Frequency Of Alternative Splicing Research Articles

The Frequency of Alternative Splicing in Multiple Myeloma Is Determined By the Non-Homologous End Joining Pathway

Introduction: Alternative Splicing (AS) plays a pivotal role in regulating cellular processes in both normal and malignant cells. Previous studies indicated mutations in the spliceosome complex, such as SF3B1, can cause aberrant AS in acute myeloid leukemia and multiple myeloma (MM) patients. However, there is also a known interaction between AS and the DNA damage response in other cancer types, which we investigate here. Materials and Methods: RNA-sequencing data of newly diagnosed MM patients (n=598) from the MMRF CoMMpass dataset were utilized to generate whole transcriptome AS profiles. For each patient, RNA-seq data was aligned to Hg38 reference genome using STAR while expression level was measured by Salmon. AS profiles were established from their alignment and expression profile using SUPPA2. Splicing level was defined by Percentage of Spliced-In (PSI). UMAP plots were generated for all patients using top 20,000 AS events ranked by variation in the sample set. High-quality differential AS events between groups of patients were obtained from the following thresholds: significant splicing level difference (|PSI|>10% and P <0.05 from T-test), sufficient expression level (TPM>1), not similarly spliced (|PSI|>10% and P-value<0.05 from T-test) in normal bone marrow plasma cells (N=5). Patient samples were annotated for cytogenetic, mutation, copy number and structural variation status. Pathway analysis was conducted for differentially expressed genes (DEGs). Patients' overall survival and progression-free survival was displayed with Kaplan-Meier curves. Survival difference was measured by log-rank test and cox-regression (when other well-known risk factors were incorporated). Results: We examined differential AS by defining and comparing high and low activity of key myeloma KEGG pathways including the spliceosome, proteasome, DNA repair and ubiquitination pathways. The DNA repair pathway generated significantly more (P<0.05) differential AS events (N=1,197) than the other pathways. To examine this further, we performed the analysis for the three main DNA repair mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) pathways. The number of differential AS events (N=2,999) between NHEJ-high (N=121) and -low (N=36) samples was significantly more than for the other DNA repair pathways (P<0.05). NHEJ-high samples were significantly enriched for TP53 mutations (15% vs. 6%, P=2x10-5, Fisher's exact test), biallelic-TP53 events (10% vs. 4%, P=3x10-5), and biallelic-TENT5C (21% vs. 10%, P=1x10-9) events (Figure 1). Additionally, expression of LIG4 and XRCC4, encoding DNA IV ligase complex, a key component of the NHEJ pathway, correlated highly (, Spearman correlation) with the number of differential AS events. The NHEJ-high group also harbored significantly more structural abnormalities than the NHEJ-low group (mean 87 vs. 19, P=6x10-4, Mann-Whitney U test), indicating their different status of DNA stability that was highly associated with this pathway. Survival analysis indicated that patients in the NHEJ-high group showed a significant worse progression-free survival (PFS) compared to others (Logrank statistic=7.3, P=0.007). In a multivariate analysis including other high-risk factors such as MGP-Double-Hit, t(4;14), t(14;16), TP53 mutation, 17p del, 1q gain/amp, and ISS=III, the NHEJ-high group retained significance (Hazard Ratio=1.8, P=1.6x10-5). To incorporate AS events we further applied a backward elimination approach to automatically select an optimal prognostic model from top 200 highly variant AS events ranked by variation and 7 well-known risk factors along with high NHEJ activity in PFS for the entire population. This model contained 16 significant risk factors (P<0.05, Multivariate cox regression) and 12 of them were AS events. NHEJ-high samples were significantly enriched (P<0.05, Fisher exact test) for eight of the AS events that were associated with poor prognosis. Conclusion: AS frequency is associated with the NHEJ pathway in MM. NHEJ activity is also associated with DNA instability through increased number of structural rearrangements and is a high-risk factor. Figure 1. High expression of the NHEJ pathways is associated with TP53 and TENT5C abnormalities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CDK6-PI3K signaling axis is an efficient target for attenuating ABCB1/P-gp mediated multi-drug resistance (MDR) in cancer cells

