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

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.

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

Blood ◽

10.1182/blood.v124.21.4714.4714 ◽

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.

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MLL-Fusion Leukemia Dependence on MBNL1 Is Associated with Alternative Splicing of Oncogenic Proteins

Blood ◽

10.1182/blood-2018-99-112349 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3883-3883

Author(s):

Arun Gurunathan ◽

Lana S Itskovich ◽

Jason Clark ◽

Matthew Burwinkel ◽

Nathan Salomonis ◽

...

Keyword(s):

Alternative Splicing ◽

Cell Lines ◽

Rna Binding ◽

Conflicts Of Interest ◽

Intron Retention ◽

Leukemia Cell ◽

Human Cells ◽

Leukemic Cells ◽

Leukemia Cell Line ◽

Infant Leukemia

Abstract Leukemia is the most common childhood cancer, and while outcomes for most children have improved significantly, the prognosis in infant leukemia remains dire. The majority of infant leukemia, either acute myeloid (AML) or acute lymphoid (ALL), is caused by reciprocal translocations of the MLL-gene. Prior studies show that one of the most consistently overexpressed genes in these leukemias (compared to all other leukemias) is the RNA binding protein muscleblind-like 1 (MBNL1). We found that MBNL1 knockdown significantly impairs propagation of MLL-rearranged (MLLr) leukemic cells in vitro and in vivo using human cell lines and transformed murine cells. To further characterize the role of MBNL1 in acute leukemia, we performed shRNA knockdown experiments in MLLr and non-MLLr leukemia cell lines and in primary patient samples. While MBNL1 knockdown does also impair growth of non-MLLr leukemic cells, the effect is less pronounced. In a 5-day growth experiment MBNL1-knockdown MLLr cells (THP-1) displayed a median 71% reduced growth compared to controls, whereas non-MLLr cells (HL-60) displayed only a median 32% growth reduction (p=0.0001). Cells from two patients with MLLr AML (one with MLL-AF9 and one with MLL-AF10 fusion) underwent shNT (non-targeting) or shMBNL1 transduction.. Unsorted cells were transplanted into NSGS mice. Mice were observed until showing signs of distress and then analyzed for engraftment of human cells and abundance of transduced cells (venus-positive). In the shNT group there was robust persistence of transduced cells (7%-98% of human cells), whereas shMBNL1-transduced cells were not detected or comprised <1% of human cells in most of the recipient mice. Given that MBNL1 is known to regulate alternative splicing, we used unbiased RNAseq along with a novel analytic splice-junction and intron-quantification toolkit (AltAnalyze) to determine splicing changes induced by knockdown of MBNL1 in the MLLr leukemia cell line MOLM-13. In a parallel analysis, we determined splicing differences between MLLr and cytogenetically-normal (CN) AML patient samples. We then compared these two results to determine the splicing events regulated by MBNL1 and assess the contribution of MBNL1 to splicing events observed in primary MLLr leukemias. Strikingly, this comparative analysis found that 88% of overlapping differentially expressed splicing events (75 out of 85) were concordant between patient MLLr and CN-AML as compared to control versus MBNL1 knockdown. The most common class of splicing event that occurred with MBNL1 knockdown was intron retention. Specifically, our findings suggest that MBNL1 knockdown restores intron retention, and that MBNL1 overexpression promotes expression of protein-coding genes that would otherwise be suppressed through intron retention-introduced premature termination codons. Several genes whose transcripts are alternatively spliced by MBNL1 have prior associations with cancer, most notably DOT1L and SETD1A which are specifically implicated in MLLr leukemia. Splicing validation through RT-PCR confirmed increased intron retention in DOT1L and SETD1A transcripts after MBNL1 knockdown. Interestingly, one target of MBNL1 is the MBNL1 mRNA itself, with resultant exclusion of exon 5. MBNL1 lacking exon 5 has a stronger affinity to RNA. In summary, our data suggests that MBNL1 plays a key role in the pathogenesis of MLL-fusion leukemia, wherein it stabilizes the transcripts of multiple leukemogenic genes including DOT1L and SETD1A. Proteins such as DOT1L are critical for transcriptional activation of downstream targets of the MLL-fusion protein (including activation of MBNL1, creating a positive feedback loop). Additionally, high levels of MBNL1 protein may alter splicing in ways that enhance MBNL1 functionality. Disclosures No relevant conflicts of interest to declare.

