Bone Marrow Microenvironment Regulates Alternative Splicing Events in Myeloma Cells through Downregulation of RNA Binding Protein Fox2

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4714-4714
Author(s):  
Weihua Song ◽  
Chaolin Zhang ◽  
Yiguo Hu ◽  
Maria Gkotzamanidou ◽  
Parantu Shah ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alternative Splicing ◽  
Cell Lines ◽  
Actin Polymerization ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Splicing Factor ◽  
Pcr Analysis ◽  
Rna Seq ◽  
Protein Coding

Abstract Alternative splicing is a crucial mechanism for gene regulation, which enhances the diversity of transcriptome and proteome. Misregulation of alternative splicing has been implicated in number of disease processes including cancer. Our data utilizing exon array profile from 170 uniformly treated newly diagnosed patients with MM confirms clinical relevance of splicing as demonstrated by impact of level and extent of alternate splicing on both progression free and overall survival. Fox2, a RNA splicing factor, is one of the most important genes predicting clinical outcome in these patients. We confirmed Fox2 expression in 10 MM cell lines at both RNA and protein levels. Immunohistochemistry staining showed a predominant nuclear localization of Fox2. Importantly, we also observed that MM cell - bone marrow stromal cells (BMSC) interaction led to significant inhibition of Fox2 expression in MM cells. Similar response was also observed using BMSC supernatants. While IL6 treatment significantly downregulated the expression of Fox2 in MM1S and RPMI8226 cells in a dose-dependent manner, IGF-1 treatment had no significant impact on Fox2 expression in MM cell lines. Since Fox2 has been described to plays a role in the maintenance of cell cytoskeleton, we therefore evaluated whether Fox2 might influence the migration and adhesion in MM cells. Transwell migration assay showed enhanced migration rate of Fox2-knocking down- MM1S and RPMI8226 cells versus controls. We also observed the increased cell adhesion to fibronetin in both cell lines upon Fox2 knockdown. Actin polymerization evaluated by Alexa488-conjugated phalloidin staining and confocal microscope analysis showed Fox2 knocking down cells with increased actin polymerization in both MM1S and RPMI8226 cell lines. Interstingly, we observed that Fox2 knockdown in MM cell lines did not affect the cell proliferation and survival. As Fox-2 is a splicing factor, we further evaluated the molecular impact of Fox2 expression in multiple myeloma by RNA-seq analysis. Our data revealed that Fox2 functions in regulating both protein-coding and non-coding RNA alternative splicing. Knockdown of Fox2 resulted in significant isoform up-regulation (60 in MM1S and 151 in RPMI8226) and down-regulation (70 in MM1S and 69 in RPMI8226). Gene enrichment analysis showed these genes are clustered in cell cytoskeleton regulation, microtubule-based movement, ATP binding, amongst others. Our study then focused on Fox2 knockdown-induced significant isoform switch in MM cell lines. We designed the primers testing the spliced exons and confirm the isoform switch in MM cells by PCR analysis (e.g. Pyk2 and PFDN6). Importantly, our RNA seq data showed that Fox2 regulates the expression of a series of microRNAs and long-noncoding RNAs (e.g. MALAT1 RPMI8226 CNT (58) vs Fox2 knockdown (108)), which provides us a new insight into impact of Fox2 on non-protein coding RNAs. We have also validated the RNA-seq data by Q-PCR analysis. In summary, our results identify Fox2 as a biologically important RNA binding protein that is regulated by bone marrow microenvironment interaction and with essential function and potential clinical implications in multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Bone Marrow Microenvironment Affects The Pathogenesis Of Multiple Myeloma Through Downregulation Of Alternative Splicing Factor Fox2 In Myeloma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3085-3085
Author(s):  
Weihua Song ◽  
Chaolin Zhang ◽  
Yiguo Hu ◽  
Maria Gkotzamanidou ◽  
Parantu Shah ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Alternative Splicing ◽  
Cell Lines ◽  
Actin Polymerization ◽  
Bone Marrow Microenvironment ◽  
Splicing Factor ◽  
Expression Level ◽  
Rna Seq ◽  
Alternate Splicing

