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

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.

scholarly journals Position Specific Alternative Splicing and Gene Expression Profiles Along the Tonotopic Axis of Chick Cochlea

2021 ◽  
Vol 8 ◽  
Author(s):  
Heiyeun Koo ◽  
Jae Yeon Hwang ◽  
Sungbo Jung ◽  
Hyeyoung Park ◽  
Jinwoong Bok ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Profiles ◽  
Single Gene ◽  
Gene Expression Profiles ◽  
Binding Motif ◽  
Dependent Manner ◽  
Mrna Isoforms

Alternative splicing (AS) refers to the production of multiple mRNA isoforms from a single gene due to alternative selection of exons or splice sites during pre-mRNA splicing. It is a primary mechanism of gene regulation in higher eukaryotes and significantly expands the functional complexity of eukaryotic organisms, contributing to animal development and disease. Recent studies have shown that AS also influences functional diversity by affecting the transcriptomic and proteomic profiles in a position-dependent manner in a single organ. The peripheral hearing organ, the cochlea, is organized to detect sounds at different frequencies depending on its location along the longitudinal axis. This unique functional configuration, the tonotopy, is known to be facilitated by differential gene expression along the cochlear duct. We profiled transcriptome-wide gene expression and AS changes that occur within the different positions of chick cochlea. These analyses revealed distinct gene expression profiles and AS, including a splicing program that is unique to tonotopy. Changes in the expression of splicing factors PTBP3, ESRP1, and ESRP2 were demonstrated to contribute to position-specific AS. RNA-binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS at different positions by different RNA-binding proteins. These data, along with gene ontology (GO) analysis, represent a comprehensive analysis of the dynamic regulation of AS at different positions in chick cochlea.

scholarly journals RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Oncogene ◽  
2021 ◽  
Author(s):  
Qiuxia Yan ◽  
Peng Zeng ◽  
Xiuqin Zhou ◽  
Xiaoying Zhao ◽  
Runqiang Chen ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Aerobic Glycolysis ◽  
Histological Grade ◽  
Migration And Invasion ◽  
Binding Motif ◽  
Hnrnp A1

AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

Arginine Methylation Of RBM15 By PRMT1 Controls Alternative Splicing Of Genes Involved In Megakaryocyte Differentiation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2443-2443
Author(s):  
Xinyang Zhao ◽  
Li Zhang ◽  
Rui Wang ◽  
Ngoc Tung Trans ◽  
Hairui Su ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Chromosome Translocation ◽  
Arginine Methylation ◽  
Mass Spectrometry Analysis ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Spectrometry Analysis ◽  
Megakaryocyte Differentiation

Abstract More than 90% of under one year old infants with acute megakaryoblastic leukemia (AMKL) have chromosome translocation t(1;22)(p13;q13) with RBM15 fused to MKL1. RBM15 encodes an RNA binding protein important for hematopoietic stem cell self-renewal and differentiation. In agreement with its roles in AMKL, RBM15 is required for normal megakaryocyte differentiation. We found that higher expression of PRMT1 (Protein Arginine Methyltransferase) is commonly seen in M7 leukemia patient samples than other types of myeloid leukemia and that RBM15 is a bona fide methylation target for PRMT1. Using mass spectrometry analysis, we mapped the PRMT1 mediated mono-methylated site. The enzymatic activity of the PRMT1 V2 isoform is required for RBM15 degradation, as both shRNA molecules knocking down PRMT1 and small chemical PRMT1 inhibitors stabilize the RBM15 protein. Mutation of the methylation site to lysine blocks the ubiquitylation mediated degradation. Thus the degradation is a methylation dependent process. We identified the E3 ligase responsible for the degradation. Down-regulation of the RBM15 protein changes the isoform ratio of genes including GATA1 critical megakaryocyte differentiation. We found that RBM15 regulates its interaction with SF3B1A in methylation dependent manner during alternative splicing of GATA1 pre-mRNA. Thus, via methylation triggered RBM15 degradation, the megakaryocyte progenitor cells maintain a delicate balance between differentiation and proliferation by keeping the proper ratio of GATA1s and GATA1-full length mRNA. SF3B1A has been shown to be mutated in myeloid dysplasia syndrome and in several different types of leukemia. Methylation by PRMT1 links the two types of leukemic genes into a single pathway. Our results imply that targeting PRMT1/RBM15 pathway might be a potential therapy for AMKL and other blood malignancies. Disclosures: No relevant conflicts of interest to declare.