BackgroundMultidrug resistance (MDR) mediated by ATP binding cassette subfamily B member 1 (ABCB1/P-gp) is a major cause of cancer chemotherapy failure, but the regulation mechanisms are largely unknown.MethodsBased on single gene knockout, we studied the regulation of CDK6-PI3K axis on ABCB1-mediated MDR in human cancer cells. CRISPR/Cas9 technique was performed in KB-C2 cells to knockout cdk6 or cdk4 gene. Western blot, RT-PCR and transcriptome analysis were performed to investigate target gene deletion and expression of critical signaling factors. The effect of cdk4 or cdk6 deficiency on cell apoptosis and the cell cycle was analyzed using flow cytometry. In vivo studies were performed to study the sensitivity of KB-C2 tumors to doxorubicin, tumor growth and metastasis.ResultsDeficiency of cdk6 led to remarkable downregulation of ABCB1 expression and reversal of ABCB1-mediated MDR. Transcriptomic analysis revealed that CDK6 knockout regulated a series of signaling factors, among them, PI3K 110α and 110β, KRAS and MAPK10 were downregulated, and FOS-promoting cell autophagy and CXCL1-regulating multiple factors were upregulated. Notably, PI3K 110α/110β deficiency in-return downregulated CDK6 and the CDK6-PI3K axis synergizes in regulating ABCB1 expression, which strengthened the regulation of ABCB1 over single regulation by either CDK6 or PI3K 110α/110β. High frequency of alternative splicing (AS) of premature ABCB1 mRNA induced by CDK6, CDK4 or PI3K 110α/110β level change was confirmed to alter the ABCB1 level, among them 10 common skipped exon (SE) events were found. In vivo experiments demonstrated that loss of cdk6 remarkably increased the sensitivity of KB-C2 tumors to doxorubicin by increasing drug accumulation of the tumors, resulting in remarkable inhibition of tumor growth and metastasis, as well as KB-C2 survival in the nude mice.ConclusionsCDK6-PI3K as a new target signaling axis to reverse ABCB1-mediated MDR is reported for the first time in cancers. Pathways leading to inhibition of cancer cell proliferation were revealed to be accompanied by CDK6 deficiency.

Pretumor microenvironment of hepatocellular carcinoma: Cancerization or anticancerization?

BackgroundTumor microenvironment (TM) has been deeply concerned. However, the pretumor microenvironment (PTM) was poorly understood. The purpose in this study was to explore the possible pathophysiological features of PTM before hepatocellular carcinoma (HCC) appearance. MethodsMouse livers with no swelling but with tumors present elsewhere in the body after subcutaneous injection of H22 in the fore underarm were considered a PTM, HCC tumors presenting far away from the PTM were regarded as a TM, and the healthy livers of mice without injection of H22 were regarded as a physiological microenvironment (PM). The transcriptomes of samples were generated using RNA-seq and validated using RT-qPCR. ResultsOverall, 4483 differentially expressed genes (DEGs) were found in the TM compared with the PTM (TM/PTM), but only 194 were altered in the PTM compared with the PM (PTM/PM). Among those 194 DEGs, 104 displayed upregulation and 90 downregulation. Some of these DEGs could promote the ability to resist cancerization or facilitate cancer metastasis, while others indicated liver impairment. The DEGs were involved in 16 relevant pathways. Additionally, the frequency of alternative splicing (AS) in the DEGs in various samples was positively related to the expression of those DEGs. ConclusionsThe PTM initiatively armed itself to combat cancerization when its indications appeared although the PTM did not manifest any tissue swelling. However, the cancer cells were negatively influencing immunity to prevent clearance and positively promoting transformation to construct a suitable environment. During transformation by cancer cells, some genes with acquired AS participated in the construction of the PTM. This alteration created an invadable space and an appropriate environment for cancer cells.

Prp8 impacts cryptic but not alternative splicing frequency

Pre-mRNA splicing must occur with extremely high fidelity. Spliceosomes assemble onto pre-mRNA guided by specific sequences (5' splice site, 3' splice site, and branchpoint). When splice sites are mutated, as in many hereditary diseases, the spliceosome can aberrantly select nearby pseudo- or "cryptic" splice sites, often resulting in nonfunctional protein. How the spliceosome distinguishes authentic splice sites from cryptic splice sites is poorly understood. We performed a Caenorhabditis elegans genetic screen to find cellular factors that affect the frequency with which the spliceosome uses cryptic splice sites and identified two alleles in core spliceosome component Prp8 that alter cryptic splicing frequency. Subsequent complementary genetic and structural analyses in yeast implicate these alleles in the stability of the spliceosome's catalytic core. However, despite a clear effect on cryptic splicing, high-throughput mRNA sequencing of these prp-8 mutant C. elegans reveals that overall alternative splicing patterns are relatively unchanged. Our data suggest the spliceosome evolved intrinsic mechanisms to reduce the occurrence of cryptic splicing and that these mechanisms are distinct from those that impact alternative splicing.