Abstract Alternate splicing is an important post translational change that alters specificity of gene function. Misregulation of alternative splicing has been implicated in number of disease processes including cancer. We have analyzed alternate splicing in myeloma using high throughput GeneChip Human Exon 1.0 ST Arrays in 170 uniformly treated patients and identified pattern of splicing as well as their impact on both overall and event free survival in myeloma. We have now further analyzed this data and identified Fox2, a RNA alternative splicing regulator, as one of the most important genes predicting clinical outcome in these patients. We observe that the expression level of Fox2 correlates with the frequency of RNA splicing and disease prognosis in MM patients. We have now further investigated the molecular role of Fox2 in myeloma. Fox2 expression was detected in all 10 MM cell lines tested at both RNA and protein levels. Immunohistochemistry staining showed a predominant nuclear localization of Fox2. We next evaluated impact of IL-6 on Fox2 expression in MM1S and RPMI8226 MM cell lines and observed dose-dependent reduction in Fox2 expression. Importantly, MM cell - bone marrow stromal cells (BMSC) interaction also led to significant inhibition of Fox2 expression in MM1S and RPMI8226 cells. Similar response was also observed using BMSC supernatants. On the other hand, IGF-1 stimulation showed slight upregulation of Fox2 in MM cell lines. We have also evaluated impact of IL-6 on Fox2 and splicing using genomewide RNA-seq and confirmed the results. Fox2 was downregulated 33% in MM1S and 37% in RPMI8226 at gene expression level. To study its role in MM, we knocked down the expression of Fox2 in MM1S and RPMI8226 cell lines by using Fox2-directed siRNA. Compared to control cell lines, Fox2 knockdown in MM cell lines did not affect the cell proliferation and survival, as measured by cell titer glo luminescent cell viability assay and annexin V and PI staining respectively. Since Fox2 has been described to plays a role in the maintenance of cell cytoskeleton, we therefore evaluated whether Fox2 might influence the migration and adhesion in MM cells. Transwell migration assay showed enhanced migration rate of Fox2-knocking down- MM1S and RPMI8226 cells versus controls. We also observed the increased cell adhesion to fibronetin in both cell lines upon Fox2 knockdown. Actin polymerization evaluated by Alexa488-conjugated phalloidin staining and confocal microscope analysis showed Fox2 knocking down cells with increased actin polymerization in both MM1S and RPMI8226 cell lines. Currently, RNA seq data following Fox2 knock down in MM cell lines is being evaluated to define the molecular mechanisms of bone marrow microenvironment-mediated Fox2-regualted alternative splicing events in MM. In summary, our results identify Fox2 as a biologically important splicing factor with essential function and potential clinical implications in multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals RNA-Seq Analysis Reveals Localization-Associated Alternative Splicing across 13 Cell Lines

Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 820 ◽  
Author(s):  
Chao Zeng ◽  
Michiaki Hamada
Keyword(s):  
Alternative Splicing ◽  
Subcellular Localization ◽  
Cell Lines ◽  
Biological Diversity ◽  
Rna Binding ◽  
Regulatory Mechanism ◽  
Intron Retention ◽  
Rna Seq ◽  
Motif Analysis ◽  
Structurally Stable

Alternative splicing, a ubiquitous phenomenon in eukaryotes, is a regulatory mechanism for the biological diversity of individual genes. Most studies have focused on the effects of alternative splicing for protein synthesis. However, the transcriptome-wide influence of alternative splicing on RNA subcellular localization has rarely been studied. By analyzing RNA-seq data obtained from subcellular fractions across 13 human cell lines, we identified 8720 switching genes between the cytoplasm and the nucleus. Consistent with previous reports, intron retention was observed to be enriched in the nuclear transcript variants. Interestingly, we found that short and structurally stable introns were positively correlated with nuclear localization. Motif analysis reveals that fourteen RNA-binding protein (RBPs) are prone to be preferentially bound with such introns. To our knowledge, this is the first transcriptome-wide study to analyze and evaluate the effect of alternative splicing on RNA subcellular localization. Our findings reveal that alternative splicing plays a promising role in regulating RNA subcellular localization.

scholarly journals A Prognostic Human Splicing Signature That Precurses Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 877-877 ◽  
Author(s):  
Meenakshi Venkatasubramanian ◽  
Xiaoting Chen ◽  
Kashish Chetal ◽  
Aishwarya Kulkarni ◽  
Kasiani C. Myers ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alternative Splicing ◽  
Board Of Directors ◽  
Protein Isoforms ◽  
Splicing Factor ◽  
Healthy Population ◽  
Rna Seq ◽  
Advisory Committees ◽  
Factor Expression ◽  
Pediatric Aml

