Single molecule analysis of c-myb alternative splicing reveals novel classifiers for precursor B-ALL.
ABSTRACT: The c-Myb transcription factor, a key regulator of proliferation and differentiation in hematopoietic and other cell types, has an N-terminal DNA binding domain and a large C-terminal domain responsible for transcriptional activation, negative regulation and determining target gene specificity. Overexpression and rearrangement of the c-myb gene (MYB) has been reported in some patients with leukemias and other types of cancers, implicating activated alleles of c-myb in the development of human tumors. Alternative RNA splicing can produce variants of c-myb with qualitatively distinct transcriptional activities that may be involved in transformation and leukemogenesis. Here, by performing a detailed, single molecule assay we found that c-myb alternative RNA splicing was elevated and much more complex in leukemia samples than in cell lines or CD34+ hematopoietic progenitor cells from normal donors. The results revealed that leukemia samples express more than 60 different c-myb splice variants, most of which have multiple alternative splicing events and were not detectable by conventional microarray or PCR approaches. For example, the single molecule assay detected 21 and 22 splice variants containing the 9B and 9S exons, respectively, most of which encoded unexpected variant forms of c-Myb protein. Furthermore, the detailed analysis identified some splice variants whose expression correlated with poor survival in a small cohort of precursor B-ALL samples. Our findings indicate that single molecule assays can reveal complexities in c-myb alternative splicing that have potential as novel biomarkers and could help explain the role of c-Myb variants in the development of human leukemia.
Project description:The c-Myb transcription factor regulates the proliferation and differentiation of hematopoietic cells, and activated alleles of c-myb induce leukemias and lymphomas in animals. Relatively minor changes in the structure of c-Myb protein change the genes that it regulates and can unleash its latent transforming activities. Here, quantitative assays were used to analyze the alternative splicing of human c-myb transcripts. We identified an array of variant transcripts, expressed in highly regulated, lineage-specific patterns, that were formed through the use of alternate exons 8A, 9A, 9B, 10A, 13A, and 14A. Expression levels of the different splice variant transcripts were regulated independently of one another during human hematopoietic cell differentiation, and the alternative splicing of c-myb mRNAs was increased in primary leukemia samples. The alternatively spliced c-myb transcripts were associated with polysomes and encoded a series of c-Myb proteins with identical DNA binding domains but unique C-terminal domains. In several types of assays, the variant c-Myb proteins exhibited quantitative and qualitative differences in transcriptional activities and specificities. The results suggest that the human c-myb gene encodes a family of related proteins with different transcriptional activities. Enhanced alternative splicing may be a mechanism for unmasking the transforming activity of c-myb in human leukemias.
Project description:We have recently established that several mRNAs encode the PR264/SC35 splicing factor and that their expression is related to that of c-Myb in human hematopoietic cells. We now report that the various PR264 mRNAs whose expression is transactivated by c-Myb proteins encode an identical PR264/SC35 polypeptide. These mRNAs differ only in their 3' untranslated sequences and exhibit significantly different half-lives. We also show that there is a direct correlation between the length of the 3' untranslated region and the stability of the mRNA species. Given that 5' and 3' splice site selection mediated by the PR264/SC35 splicing factor is concentration-dependent, we postulate that alternative splicing of the 3' untranslated sequences might represent another key element in regulating the levels of PR264/SC35 expression during hematopoietic differentiation.
Project description:The circadian clock regulates numerous biological processes in plants, especially development and stress responses. CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) is one of the core components of the day-night rhythm response and is reportedly associated with ambient temperature in Arabidopsis thaliana. However, it remains unknown if alternative splicing of ZmCCA1 is modulated by external stress in maize, such as drought stress and photoperiod. Here, we identified three ZmCCA1 splice variants in the tropical maize line CML288, which are predicted to encode three different protein isoforms, i.e., ZmCCA1.1, ZmCCA1.2, and ZmCCA1.3, which all retain the MYB domain. In maize, the expression levels of ZmCCA1 splice variants were influenced by photoperiod, tissue type, and drought stress. In transgenic A. thaliana, ZmCCA1.1 may be more effective than ZmCCA1.3 in increasing drought tolerance while ZmCCA1.2 may have only a small effect on tolerance to drought stress. Additionally, although CCA1 genes have been found in many plant species, alternative CCA1 splicing events are known to occur in species-specific ways. Our study provides new sight to explore the function of ZmCCA1 splice variants' response to abiotic stress, and clarify the linkage between circadian clock and environmental stress in maize.