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An Erythroid-Specific Intron Retention Program Regulates Expression of Selected Genes during Terminal Erythropoiesis

Blood ◽

10.1182/blood.v124.21.449.449 ◽

2014 ◽

Vol 124 (21) ◽

pp. 449-449

Author(s):

Harold Pimentel ◽

Marilyn Parra ◽

Sherry Gee ◽

Narla Mohandas ◽

Lior Pachter ◽

...

Keyword(s):

Alternative Splicing ◽

Iron Homeostasis ◽

Rna Binding ◽

Conflicts Of Interest ◽

Intron Retention ◽

Great Majority ◽

Protein Translation ◽

Membrane Properties ◽

Disease Genes ◽

Rna Seq

Abstract Erythroid RNAs, like their nonerythroid counterparts, are subject to post-transcriptional processing events that critically impact their coding capacity for the erythroid proteome. Previous studies have shown that differentiating human and mouse erythroblasts execute an extensive and dynamic alternative splicing program involving regulation of numerous alternative exons. Here we report that controlled excision of selected introns is also an important component of the erythroblast alternative splicing program. Intron retention (IR) patterns in differentiating human erythroblasts were determined via RNA-seq analysis of FACS-purified erythroblast populations. Comparison of IR among erythroblast populations and between erythroblasts and other hematopoietic cells suggests that regulation of IR occurs in a differentiation stage- and tissue-specific manner. For example, there was little overlap of intron retention events in erythroblasts with those reported in differentiating granulocytes. Moreover, the IR profile of proerythroblasts differed substantially from that in orthochromatic erythroblasts, with IR generally increasing in the more mature cells that are preparing for enucleation. IR in erythroblasts affected numerous genes functioning in RNA processing, iron homeostasis and heme biosynthesis, protein translation, and membrane properties. Mature erythroblasts exhibited retention of introns in several human disease genes including SF3B1, a splicing factor often mutated in myelodysplasia; TFR2, encoding transferrin receptor 2 that is mutated in a form of hemochromatosis; and FUS, an RNA binding protein implicated in ALS. Inspection of intronic RNA-seq reads in >60 genes with IR revealed that single or multiple introns can be retained within a transcript; however, other introns within the same genes, and indeed the great majority of introns in erythroblast-expressed genes, are efficiently spliced with minimal or no IR. Retained introns may be flanked by either constitutively or alternatively spliced exons, suggesting different regulatory mechanisms. Ongoing studies will explore whether IR in some transcripts might function to down-regulate gene expression by introduction of premature termination codons that induce nonsense-mediated decay, or alternatively, whether IR transcripts could represent a reserve of nearly-completed mRNAs that can be processed in response to appropriate physiological stimuli. In sum, these results suggest that a highly regulated IR program plays an important role in erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

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Plant buffering against the high-light stress-induced accumulation of CsGA2ox8 transcripts via alternative splicing to finely tune gibberellin levels and maintain hypocotyl elongation

Horticulture Research ◽

10.1038/s41438-020-00430-w ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Bin Liu ◽

Shuo Zhao ◽

Pengli Li ◽

Yilu Yin ◽

Qingliang Niu ◽

...

Keyword(s):

Alternative Splicing ◽

Light Intensity ◽

Intron Retention ◽

Light Stress ◽

Photosynthetic Photon Flux Density ◽

Photon Flux ◽

Photon Flux Density ◽

Rna Seq ◽

Multiple Transcripts ◽

Novel Transcript

AbstractIn plants, alternative splicing (AS) is markedly induced in response to environmental stresses, but it is unclear why plants generate multiple transcripts under stress conditions. In this study, RNA-seq was performed to identify AS events in cucumber seedlings grown under different light intensities. We identified a novel transcript of the gibberellin (GA)-deactivating enzyme Gibberellin 2-beta-dioxygenase 8 (CsGA2ox8). Compared with canonical CsGA2ox8.1, the CsGA2ox8.2 isoform presented intron retention between the second and third exons. Functional analysis proved that the transcript of CsGA2ox8.1 but not CsGA2ox8.2 played a role in the deactivation of bioactive GAs. Moreover, expression analysis demonstrated that both transcripts were upregulated by increased light intensity, but the expression level of CsGA2ox8.1 increased slowly when the light intensity was >400 µmol·m−2·s−1 PPFD (photosynthetic photon flux density), while the CsGA2ox8.2 transcript levels increased rapidly when the light intensity was >200 µmol·m−2·s−1 PPFD. Our findings provide evidence that plants might finely tune their GA levels by buffering against the normal transcripts of CsGA2ox8 through AS.