Abstract Alternative splicing is a primary mechanism used to achieve mRNA transcript and proteomic diversity in higher-order eukaryotes. While alternative splicing is a recognized oncogenic driver in a small percentage of adult acute myeloid leukemia (AML) cases (~10-15%), splicing factor mutations are rarely found in pediatric AML. To discover splicing heterogeneity within diverse cancers in the absence of known causal mutations we developed a new computational workflow called OncoSplice. This workflow incorporates unsupervised splicing pattern analysis to iteratively identify novel patient splicing-defined subtypes (splice-ICGS algorithm), in conjunction with new integrative methods for cis-regulatory motif, CLIP-Seq binding and splicing factor expression data analysis (Fig. 1a). Analysis of RNA-Seq data from over 800 AML diagnosis samples identified subtypes associated with nearly all known splicing factor mutations in addition to over a dozen novel splicing-defined subtypes. Novel splicing subtypes were confirmed in independent patient cohorts and were associated with the expression or mutation of oncogenes (MYC, TP53, NPM1), oncofusions (CBFB-MYH11, MLL, PML-RAR, RUNX1), or the regulation of diverse splicing factors (U2AF1, SRSF2, HNRNPK). Both adult and pediatric AML are most frequently characterized by a single predominant splicing signature which divides the majority of AML patients into subtypes with splicing events overlapping those found in patients with mutations in U2AF1 or SRSF2 (Figure 1b). These "U2AF1-covarying" or "SRSF2-covarying" (CV) occur independently of splicing-factor mutations and were principally linked to mis-splicing rather than differential gene expression. Unlike patients with U2AF1-S3F mutations, U2AF1-CV splicing events are associated with canonical rather than altered U2AF1 binding specificity (Fig. 1c). In both adult and pediatric AML, U2AF1-CV splice events result in a shift towards longer protein isoforms associated with stem and progenitor programs, have significantly worse outcomes (poor survival and increased relapse) and are persistent during relapse in adults (Fig. 1d). Survival correlated U2AF1-CV splicing events were statistically enriched in genes required for leukemic growth based on a published CRISPR dropout screen. U2AF1-CV splicing was not associated with prior described epigenetic AML subtypes, but appears to be dependent on the expression of MYC and downstream splicing regulators (WDR77 and PRMT5). Surprisingly, RNA-Seq analysis of CD34+ bone marrow progenitors in healthy donors finds the same overall skewing in a subset of U2AF1-CV or SRSF2-CV splicing events as those found in AML. Distinct sorted progenitor populations (HSC, Multi-Lin, CMP, GMP, MEP) further show consistent U2AF1-CV or SRSF2-CV skewing within progenitors from the same donors, indicating that this is not a cell-type associated splicing difference. Hence, our data suggest that healthy bone marrow is skewed in the healthy population towards either U2AF1 or SRSF2 splicing pathways and that these splicing profiles impact future oncogenic transformation and patient survival in AML. These data further suggest a paradigm shifting model, in which widespread coordinated pathogenic splicing occurs across cancers, likely via imbalances in splicing factor expression, regulation or mutation. Figure 1. a) Steps implemented in the OncoSplice splicing subtype discovery workflow for splicing event quantification (MultiPath-PSI), iterative unsupervised splicing subtype discovery (splice-ICGS), supervised splicing subtype discovery (Bridger) and RNA-regulatory splicing subtype prediction (RBP-Finder). b) Comparison of splicing events enriched in adult AML with splicing factor mutations identifies their coincidence with U2AF1-CV and SRSF2-CV splicing events. c) Analysis of U2AF1 binding-site preferences at the e-3 splice-site position for cassette-exon splicing events (U2AF1-S34-specific but not U2AF1-CV; occurring in U2AF1-S34; occurring in U2AF1-CV; U2AF1-CV-specific but not U2AF1-S34). d) Kaplan-Meier curves for overall survival in patients from TCGA (top) and TARGET (bottom) with associated coxph p-values (all splice-ICGS stringently classified U2AF1-CV versus all other considered AMLs). Analysis of TCGA was restricted to cytogenetically normal AMLs with no splicing factor mutations and under 60 years of age. Figure 1. Figure 1. Disclosures Myers: Bellicum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Identification and Expression of Two microRNA Primary Transcripts, pri-miR-223 and pri-miR-10a, in Hematopoietic Cell Lines during Cytokine-Stimulated Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1747-1747
Author(s):  
Nobuyoshi Kosaka ◽  
Keiichi Sugiura ◽  
Nami Nogawa ◽  
Norio Komatsu ◽  
Takashi Kato
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Target Genes ◽  
Cell Lineage ◽  
Gene Clusters ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Protein Coding ◽  
Splicing Variants ◽  
Cytokine Stimulation