Regulation of CD19 Exon 2 Inclusion in B-Lymphoid Cells By Splicing Factors and Epigenetic Marks

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2425-2425 ◽  
Author(s):  
Kathryn Black ◽  
Elena Sotillo ◽  
Nicole Martinez ◽  
Matthew Gazzara ◽  
Alejandro Barrera ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Lymphoid Cells ◽  
Splicing Factors ◽  
Great Success ◽  
Dependent Manner ◽  
Exon 2 ◽  
Hnrnp C

Abstract CD19 is expressed broadly on the surface of B-cells during normal development and malignant growth, making it a good target for immunotherapy. While immunotherapies targeting CD19 have had great success against pediatric B-cell acute lymphoblastic leukemia (B-ALL), relapses lacking the CD19 epitope still occur (Maude et al., 2014). We have discovered that alternative splicing of CD19, in particular the skipping of exon 2, is responsible for the loss of CD19 extracellular domains, causing resistance to therapy (Sotillo et al., 2015). Here we investigate the molecular mechanism of CD19 exon 2 skipping. The sequence-based algorithm AVISPA (Barash et al., 2013) predicts several splicing factors (SF) to bind near exon 2. We used RNA crosslink immunoprecipitation (CLIP) in nuclear lysates from Nalm-6 B-ALL cells to test the direct binding to exon 2 of 9 AVISPA-predicted SFs and 6 SFs commonly involved in exon skipping. This allowed us to identify SRSF3, hnRNP-A, and hnRNP-C as CD19 exon 2-bound proteins. Subsequent siRNA knockdown experiments reveled that downregulation of SRSF3, but not hnRNP-C, increases the frequency of exon 2 skipping in a dose dependent manner, suggesting that SRSF3 promotes the inclusion of exon 2. To further validate the role of SRSF3 in CD19 splicing we mined the publicly available GSE52834 dataset where 22 RNA binding proteins were knocked down in the GM19238 lymphoblastoid cell line. Of all siRNAs tested, only the anti-SRSF3 siRNA caused an increase in exon 2 skipping, suggesting that SRSF3 is indeed the key regulator of CD19 splicing. Interestingly, SRSF3 has been shown to interact with PSIP1, a cofactor known to "read" modified histone H3K36me3 (Pradeepa et al., 2012), suggesting a convergence of splicing-based and epigenetics mechanisms. Indeed, exonic regions in genomic DNA are enriched for H3K36me3, and knockdown of Setd2, the H3K36 methyltransferase, results in changes in exon inclusion (Luco et al., 2010; Brown et al., 2012; Hnilicova and Stanek, 2011). Thus, we are currently investigating the connection between the H3K36me3 marks in the CD19 locus and alternative splicing of CD19. Our data could suggest a method of restoring full-length CD19 expression in immunotherapy-resistant cancers using epigenetic drugs. Maude, S L, Noelle, F, Shaw, PA, Aplenc, R, Barrett, DM, Bunin, NJ, Chew, A, Gonzalez, VE, Zheng, Z, Lacey, SF, Mahnke, YD, Melenhorst, JJ, Rheingold, SR, Shen, A, Teachey, DT, Levine, BL, June CH, Porter, DL, and Grupp, SA. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014; 371: 1507-1517. Sotillo, E, Barrett, D, Bagashev, A, Black, K, Lanauze, C, Oldridge, D, Sussman, R, Harrington, C, Chung, EY, Hofmann, TJ, Maude, SL, Martinez, NM, Raman, P, Ruella, M, Allman, D, Jacoby, E, Fry, T, Barash, Y, Lynch, KW, Mackall, C, Maris, J, Grupp, SA, and Thomas-Tikhonenko, A. Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope andcontributes to treatment failure. 2015AACR Annual Meeting, Philadelphia. Barash Y, Vaquero-Garcia J, González-Vallinas J, Xiong HY, Gao W, Lee LJ, and Frey BJ. AVISPA: a web tool for the prediction and analysis of alternative splicing. Genome Biol 2013; 14(10):R114. Pradeepa, MM, Sutherland, HG, Ule, J, Grimes, GR, and Bickmore, WA. Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing. PLOS Genets 2012; 8:e1002717. Luco, RF, Pan, Q, Tominaga, K, Blencowe, BJ, Pereira-Smith, OM, Misteli, T. Regulation of alternative splicing by histone modifications. Science 2010; 327: 996-1000. Brown, SJ, Stoilov, P, and Xing, Y. Chromatin and epigenetic regulation of pre-mRNA processing. Human Mol Genets 2012; 21:R90-R96. Hnilicova, J, and Stanek, D. Where splicing joins chromatin. Nucleus 2011; 2:182-188. Disclosures No relevant conflicts of interest to declare.