Project description:Complete information regarding transcriptional and posttranscriptional gene regulation in stem cells is necessary to understand the regulation of self-renewal and differentiation. Alternative splicing is a prevalent mode of posttranscriptional regulation, and occurs in approximately one half of all mammalian genes. The frequency and functional impact of alternative splicing in stem cells are yet to be determined. In this study we combine computational and experimental methods to identify splice variants in embryonic and hematopoietic stem cells on a genome-wide scale. Using EST collections derived from stem cells, we detect alternative splicing in >1,000 genes. Systematic RT-PCR and sequencing studies show confirmation of computational predictions at a level of 80%. We find that alternative splicing can modify multiple components of signaling pathways important for stem cell function. We also analyze the distribution of splice variants across different classes of genes. We find that tissue-specific genes have a higher tendency to undergo alternative splicing than ubiquitously expressed genes. Furthermore, the patterns of alternative splicing are only weakly conserved between orthologous genes in human and mouse. Our studies reveal extensive modification of the stem cell molecular repertoire by alternative splicing and provide insights into its overall role as a mechanism of generating genomic diversity.
Project description:Age-related human hematopoietic stem cell (HSC) exhaustion and myeloid-lineage skewing promote oncogenic transformation of hematopoietic progenitor cells into therapy-resistant leukemia stem cells (LSCs) in secondary acute myeloid leukemia (AML). While acquisition of clonal DNA mutations has been linked to increased rates of secondary AML for individuals older than 60 years, the contribution of RNA processing alterations to human hematopoietic stem and progenitor aging and LSC generation remains unclear. Comprehensive RNA sequencing and splice-isoform-specific PCR uncovered characteristic RNA splice isoform expression patterns that distinguished normal young and aged human stem and progenitor cells (HSPCs) from malignant myelodysplastic syndrome (MDS) and AML progenitors. In splicing reporter assays and pre-clinical patient-derived AML models, treatment with a pharmacologic splicing modulator, 17S-FD-895, reversed pro-survival splice isoform switching and significantly impaired LSC maintenance. Therapeutic splicing modulation, together with monitoring splice isoform biomarkers of healthy HSPC aging versus LSC generation, may be employed safely and effectively to prevent relapse, the leading cause of leukemia-related mortality.
Project description:Chronic myeloid leukemia (CML) is a malignancy that evolves through a multi-step process. Alternative splicing of several genes has been linked to the progression of the disease, but involvement of alternations in splicing profiles has not been reported. RNA-seq of peripheral blood mononuclear cell (PBMC) samples characterized the differentially expressed and spliced transcripts in five CML chronic phase (CP) and five blast phase (BP) patients, and five healthy controls. Global splicing alteration analysis detected 6474 altered splicing events altered between CML and healthy samples, including many of the previously reported splicing variants and showing a more profound altered splicing deregulation in BP samples. Functional clustering of differentially spliced genes in CP revealed a preferred enrichment relating to cell signaling, while the spliceosome pathway was most overrepresented in BP samples. One differentially spliced spliceosome gene hnRNPA1 showed two splice isoforms; the longer isoform contained exon 8 was preferentially expressed in the BP patients, and the short one excluding exon 8 was specific to healthy controls. Our findings suggested that alternative splicing deregulation played a central role during the progression of CML from CP to BP, and the longer isoform of hnRNPA1 might represent a diagnostic marker and therapeutic target for CML.