Abstract MicroRNAs (miRNAs) are small RNA molecules involved in various biological processes including cell differentiation, apoptosis, and oncogenesis. MiRNAs are generated from non-protein coding transcripts that are known as primary miRNA (pri-miRNA). Recently, some miRNAs were identified in hematopoietic cells. However, the relationship between pri-miRNA/miRNA expression and cytokine-induced cell proliferation/differentiation remains unclear. It has been reported that miR-223 is one of the miRNAs specifically expressed in murine myeloid lineages. We identified two splicing variants (672-nt and 437-nt) originated from its precursor pri-miR-223 (3256-nt). RT-PCR analysis by using oligo-dT primer and splice specific primer pair was performed to detect their expressions, and it revealed that pri-miR-223 was expressed in lung, brain, spleen and bone marrow but not in heart, liver or muscle. Notably each splicing variant displayed different tissue-specific expressions. Pri-miR-223 was more broadly expressed compared to the mature miR-223 expressed in bone marrow, spleen and thymus (Chen et al, Science, 2004), indicating that the generation of miR-223 is partly regulated by the miRNA processing pathway. In mouse promyelocytic NFS-60 cells, both pri-miR-223 splicing variants were expressed substantially when the cells were maintained in G-CSF (1ng/mL) exhibited granulocytic phenotype with myeloperoxidase expression, but the expression of the variants decreased on IL-3 (1ng/mL) stimulation: while the addition of G-CSF (10ng/mL) did not increase the expression of pri-miR-223. In other hematopoietic lineages, erythrocytic and megakaryocytic cells, the expressions of miRNAs had not been examined yet. As a first step to detect the miRNAs specific to erythrocytic and megakaryocytic cells, pri-miR-10a located within Hox gene clusters, was examined in the human cell lines established from the parent UT-7 cells, UT-7/GM, UT-7/EPO and UT-7/TPO. Their survival, growth and differentiation are known to be dependent on specific cytokine stimulations. Firstly, we identified the full-length sequence of human pri-miR-10a (482-nt) from EST database. RT-PCR analysis as with that for pri-miR-223 was then performed in each UT-7 cell line. Interestingly, pri-miR-10a was highly expressed in UT-7/EPO cells stimulated with erythropoietin but not in UT-7/TPO and UT-7/GM cells; though the expression of pri-miR-10a did not elevate by respective stimulation of erythropoietin (10U/mL), thrombopoietin (100ng/mL), or GM-CSF (10ng/mL) in UT-7/EPO, UT-7/TPO and UT-7/GM cells. These results showed that miR-223 and miR-10a expression was not regulated directly by cytokine stimulation, while their expression levels were altered by cytokine-dependent cell lineage commitment. The observations prompted us the hypothesis that lineage-specific miRNAs suppress the expression of their target genes that inhibit master transcription factors to direct gene silencing processes critical for differentiation. Since little information is available on transcription and processing of miRNAs specific to hematopoetic prolifiration and differentiation, our results provide a new approach to understand vital issues unsolved.

scholarly journals Mild hypothermia causes a shift in the alternative splicing of cold-inducible RNA-binding protein transcripts in Syrian hamsters

2019 ◽  
Vol 317 (2) ◽  
pp. R240-R247 ◽  
Author(s):  
Yuuki Horii ◽  
Hiroki Shimaoka ◽  
Kazuhiro Horii ◽  
Takahiko Shiina ◽  
Yasutake Shimizu
Keyword(s):  
Alternative Splicing ◽  
Body Temperature ◽  
Rna Binding ◽  
Mild Hypothermia ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Pcr Analysis ◽  
Central Administration ◽  
Syrian Hamsters ◽  
Splicing Pattern