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A Novel Long Non-Coding RNA, SNHG4 Complex With Eukaryotic Initiation Factor-4E and Regulate Aberrant Protein Translation In Mantle Cell Lymphoma: Implications For Novel Biomarker

Blood ◽

10.1182/blood.v122.21.81.81 ◽

2013 ◽

Vol 122 (21) ◽

pp. 81-81

Author(s):

Guangzhen Hu ◽

Thomas E Witzig ◽

Mamta Gupta

Keyword(s):

B Cell ◽

Cell Lines ◽

Rna Binding ◽

Cell Lymphoma ◽

Protein Translation ◽

Rna Seq ◽

Lymphoma Cells ◽

Binding Motifs ◽

Mantle Cell

Abstract Long noncoding RNAs (lncRNAs) are defined as RNA-like transcripts that are over 200 nucleotides and lack significant open reading frames. Some lncRNAs such as HOTAIR, MALAT1 and H19 have been found to be associated with clinical prognosis and are potential drivers of cancer progression in cancers of the breast, lung, and liver respectively. The role of lncRNAs in lymphoma is unknown. Dysregulation of eIF4E (a key component of the translation initiation complex eIF4F) influences global protein translation, especially the translation of “weak” mRNAs that can be malignancy-related. We and others have found that eIF4E is dysregulated in B-cell lymphoma. The aim of this study is to identify eIF4E-associated lncRNAs through next generation RNA-Sequencing (NGS RNA-Seq) and delineate their role in protein translation in lymphoma. RNA-immunoprecipitation (RNA-IP) was used to pull down eIF4E-bound lncRNA in lymphoma cells. eIF4E-bound lncRNAs were immunoprecipitated with eIF4E antibody or IgG control in Jeko, a mantle cell lymphoma (MCL) cell line and sent for microarray analysis and NGS-RNA-Seq for identification of lncRNAs. The microarray analysis showed that several lncRNAs were enriched with eIF4E antibody compared to IgG control. These included SNHG4 (13.6 fold), SNHG12 (4.8 fold), NCRNA00171 (4.8 fold) and IPW (4.6 fold), GNASAS (3.5 fold), SNHG7 (3.3 fold), NCRNA00182 (2.7 fold), NCRNA00094 (2.6 fold), NCRNA00188 (2.4 fold) and NCRNA00201 (2.1 fold). The binding of these lncRNAs to eIF4E was further confirmed by RT-PCR in Jeko, Mino and Granta MCL cell lines. Next, we looked the expression of these lncRNAs by qRT- PCR in the MCL cell lines and normal controls. We found SNHG4 and IPW to be overexpressed in all the MCL cell lines, while SNHG12 and NCRNA00201 were overexpressed in the selected cell lines. No significant difference was found for the expression of NCRNA00171 and NCRNA00182 in any of the MCL cell lines compared to controls. Overall, these data suggest that several lncRNA have altered expression in malignant B-cells. Considering that the microarray assay only covered a limited number of lncRNAs, we further confirmed eIF4E bound lncRNA by NGS RNA-Seq in Jeko MCL and normal control. The binding of 10/13 lncRNA mentioned above with eIF4E were found upregulated by NGS-RNA-Seq. In addition several novel lncRNAs such as SNHG1 (161.6), AC091814.2 (98.8) and RP11-304L19.5 (64.2) showed up in NGS-RNA-Seq data. These data suggest that lncRNAs, such as SNHG12, SNHG4, and SNHG1 bind to eIF4E with high affinity in malignant B-cells and might play a role in protein translation. We knocked down the expression of SNHG4 through siRNA and demonstrated that cell proliferation and global protein translation was inhibited in lymphoma cells. To further confirm the role of SNHG4 in translation regulation, a plasmid, which contains a renilla luciferase driven by SV40 promoter, was co-transfected with SNHG4 siRNA into Mino cells. The luciferase signal, decreased compared with the cells transfected with nontargeting siRNA. These data suggest that SNHG4 is involved in the regulation of protein translation. In order to clarify the mechanism of lncRNAs bound to eIF4E we searched for RNA binding sites or motifs in eIF4E protein using the web-based tools, BindN and PPRInt. Interestingly two RNA binding motifs, KNKRGGRWLITLNKQQRRS and SHADTATKSGSTTKNR, were found in eIF4E based on the prediction. To examine whether lncRNAs bind with eIF4E through these RNA binding motifs, an eIF4E mutant plasmid with both RNA binding motifs deleted (eIF4EDel), was constructed and transfected transiently into HEK-293T cells along with eIF4EWT plasmid. RNA-IP data showed that the lncRNAs SNHG12, SNHG4 and SNHG1 were not able to bind with eIF4E in eIF4EDel-transfected cells compared with that of eIF4EWT, suggesting that these lncRNAs complex with eIF4E through RNA-binding motifs within the eIF4E. Overall, our results show that the lncRNAs, SNHG1 and SNHG4 are able to bind with eIF4E and regulate protein translation. Since lncRNAs had been found to play roles in the regulation of gene expression, including transcription, splicing and mRNA stability, our results may broaden the view of the functional role of lncRNAs in translation in lymphoma cells and in other cancers. Furthermore, our results also suggested that SNHG4 lncRNAs might be served as potential biomarkers for MCL and other B cell lymphomas for translation therapy. Disclosures: No relevant conflicts of interest to declare.

