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

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

Genes ◽

10.3390/genes11070820 ◽

2020 ◽

Vol 11 (7) ◽

pp. 820 ◽

Cited By ~ 1

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.

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A Dynamic Alternative Splicing Program Regulates Gene Expression In A Differentiation Stage-Specific Manner During Terminal Erythropoiesis

Blood ◽

10.1182/blood.v122.21.3413.3413 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3413-3413 ◽

Cited By ~ 1

Author(s):

Harold Pimentel ◽

Marilyn Parra ◽

Jie Li ◽

Sherry Gee ◽

Dana Ghanem ◽

...

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Rna Binding ◽

Conflicts Of Interest ◽

Globin Gene ◽

Structure And Function ◽

Rna Seq ◽

Arginine Residues ◽

Alternative Splicing Events ◽

And Function

Abstract Spatio-temporal regulation of switches in alternative pre-mRNA splicing modulate exon usage, critically remodeling the transcriptome during development and differentiation of many tissues, while aberrant regulation of alternative splicing disrupts these processes and plays a role in numerous human diseases. Recently, the discovery of splicing factor mutations in myelodysplasia has increased interest in splicing regulation in hematology. Previously, a functionally critical erythroid splicing switch in protein 4.1 pre-mRNA has been reported, in which activation of alternative exon 16 splicing in late erythroblasts is required for assembly of a mechanically stable red cell membrane. To explore globally the landscape of important alternative splicing events in the erythroid lineage, we applied RNA-seq analysis to five highly FACS-purified populations of human erythroblasts, cultured from CD34+ cord blood progenitors, representing proerythroblasts, early and late basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatophilic erythroblasts. Alternative splicing events predicted by computational analysis were filtered to remove low expression genes and low frequency splicing events, to derive a list of >3000 ‘major’ alternative splicing events of potential importance in erythroid biology. Many of these were validated by inspection of RNA-seq reads mapped on the human genome, and/or by RT-PCR analysis. In this unique differentiation system we found an extensive and dynamic alternative splicing program enriched in genes that function in cell cycle regulation, organelle organization, chromatin structure and function, and RNA processing. For example, we identified alternative splicing events in ∼25 genes encoding chromatin modifying enzymes that methylate, demethylate, or acetylate specific lysine or arginine residues in histones; in transcription modulators such as ATRX and BCL11A that regulate normal globin gene expression; and in ∼50 RNA binding proteins with various roles in post-transcriptional gene regulation. Comparison of PSI (percent spliced in) values across the differentiation series revealed that dozens of alternative exons exhibit substantial switches in splicing efficiency during terminal erythropoiesis. The majority of splicing switches occur in late-stage polychromatophilic and orthochromatophilic erythroblasts, temporally correlated with changes in transcript abundance for many splicing factors and with substantial cell remodeling prior to enucleation. One of the biggest switches in late erythroblasts involves inclusion of a 35nt exon in the NDEL1 (nuclear distribution factor E-homolog-like1) gene, which alters C-terminal structure of a protein that functions in nuclear migration and nucleokinesis in nonerythroid cells and may have a role in erythroblast enucleation. Most of the regulated splicing events insert or delete sequences predicted to modulate protein structure and function in late erythroblasts. However, a subset of altered splicing events have a different effect on gene expression by introducing premature translation termination codons (PTCs), leading us to hypothesize that alternative splicing-coupled nonsense-mediated-decay (AS-NMD) contributes to stage-specific down-regulation of numerous erythroid transcripts. Consistent with such a model, most genes that up-regulate PTC exons in late erythroblasts exhibit reduction in overall expression levels, and inhibition of NMD increases the apparent expression of PTC isoforms. In contrast, genes that up-regulate coding exons are not preferentially down-regulated in late erythroblasts. We conclude that a dynamically regulated alternative splicing program in terminally differentiating erythroblasts plays a major post-transcriptional role in shaping gene expression as the cells transition from proliferation to differentiation, ensuring synthesis of the appropriate constellation of proteins as the cells prepare for enucleation and production of mature red cells. Disclosures: No relevant conflicts of interest to declare.

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SF3B1 Gene Expression in Erythroid Cells Is Regulated By Intron Retention Via a Posttranscriptional Mechanism Involving Cryptic Exons Proposed to Function As Splicing Decoys

Blood ◽

10.1182/blood.v130.suppl_1.931.931 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 931-931

Author(s):

Marilyn Parra ◽

Benjamin W Booth ◽

Gene W Yeo ◽

Richard Weiszmann ◽

Susan E Celniker ◽

...