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

2019 ◽  
Vol 39 (12) ◽  
pp. 2355-2367 ◽  
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.

scholarly journals Synovial Sarcoma Translocation (SYT) Encodes a Nuclear Receptor Coactivator

Endocrinology ◽  
2005 ◽  
Vol 146 (9) ◽  
pp. 3892-3899 ◽  
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.

scholarly journals QKI-5 regulates the alternative splicing of cytoskeletal gene ADD3 in lung cancer

2020 ◽  
Author(s):  
Jin-Zhu Wang ◽  
Xing Fu ◽  
Zhaoyuan Fang ◽  
Hui Liu ◽  
Feng-Yang Zong ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Dependent Manner ◽  
Genome Wide ◽  
Intron Region ◽  
And Migration ◽  
Nucleotide Resolution ◽  
Anti Tumor Activity

Abstract Accumulating evidence indicates that the alternative splicing program undergoes extensive changes during cancer development and progression. The RNA-binding protein QKI-5 is frequently down-regulated and exhibits anti-tumor activity in lung cancer. However, little is known about the functional targets and regulatory mechanism of QKI-5. Here, we report that up-regulation of exon 14 inclusion of cytoskeletal gene Adducin 3 (ADD3) significantly correlates with a poor prognosis in lung cancer. QKI-5 inhibits cell proliferation and migration in part through suppressing the splicing of ADD3 exon 14. Through genome-wide mapping of QKI-5 binding sites in vivo at nucleotide resolution by iCLIP-seq analysis, we found that QKI-5 regulates alternative splicing of its target mRNAs in a binding position-dependent manner. By binding to multiple sites in an upstream intron region, QKI-5 represses the splicing of ADD3 exon 14. We also identified several QKI mutations in tumors, which cause dysregulation of the splicing of QKI targets ADD3 and NUMB. Taken together, our results reveal that QKI-mediated alternative splicing of ADD3 is a key lung cancer-associated splicing event, which underlies in part the tumor suppressor function of QKI.

scholarly journals Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

2015 ◽  
Vol 112 (50) ◽  
pp. 15444-15449 ◽  
Author(s):  
Frida Holm ◽  
Eva Hellqvist ◽  
Cayla N. Mason ◽  
Shawn A. Ali ◽  
Nathaniel Delos-Santos ◽  
...  
Keyword(s):  
Stem Cell ◽  
Alternative Splicing ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Rna Binding ◽  
Combined Treatment ◽  
Embryonic Stem ◽  
Chronic Phase ◽  
Dependent Manner ◽  
Splice Isoform

Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8–10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation–related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.

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