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Alternative Splicing in the Obligate Biotrophic Oomycete Pathogen Pseudoperonospora cubensis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-09-14-0300-fi ◽

2015 ◽

Vol 28 (3) ◽

pp. 298-309 ◽

Cited By ~ 10

Author(s):

Alyssa Burkhardt ◽

Alex Buchanan ◽

Jason S. Cumbie ◽

Elizabeth A. Savory ◽

Jeff H. Chang ◽

...

Keyword(s):

Alternative Splicing ◽

Intron Retention ◽

Draft Genome ◽

Pseudoperonospora Cubensis ◽

Rna Seq ◽

Transcriptome Regulation ◽

Oomycete Pathogen ◽

Alternatively Spliced ◽

Genome Annotations ◽

Obligate Pathogen

Pseudoperonospora cubensis is an obligate pathogen and causative agent of cucurbit downy mildew. To help advance our understanding of the pathogenicity of P. cubensis, we used RNA-Seq to improve the quality of its reference genome sequence. We also characterized the RNA-Seq dataset to inventory transcript isoforms and infer alternative splicing during different stages of its development. Almost half of the original gene annotations were improved and nearly 4,000 previously unannotated genes were identified. We also demonstrated that approximately 24% of the expressed genome and nearly 55% of the intron-containing genes from P. cubensis had evidence for alternative splicing. Our analyses revealed that intron retention is the predominant alternative splicing type in P. cubensis, with alternative 5′- and alternative 3′-splice sites occurring at lower frequencies. Representatives of the newly identified genes and predicted alternatively spliced transcripts were experimentally validated. The results presented herein highlight the utility of RNA-Seq for improving draft genome annotations and, through this approach, we demonstrate that alternative splicing occurs more frequently than previously predicted. In total, the current study provides evidence that alternative splicing plays a key role in transcriptome regulation and proteome diversification in plant-pathogenic oomycetes.

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Abstract 186: The Cardiac Specific Splicing Regulator Rbm20 decreases in Specific Forms of Acquired Heart Disease

Circulation Research ◽

10.1161/res.113.suppl_1.a186 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Maarten van den Hoogenhof ◽

Abdelaziz Beqqali ◽

Inge van der Made ◽

Dirk Duncker ◽

Jolanda van der Velden ◽

...

Keyword(s):