Cold-shock proteins are thought to participate in the cold-tolerant nature of hibernating animals. We previously demonstrated that an alternative splicing may allow rapid induction of functional cold-inducible RNA-binding protein (CIRBP) in the hamster heart. The purpose of the present study was to determine the major cause of the alternative splicing in Syrian hamsters. RT-PCR analysis revealed that CIRBP mRNA is constitutively expressed in the heart, brain, lung, liver, and kidney of nonhibernating euthermic hamsters with several alternative splicing variants. In contrast, the short variant containing an open-reading frame for functional CIRBP was dominantly found in the hibernating animals. Keeping the animals in a cold and dark environment did not cause a shift in the alternative splicing. Induction of hypothermia by central administration of an adenosine A1-receptor agonist reproduced the shift in the splicing pattern. However, the agonist failed to shift the pattern when body temperature was kept at 37°C, suggesting that central adenosine A1 receptors are not directly linked to the shift of the alternative splicing. Rapid reduction of body temperature to 10°C by isoflurane anesthesia combined with cooling did not alter the splicing pattern, but maintenance of mild hypothermia (~28°C) for 2 h elicited the shift in the pattern. The results suggest that animals need to be maintained at mild hypothermia for an adequate duration to induce the shift in the alternative splicing. This is applicable to natural hibernation because hamsters entering hibernation show a gradual decrease in body temperature, being maintained at mild hypothermia for several hours.

scholarly journals U2AF1 Mutations Enhance Stress Granule Response in Myeloid Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 321-321
Author(s):  
Giulia Biancon ◽  
Poorval Joshi ◽  
Joshua T Zimmer ◽  
Torben Hunck ◽  
Yimeng Gao ◽  
...  
Keyword(s):  
Cell Lines ◽  
Rna Binding ◽  
Stress Granules ◽  
Stress Granule ◽  
Confocal Imaging ◽  
Splicing Factor ◽  
Immunofluorescent Staining ◽  
Rna Seq ◽  
Wild Type ◽  
Granule Formation

Abstract Somatic mutations in splicing factor genes are drivers of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The splicing factors U2AF1 and U2AF2 form the U2AF heterodimer that is critical in the 3' splice site (3'SS) recognition and in the recruitment of U2 small nuclear ribonucleoproteins for the activation of the spliceosome complex. U2AF1 carries hotspot mutations in its two RNA binding motifs; yet the molecular mechanisms affecting the splicing process and promoting clonal advantage remain unclear, albeit necessary to develop effective targeted therapies. We applied a multi-omics approach comparing the activities of two U2AF1 mutants (S34F and Q157R) in MDS/AML cell lines and primary samples. Using a novel approach of fractionated enhanced crosslinking immunoprecipitation coupled with deep RNA-sequencing (freCLIP-seq), we mapped transcriptome-wide binding at nucleotide resolution and we identified conformational changes in mutant vs wild-type U2AF1 binding. Specifically, we observed an emergent peak in position -3 of the 3'SS for the S34F mutant and in position +1 for the Q157R mutant, matching the critical positions observed by differential splicing analysis on RNA-seq data. Altered U2AF1-RNA binding compromised U2AF2-RNA interactions, resulting predominantly in exon exclusion and intron retention. Combined binding-splicing analysis showed that while the Q157R mutant mainly exhibits loss of binding, the S34F mutant follows a gain-of-binding pattern, where splicing progression appears impaired by increased mutant binding. Functional analysis of genes affected by both binding and splicing alterations revealed that U2AF1 mutants alter RNA granule biology, affecting in particular stress granule-enriched transcripts and proteins. Stress granules are membrane-less cytoplasmic assemblies of RNAs and RNA binding proteins that improve cellular adaptation in response to stress conditions. Increased stress granule formation has been linked to tumorigenesis as a strategy exploited by cancer cells to regulate gene expression and signal transduction, enhancing their fitness under stress. To probe how aberrant binding and splicing of stress granule components affected stress granule biology, we assessed stress granule formation in U2AF1 mutant vs wild-type cells at steady state and after stress induction with sodium arsenite treatment. Immunofluorescent staining followed by confocal imaging demonstrated that U2AF1 mutations enhance stress granule formation upon arsenite stress in both cell lines and primary samples. RNA turnover analysis by TimeLapse-seq confirmed that U2AF1 S34F and Q157R mutations promote stability/synthesis of transcripts that are enriched in stress granules and determine degradation/shutdown of transcripts that are depleted in stress granules, providing a molecular explanation for the increase in stress granules observed by imaging. Finally, we were able to corroborate our observations by single-cell RNA-seq in patient-derived U2AF1-mutant MDS blasts, establishing the causal link between U2AF1 mutations and upregulation of stress granule components. Collectively, this multi-omics analysis identified biological processes directly influenced by mutant U2AF1 binding and splicing, laying the foundation for a new paradigm where splicing factor mutations enhance stress granule formation by acting on the availability of their RNA and protein components. The enhanced formation of stress granules potentially fosters the stress adaptation of U2AF1-mutant cells, contributing to their clonal advantage in MDS/AML. Stress granule perturbations may therefore represent a novel therapeutic vulnerability in U2AF1-mutant MDS/AML patients and possibly in patients carrying other splicing factor mutations. Disclosures Hunck: Boehringer Ingelheim: Other: Fellowship.

