroid hormone receptor-binding protein-interacting protein using a Sos-Ras yeast two-hybrid screening system. A database search revealed that CoAA is identical with synovial sarcoma translocation (SYT)-interacting protein. Thus, we hypothesized that SYT could also function as a coactivator. Subsequently, we isolated a cDNA encoding a larger isoform of SYT, SYT-long (SYT-L), from the brain and liver total RNA using RT-PCR. SYT-L possesses an additional 31 amino acids in its C terminus compared with SYT, suggesting that these two SYT isoforms may be expressed from two mRNAs produced by alternative splicing of a transcript from a single gene. By Northern blot analysis, we found that SYT-L mRNA is expressed in several human embryonic tissues, such as the brain, liver, and kidney. However, we could not detect SYT-L in adult tissues. Glutathione-S-transferase pull-down studies showed that SYT binds to the C-terminus of CoAA, but not to the coactivator modulator. Both isoforms of SYT function as transcriptional coactivators of nuclear hormone receptors in a ligand- and dose-dependent manner in CV-1, COS-1, and JEG-3 cells. However, the pattern of transactivation was different between SYT and SYT-L among these cells. SYT synergistically activates transcription with CoAA. In addition, SYT activates transcription through activator protein-1, suggesting that SYT may function as a general coactivator. These results indicate that SYT activates transcription, possibly through CoAA, to interact with the histone acetyltransferase complex.

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Angiotensin II Influences Pre-mRNA Splicing Regulation by Enhancing RBM20 Transcription Through Activation of the MAPK/ELK1 Signaling Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms20205059 ◽

2019 ◽

Vol 20 (20) ◽

pp. 5059 ◽

Cited By ~ 2

Author(s):

Hanfang Cai ◽

Chaoqun Zhu ◽

Zhilong Chen ◽

Rexiati Maimaiti ◽

Mingming Sun ◽

...

Keyword(s):

Angiotensin Ii ◽

Protein Kinase ◽

Rna Binding ◽

Mapk Signaling ◽

Mrna Splicing ◽

Mitogen Activated Protein Kinase ◽

Binding Motif ◽

Cardiac Diseases ◽

Ang Ii ◽

The Impact

RNA binding motif 20 (RBM20) is a key regulator of pre-mRNA splicing of titin and other genes that are associated with cardiac diseases. Hormones, like insulin, triiodothyronine (T3), and angiotensin II (Ang II), can regulate gene-splicing through RBM20, but the detailed mechanism remains unclear. This study was aimed at investigating the signaling mechanism by which hormones regulate pre-mRNA splicing through RBM20. We first examined the role of RBM20 in Z-, I-, and M-band titin splicing at different ages in wild type (WT) and RBM20 knockout (KO) rats using RT-PCR; we found that RBM20 is the predominant regulator of I-band titin splicing at all ages. Then we treated rats with propylthiouracil (PTU), T3, streptozotocin (STZ), and Ang II and evaluated the impact of these hormones on the splicing of titin, LIM domain binding 3 (Ldb3), calcium/calmodulin-dependent protein kinase II gamma (Camk2g), and triadin (Trdn). We determined the activation of mitogen-activated protein kinase (MAPK) signaling in primary cardiomyocytes treated with insulin, T3, and Ang II using western blotting; MAPK signaling was activated and RBM20 expression increased after treatment. Two downstream transcriptional factors c-jun and ETS Transcription Factor (ELK1) can bind the promoter of RBM20. A dual-luciferase activity assay revealed that Ang II, but not insulin and T3, can trigger ELK1 and thus promote transcription of RBM20. This study revealed that Ang II can trigger ELK1 through activation of MAPK signaling by enhancing RBM20 expression which regulates pre-mRNA splicing. Our study provides a potential therapeutic target for the treatment of cardiac diseases in RBM20-mediated pre-mRNA splicing.

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RBM15 promotes hepatocellular carcinoma progression by regulating N6-methyladenosine modification of YES1 mRNA in an IGF2BP1-dependent manner

Cell Death Discovery ◽

10.1038/s41420-021-00703-w ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Xianlei Cai ◽

Yunhao Chen ◽

Da Man ◽

Beng Yang ◽

Xiaode Feng ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Transcriptional Activation ◽