Heart Failure ◽

Heart Disease ◽

Alternative Splicing ◽

Aortic Stenosis ◽

Western Blotting ◽

Rna Binding ◽

Intron Retention ◽

Left Ventricular ◽

Internal Diameter ◽

Acquired Heart Disease

Introduction: Rbm20 is a RNA-binding protein enriched in heart- and skeletal muscle. Mutations in Rbm20 have been described to cause heart failure. Furthermore, Rbm20 has been shown to regulate alternative splicing of titin (Ttn). This demonstrates that RBM20 has a major role in cardiac biology. However, it is unknown how Rbm20 expression changes in acquired heart disease. Therefore, we investigated expression of Rbm20 on mRNA and protein level and the degree of alternative splicing of Ttn in various forms of heart disease. Methods: We measured mRNA expression and protein levels of Rbm20 by western blotting and qPCR in transverse aorta constrictions (TAC) in mice and pigs, and in human heart disease including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), aortic stenosis (AoS) and pulmonary artery hypertension (PAH). We measured cardiac function in mice with echocardiography, and measured stress markers with qPCR. Furthermore, we analyzed Ttn splicing using qPCR and RT-PCR. Results: Real-time PCR and western blotting revealed that Rbm20 is selectively downregulated in mice after TAC and in patients with aortic stenosis. In contrast, we did not observe a difference in Rbm20 levels in patients with DCM, HCM and PAH. Furthermore, we measured functional parameters such as ejection fraction (EF), systolic left ventricular internal diameter (LVIDsys), and fractional shortening (FS), as well as ANF level in TAC mice, and correlated these to Rbm20 level. We found that the level of downregulation of Rbm20 is correlated with heart failure severity in mice after TAC. Also, we found that Ttn is differentially spliced after loss of Rbm20. Conclusions: Here we show for the first time that Rbm20 decreases in acquired forms of heart failure such as TAC in mice and pigs, and AoS in men. Furthermore, in experimental HF in mice, Rbm20 levels correlate to HF severity. Moreover, loss of Rbm20 leads to skipping of intron retention in Ttn in the region between exon 50-70. However, the biological effect of this event remains unclear. Given its major role in the heart, loss of Rbm20 may play an important role in the development of heart disease.

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

Blood ◽

10.1182/blood.v122.21.3085.3085 ◽

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.

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Long Non-Coding RNA Expression Levels Modulate Cell-Type-Specific Splicing Patterns by Altering Their Interaction Landscape with RNA-Binding Proteins

Genes ◽

10.3390/genes10080593 ◽

2019 ◽

Vol 10 (8) ◽

pp. 593 ◽

Cited By ~ 4

Author(s):

Felipe Wendt Porto ◽

Swapna Vidhur Daulatabad ◽

Sarath Chandra Janga

Keyword(s):

Alternative Splicing ◽

Cell Lines ◽

Rna Binding ◽

Cell Type ◽

Specific Manner ◽

Alternatively Spliced ◽

A Cell ◽

Specific Alternative ◽

Cell Type Specific ◽

Rna Interaction

Recent developments in our understanding of the interactions between long non-coding RNAs (lncRNAs) and cellular components have improved treatment approaches for various human diseases including cancer, vascular diseases, and neurological diseases. Although investigation of specific lncRNAs revealed their role in the metabolism of cellular RNA, our understanding of their contribution to post-transcriptional regulation is relatively limited. In this study, we explore the role of lncRNAs in modulating alternative splicing and their impact on downstream protein–RNA interaction networks. Analysis of alternative splicing events across 39 lncRNA knockdown and wildtype RNA-sequencing datasets from three human cell lines—HeLa (cervical cancer), K562 (myeloid leukemia), and U87 (glioblastoma)—resulted in the high-confidence (false discovery rate (fdr) < 0.01) identification of 11,630 skipped exon events and 5895 retained intron events, implicating 759 genes to be impacted at the post-transcriptional level due to the loss of lncRNAs. We observed that a majority of the alternatively spliced genes in a lncRNA knockdown were specific to the cell type. In tandem, the functions annotated to the genes affected by alternative splicing across each lncRNA knockdown also displayed cell-type specificity. To understand the mechanism behind this cell-type-specific alternative splicing pattern, we analyzed RNA-binding protein (RBP)–RNA interaction profiles across the spliced regions in order to observe cell-type-specific alternative splice event RBP binding preference. Despite limited RBP binding data across cell lines, alternatively spliced events detected in lncRNA perturbation experiments were associated with RBPs binding in proximal intron–exon junctions in a cell-type-specific manner. The cellular functions affected by alternative splicing were also affected in a cell-type-specific manner. Based on the RBP binding profiles in HeLa and K562 cells, we hypothesize that several lncRNAs are likely to exhibit a sponge effect in disease contexts, resulting in the functional disruption of RBPs and their downstream functions. We propose that such lncRNA sponges can extensively rewire post-transcriptional gene regulatory networks by altering the protein–RNA interaction landscape in a cell-type-specific manner.

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