scholarly journals RNA-Seq Analysis Reveals Localization-Associated Alternative Splicing across 13 Cell Lines

2019 ◽  
Author(s):  
Chao Zeng ◽  
Michiaki Hamada
Keyword(s):  
Alternative Splicing ◽  
Subcellular Localization ◽  
Cell Lines ◽  
Biological Diversity ◽  
Rna Binding ◽  
Regulatory Mechanism ◽  
Intron Retention ◽  
Rna Seq ◽  
Motif Analysis ◽  
Structurally Stable

ABSTRACTAlternative splicing, a ubiquitous phenomenon in eukaryotes, is a regulatory mechanism for the biological diversity of individual genes. Most studies have focused on the effects of alternative splicing for protein synthesis. However, the transcriptome-wide influence of alternative splicing on RNA subcellular localization has rarely been studied. By analyzing RNA-seq data obtained from subcellular fractions across 13 human cell lines, we identified 8720 switching genes between the cytoplasm and the nucleus. Consistent with previous reports, intron retention was observed to be enriched in the nuclear transcript variants. Interestingly, we found that short and structurally stable introns were positively correlated with nuclear localization. Motif analysis reveals that fourteen RNA-binding protein (RBPs) are prone to be preferentially bound with such introns. To our knowledge, this is the first transcriptome-wide study to analyze and evaluate the effect of alternative splicing on RNA subcellular localization. Our findings reveal that alternative splicing plays a promising role in regulating RNA subcellular localization.

scholarly journals A 5′-tRNA halve, tiRNA-Gly promotes cell proliferation and migration via binding to RBM17 and inducing alternative splicing in papillary thyroid cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Litao Han ◽  
Hejing Lai ◽  
Yichen Yang ◽  
Jiaqian Hu ◽  
Zhe Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Thyroid Cancer ◽  
Alternative Splicing ◽  
Papillary Thyroid Cancer ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Papillary Thyroid ◽  
Cell Proliferation And Migration ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background tRNA-derived small noncoding RNAs (sncRNAs) are mainly categorized into tRNA halves (tiRNAs) and fragments (tRFs). Biological functions of tiRNAs in human solid tumor are attracting more and more attention, but researches concerning the mechanisms in tiRNAs-mediated tumorigenesis are rarely. The direct regulatory relationship between tiRNAs and splicing-related proteins remain elusive. Methods Papillary thyroid carcinoma (PTC) associated tRNA fragments were screened by tRNA fragments deep sequencing and validated by qRT-PCR and Northern Blot in PTC tissues. The biological function of tRNA fragments were assessed by cell counting kit, transwells and subcutaneous transplantation tumor of nude mice. For mechanistic study, tRNA fragments pull-down, RNA immunoprecipitation, Western Blot, Immunofluorescence, Immunohistochemical staining were performed. Results Herein, we have identified a 33 nt tiRNA-Gly significantly increases in papillary thyroid cancer (PTC) based on tRFs & tiRNAs sequencing. The ectopic expression of tiRNA-Gly promotes cell proliferation and migration, whereas down-regulation of tiRNA-Gly exhibits reverse effects. Mechanistic investigations reveal tiRNA-Gly directly bind the UHM domain of a splicing-related RNA-binding protein RBM17. The interaction with tiRNA-Gly could translocate RBM17 from cytoplasm into nucleus. In addition, tiRNA-Gly increases RBM17 protein expression via inhibiting its degradation in a ubiquitin/proteasome-dependent way. Moreover, RBM17 level in tiRNA-Gly high-expressing human PTC tissues is upregulated. In vivo mouse model shows that suppression of tiRNA-Gly decreases RBM17 expression. Importantly, tiRNA-Gly can induce exon 16 splicing of MAP4K4 mRNA leading to phosphorylation of downstream signaling pathway, which is RBM17 dependent. Conclusions Our study firstly illustrates tiRNA-Gly can directly bind to RBM17 and display oncogenic effect via RBM17-mediated alternative splicing. This fully novel model broadens our understanding of molecular mechanism in which tRNA fragment in tumor cells directly bind RNA binding protein and play a role in alternative splicing.