Project description:BACKGROUND:PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2-without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. METHODS:Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2 c.212-1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant. RESULTS:We identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies. CONCLUSIONS:PVS1 is not necessarily warranted for splice site variants targeting four PALB2 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenic without further evidences. Our study puts a warning in up to five PALB2 genetic variants that are currently reported as pathogenic/likely pathogenic in ClinVar.
Project description:The transcription factor MYB plays key roles in hematopoietic cells and has been implicated the development of leukemia. MYB has therefore emerged as an attractive target for drug development. Recent work has suggested that targeting MYB by small-molecule inhibitors is feasible and that inhibition of MYB has potential as a therapeutic approach against acute myeloid leukemia. To facilitate the identification of small-molecule MYB inhibitors we have re-designed and improved a previously established cell-based screening assay and have employed it to screen a natural product library for potential inhibitors. Our work shows that teniposide and etoposide, chemotherapeutic agents causing DNA-damage by inhibiting topoisomerase II, potently inhibit MYB activity and induce degradation of MYB in AML cell lines. MYB inhibition is suppressed by caffeine, suggesting that MYB is inhibited indirectly via DNA-damage signalling. Importantly, ectopic expression of an activated version of MYB in pro-myelocytic NB4 cells diminished the anti-proliferative effects of teniposide, suggesting that podophyllotoxins disrupt the proliferation of leukemia cells not simply by inducing general DNA-damage but that their anti-proliferative effects are boosted by inhibition of MYB. Teniposide and etoposide therefore act like double-edged swords that might be particularly effective to inhibit tumor cells with deregulated MYB.
Project description:Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors.We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score-the Maximum-Minimum Exon Score (MMES)--designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort.After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes LAMA3, DST, VEGFC, SDHA, RASIP1, and TP63 were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed TP63 as having dominant expression of the short DeltaNp63 isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (LAMA3 and DST) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Student's t test, P<0.001).Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression.
Project description:BACKGROUND:Expression of the NPM1 gene, encoding nucleophosmin, is upregulated in cancers. Although more than ten NPM1 transcripts are known, the reports were usually limited to one predominant transcript. In leukemia, the NPM1 expression has not been widely studied so far. In acute myeloid leukemia (AML), the mutational status of the gene seems to play a pivotal role in carcinogenesis. Therefore, the aim of the study was to quantify alternative NPM1 transcripts in two types of acute leukemia, AML and ALL (acute lymphoblastic leukemia). METHODS:Using droplet digital PCR, we analyzed the levels of three protein-coding NPM1 transcripts in 66 samples collected from AML and ALL patients and 16 control samples. Using RNA-seq, we detected 8 additional NPM1 transcripts, including non-coding splice variants with retained introns. For data analysis, Welch two sample t-test, Pearson's correlation and Kaplan-Meier analysis were applied. RESULTS:The levels of the particular NPM1 transcripts were significantly different but highly correlated with each other in both leukemia and control samples. Transcript NPM1.1, encoding the longest protein (294 aa), had the highest level of accumulation and was one of the most abundant transcripts in the cell. Comparing to NPM1.1, the levels of the NPM1.2 and NPM1.3 transcripts, encoding a 265-aa and 259-aa proteins, were 30 and 3 times lower, respectively. All three NPM1 transcripts were proportionally upregulated in both types of leukemia compared to control samples. In AML, the levels of NPM1 transcripts decreased in complete remission and increased again with relapse of the disease. Low levels of NPM1.1 and NPM1.3 were associated with better prognosis. The contribution of non-coding transcripts to the total level of NPM1 gene seemed to be marginal, except for one short 5-end transcript accumulated at high levels in AML and control cells. Aberrant proportions of particular NPM1 splice variants could be linked to abnormal expression of genes encoding alternative splicing factors. CONCLUSIONS:The levels of the studied NPM1 transcripts were different but highly correlated with each other. Their upregulation in AML and ALL, decrease after therapy and association with patient outcome suggests the involvement of elevated NPM1 expression in the acute leukemia pathogenesis.