Rna Binding ◽

Mapk Pathway ◽

Mitogen Activated Protein Kinase ◽

Luciferase Reporter ◽

Binding Motif ◽

Dependent Manner ◽

Rna Immunoprecipitation ◽

Hcc Cells

AbstractThe function of the N6-methyladenosine (m6A) methyltransferase RNA-binding motif protein 15 (RBM15) in hepatocellular carcinoma (HCC) has not been thoroughly investigated. Here we determined the clinical value, biological functions, and potential mechanisms of RBM15 in HCC. Expression of RBM15 was identified using tissue microarrays and online databases. A risk-prediction model based on RBM15 was developed and validated. We determined the biological role of RBM15 on HCC cells in vitro and in vivo. RNA sequencing was used to screen candidate targets of RBM15. Subsequently, the m6A dot blot assay, methylated RNA immunoprecipitation qPCR, dual-luciferase reporter assays, RNA decay assay, and RNA immunoprecipitation qPCR were employed to explore the mechanisms of RBM15. Our study showed that RBM15 was highly expressed in HCC, and overexpression of RBM15 indicated a worse outcome. A new nomogram combining RBM15 with age and TNM stage was developed and validated to predict the outcome of HCC patients; our nomogram increased the prediction accuracy of the TNM system. Functionally, RBM15 facilitates the proliferation and invasiveness of HCC. RBM15-mediated m6A modification contributed to a post-transcriptional activation of YES proto-oncogene 1 (YES1) in an insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1)-dependent manner. In addition, YES1 was confirmed as an oncogene in HCC cells by activating the mitogen-activated protein kinase (MAPK) pathway. In conclusion, RBM15-mediated m6A modification might facilitate the progression of HCC via the IGF2BP1–YES1–MAPK axis. RBM15 may be a promising biomarker in the outcome prediction of HCC.

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RBM20 phosphorylation on serine/arginine domain is crucial to regulate pre-mRNA splicing and protein shuttling in the heart

10.1101/2020.09.15.297002 ◽

2020 ◽

Author(s):

Mingming Sun ◽

Yutong Jin ◽

Chaoqun Zhu ◽

Yanghai Zhang ◽

Martin Liss ◽

...

Keyword(s):

Protein Trafficking ◽

Rna Binding ◽

Heart Function ◽

Mrna Splicing ◽

Splicing Factors ◽

Binding Motif ◽

Amino Acid Residues ◽

Cellular Mechanisms ◽

Sr Protein ◽

Protein Protein Interaction

AbstractMolecular and cellular mechanisms of mutations of splicing factors in heart function are not well understood. The splicing of precursor mRNA is dependent on an essential group of splicing factors containing serine-arginine (SR) domain(s) that are critical for protein-RNA and protein-protein interaction in the spliceosome assembly. Phosphorylation of SR domains plays a key role in splicing control and the distribution of splicing factors in the cell. RNA binding motif 20 (RBM20) is a splicing factor predominantly expressed in muscle tissues with the highest expression level in the heart. However, its phosphorylation status is completely unknown up-to-date. In this study, we identified sixteen amino acid residues that are phosphorylated by middle-down mass spectrometry. Four of them are located in the SR domain, and two out of the four residues, S638 and S640, play an essential role in splicing control and facilitate RBM20 shuttling from the nucleus to the cytoplasm. Re-localization of RBM20 promotes protein aggregation in the cytoplasm. We have also verified that SR-protein kinases (SRPKs), cdc2-like kinases (CLKs) and protein kinase B (PKB or AKT) phosphorylate S638 and S640. Mutations of S638A and S640G reduce RBM20 phosphorylation and disrupt the splicing. Taken together, we determine the phosphorylation status of RBM20 and provide the first evidence that phosphorylation on SR domain is crucial for pre-mRNA splicing and protein trafficking. Our findings reveal a new role of RBM20 via protein shuttling in cardiac function.

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SAFA facilitates chromatin opening of immune genes through interacting with nascent antiviral RNAs

10.1101/2021.07.06.451336 ◽

2021 ◽

Author(s):

Lili Cao ◽

Yunfei Li ◽

Yujie Luo ◽

Xuefei Guo ◽

Siji Li ◽

...

Keyword(s):

Viral Infection ◽

Matrix Protein ◽

Rna Binding ◽

Expression Patterns ◽

Chromatin Accessibility ◽

Specific Gene ◽

Dependent Manner ◽

Immune Genes ◽

C Terminus ◽

A Genome

Regulation of chromatin accessibility determines the transcription activities of genes, which endow the host with function specific gene expression patterns. It remains unclear how chromatin accessibility is specifically directed, particularly, during host defense against viral infection. We previously reported that the nuclear matrix protein SAFA surveils viral RNA and regulates antiviral immune genes expression. However, how SAFA regulates the expression and what determines the specificity of antiviral immune genes remains unknown. Here, we identified that the depletion of SAFA specifically decreased the chromatin accessibility, activation and expression of virus induced genes in a genome wide scale after VSV infection. SAFA exclusively bound with antiviral related RNAs, which mediated the specific opening of the according chromatin and robust transcription of these genes. Knockdown of these associated RNAs dampened the accessibility of corresponding genes in an extranuclear signaling pathway dependent manner. Moreover, VSV infection cleaved SAFA protein at the C terminus which deprived its RNA binding ability for immune evasion. Thus, our results demonstrated that SAFA and the interacting RNA products during viral infection collaborate and remodel chromatin accessibility to facilitate anti viral innate immune response.

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