scholarly journals The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x

2007 ◽  
Vol 176 (7) ◽  
pp. 929-939 ◽  
Author(s):  
Maria Paola Paronetto ◽  
Tilman Achsel ◽  
Autumn Massiello ◽  
Charles E. Chalfant ◽  
Claudio Sette
Keyword(s):  
Alternative Splicing ◽  
Tyrosine Phosphorylation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Live Cells ◽  
Hnrnp A1 ◽  
Splice Site Selection ◽  
Subnuclear Localization ◽  
Mrna Targets

The RNA-binding protein Sam68 is involved in apoptosis, but its cellular mRNA targets and its mechanism of action remain unknown. We demonstrate that Sam68 binds the mRNA for Bcl-x and affects its alternative splicing. Depletion of Sam68 by RNA interference caused accumulation of antiapoptotic Bcl-x(L), whereas its up-regulation increased the levels of proapoptotic Bcl-x(s). Tyrosine phosphorylation of Sam68 by Fyn inverted this effect and favored the Bcl-x(L) splice site selection. A point mutation in the RNA-binding domain of Sam68 influenced its splicing activity and subnuclear localization. Moreover, coexpression of ASF/SF2 with Sam68, or fusion with an RS domain, counteracted Sam68 splicing activity toward Bcl-x. Finally, Sam68 interacted with heterogenous nuclear RNP (hnRNP) A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated Bcl-x(s) splicing. Our results indicate that Sam68 plays a role in the regulation of Bcl-x alternative splicing and that tyrosine phosphorylation of Sam68 by Src-like kinases can switch its role from proapoptotic to antiapoptotic in live cells.

scholarly journals rMAPS2: an update of the RNA map analysis and plotting server for alternative splicing regulation

2020 ◽  
Vol 48 (W1) ◽  
pp. W300-W306 ◽  
Author(s):  
Jae Y Hwang ◽  
Sungbo Jung ◽  
Tae L Kook ◽  
Eric C Rouchka ◽  
Jinwoong Bok ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
High Throughput ◽  
Splice Site ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Sequencing Data ◽  
High Throughput Sequencing Data ◽  
Retained Intron ◽  
Map Analysis

Abstract The rMAPS2 (RNA Map Analysis and Plotting Server 2) web server, freely available at http://rmaps.cecsresearch.org/, has provided the high-throughput sequencing data research community with curated tools for the identification of RNA binding protein sites. rMAPS2 analyzes differential alternative splicing or CLIP peak data obtained from high-throughput sequencing data analysis tools like MISO, rMATS, Piranha, PIPE-CLIP and PARalyzer, and then, graphically displays enriched RNA-binding protein target sites. The initial release of rMAPS focused only on the most common alternative splicing event, skipped exon or exon skipping. However, there was a high demand for the analysis of other major types of alternative splicing events, especially for retained intron events since this is the most common type of alternative splicing in plants, such as Arabidopsis thaliana. Here, we expanded the implementation of rMAPS2 to facilitate analyses for all five major types of alternative splicing events: skipped exon, mutually exclusive exons, alternative 5′ splice site, alternative 3′ splice site and retained intron. In addition, by employing multi-threading, rMAPS2 has vastly improved the user experience with significant reductions in running time, ∼3.5 min for the analysis of all five major alternative splicing types at once.

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