Keyword(s):

Rna Binding ◽

Conflicts Of Interest ◽

Early Stage ◽

Intron Retention ◽

K562 Cells ◽

Rna Seq ◽

Splice Sites ◽

Site Factors ◽

Splice Junctions ◽

Intron 4

Abstract Proper expression of the MDS-disease gene, SF3B1, ensures appropriate pre-mRNA splicing in erythroid progenitors and during terminal erythropoiesis. We previously showed that the SF3B1 gene is post-transcriptionally regulated in a differentiation stage-specific manner by intron retention (IR), such that ~50% of its transcripts in mature erythroblasts retain intron 4. Based on new mechanistic studies, we propose a model in which mostly unannotated and noncoding exons within intron 4 function as splicing decoys; i.e., they promote retention of intron 4 by interacting with, and blocking splice sites of, the adjacent exons 4 and 5. A total of six putative decoy exons were revealed via RT-PCR and RNA-seq analysis of RNA from erythroblasts treated with inhibitors of nonsense-mediated decay. That decoy exons have IR-promoting activity is suggested by several criteria. First, the frequency of interaction between constitutive exons 4 and 5 and putative decoy exons within intron 4, measured by the abundance of splice junctions in RNA-seq read data, is temporally correlated with levels of intron 4 retention during terminal erythropoiesis. Both IR and decoy splice junctions were low in early stage erythroblasts and much higher in mature erythroblasts. Second, selected decoy exons exhibited IR-promoting activity in the context of minigene splicing reporters expressing the exon 3-6 region of SF3B1 in transfected K562 cells. The wild type minigene reproduced the intron-specific retention phenotype, since it was fully spliced at introns 3 and 5 but exhibited substantial retention of intron 4, whereas deletion of decoy exon 4e, or mutation of its splice sites, substantially decreased IR. Third, RBP (RNA binding protein) cross-linking data from K562 cells show that 3' splice site factors including U2AF1 and U2AF2 can bind specifically to 3' splice sites of intron 4's decoy exons. Finally, several experiments showed that IR-promoting activity of decoy exons is a more general phenomenon that likely governs IR in other erythroid genes. We observed not only that SF3B1 intron 4 decoy exons could promote IR in heterologous contexts, but also that predicted decoy exons from other erythroblast transcripts could promote IR in the SF3B1 minigene. Apart from this experimental data, comparative genomics revealed that the SF3B1 decoy exons are extremely conserved among vertebrate genomes, with two of the exons being essentially identical from fish to humans. Together this data supports the hypothesis that a subset of up-regulated IR events in late erythroblasts are controlled by decoy exons that block productive splicing at the flanking exons. We propose that regulated IR is an important post-transcriptional mechanism for adjusting cellular splicing capacity during terminal erythropoiesis by regulating expression of key splicing factors such as SF3B1. 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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RNA-Seq Analysis Reveals Localization-Associated Alternative Splicing across 13 Cell Lines

10.1101/860783 ◽

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.

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

Blood ◽

10.1182/blood.v122.21.2443.2443 ◽

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.

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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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Functional Consequences of Spliceosome Mutations

Blood ◽

10.1182/blood.v128.22.sci-18.sci-18 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-18-SCI-18

Author(s):

Stephanie Halene

Keyword(s):

Alternative Splicing ◽

Alternative Splice ◽

Rna Binding ◽

Conflicts Of Interest ◽

Cellular Transformation ◽

Cellular Function ◽

Splicing Factor ◽

Common Mechanism ◽

Small Subset ◽

Protein Levels

Abstract Mutations in key factors of the spliceosome occur in over 50% of patients with myelodysplastic syndromes (MDS) and thus splicing represents the most frequently affected pathway in MDS. The vast majority of mutations cluster in four genes: SF3B1, SRSF2, U2AF1, and ZRSR2. Mutations occur in a mutually exclusive manner suggesting a common mechanism by which they cause disease, and yet they segregate to specific subtypes of myeloid malignancies suggesting unique, mutation-specific functions. With the exclusion of ZRSR2, mutations occur at highly restricted residues and are strictly heterozygous, suggestive of gain of function mutations. We and others have shown that mutations affect RNA binding affinity and specificity, and can lead to a variety of outcomes including exon inclusion or skipping, alternative splice site selection, intron retention, and most recently alternative polyadenylation. Overall, these splice changes are subtle: only a small subset of all splice events is affected by these mutations and changes in isoform usage are < 10% for most alternative splice events identified. Furthermore, a direct connection between alternatively spliced transcripts and differentially expressed genes is often not seen. However, these alternative splice events affect genes critical to cellular function and hematopoiesis. Examples include EZH2, ABCB7, macroH2A1, and ATG7. Mutations in SRSF2 result in inclusion of a poison exon in EZH2 resulting in reduced protein levels of this critical epigenetic regulator. Mutations in SF3B1 cause alternative splicing of ABCB7 slating its RNA for non-sense mediated decay. Intriguingly, mutations in U2AF1 promote usage of a distal polyadenylation site in an essential autophagy factor, ATG7, resulting in inefficient translation and decreased protein levels. As a consequence, autophagy is impaired, which can promote cellular transformation, providing a possible explanation for the clonal advantage of cells with splicing factor mutations. Additional research is critical to further determine how alternative splicing contributes to the MDS phenotype with impaired hematopoietic differentiation and cellular function and eventual malignant transformation. Latest findings will be presented here with a focus on mechanistic, functional, and biologic effects of splicing factor mutations. 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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