Cascade: an RNA-seq visualization tool for cancer genomics.

BackgroundCancer genomics projects are producing ever-increasing amounts of rich and diverse data from patient samples. The ability to easily visualize this data in an integrated an intuitive way is currently limited by the current software available. As a result, users typically must use several different tools to view the different data types for their cohort, making it difficult to have a simple unified view of their data.ResultsHere we present Cascade, a novel web based tool for the intuitive 3D visualization of RNA-seq data from cancer genomics experiments. The Cascade viewer allows multiple data types (e.g. mutation, gene expression, alternative splicing frequency) to be simultaneously displayed, allowing a simplified view of the data in a way that is tuneable based on user specified parameters. The main webpage of Cascade provides a primary view of user data which is overlaid onto known biological pathways that are either predefined or added by users. A space-saving menu for data selection and parameter adjustment allows users to access an underlying MySQL database and customize the features presented in the main view.ConclusionsThere is currently a pressing need for new software tools to allow researchers to easily explore large cancer genomics datasets and generate hypotheses. Cascade represents a simple yet intuitive interface for data visualization that is both scalable and customizable.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2389-8) contains supplementary material, which is available to authorized users.

Alternative mRNA processing increases the complexity of microRNA‐based gene regulation in Arabidopsis

MicroRNAs (miRNAs) represent an important class of sequence-specific, trans-acting endogenous small RNA molecules that modulate gene expression at the post-transcriptional level. They function by binding to partial complementary cis-regulatory sites (miRNA binding sites) in their target mRNAs. Based on two recent observations from plant genome studies, namely that alternative splicing is a common phenomenon and that miRNA regulates a significant proportion of the transcriptome, we hypothesize that there may be a mechanism for gene regulation that involves both processes. In the present study, we performed a systemic search in the model plant Arabidopsis thaliana using annotated gene models as well as publically available high-throughput RNA sequencing data with a total of 570 million reads. Of the 354 high-confidence miRNA binding sites identified in Arabidopsis, at least 44 (12.4%) were affected by alternative splicing such that mRNA isoforms of the same miRNA target gene differ in the sequences encoding the miRNA binding sites. By simulation, we found that the frequency of alternative splicing at miRNA binding sites is significantly higher than at other regions. Comparative and functional analyses further indicated that the alternative splicing events are important for target gene expression and miRNA action. Together our results show that alternative splicing of miRNA binding sites is a plausible mechanism for attenuating miRNA-mediated gene regulation.

The adaptive significance of unproductive alternative splicing in primates

Alternative gene splicing is pervasive in metazoa, particularly in humans, where the majority of genes generate splice variant transcripts. Characterizing the biological significance of alternative transcripts is methodologically difficult since it is impractical to assess thousands of splice variants as to whether they actually encode proteins, whether these proteins are functional, or whether transcripts have a function independent of protein synthesis. Consequently, to elucidate the functional significance of splice variants and to investigate mechanisms underlying the fidelity of mRNA splicing, we used an indirect approach based on analyzing the evolutionary conservation of splice variants among species. Using DNA polymerase β as an indicator locus, we cloned and characterized the types and frequencies of transcripts generated in primary cell lines of five primate species. Overall, we found that in addition to the canonical DNA polymerase β transcript, there were 25 alternative transcripts generated, most containing premature terminating codons. We used a statistical method borrowed from community ecology to show that there is significant diversity and little conservation in alternative splicing patterns among species, despite high sequence similarity in the underlying genomic (exonic) sequences. However, the frequency of alternative splicing at this locus correlates well with life history parameters such as the maximal longevity of each species, indicating that the alternative splicing of unproductive splice variants may have adaptive significance, even if the specific RNA transcripts themselves have no function. These results demonstrate the validity of the phylogenetic conservation approach in elucidating the biological significance of alternative splicing.

An Alternative Cyclin-D1 Splice Site is Not Linked to Inflammatory Bowel Disease-Associated Neoplasia

Inflammatory bowel diseases (IBD) encompass inflammatory disorders affecting the gastrointestinal tract, primarily ulcerative colitis (UC) and Crohn's disease (CD). The risk of developing colorectal cancer (CRC) is increased in patients with IBD. The CCND1 protein is the regulatory subunit of an enzyme that inactivates the retinoblastoma protein, a tumor suppressor protein, and promotes progression through the G1-S phase of the cell cycle. The CCND1 870G-A gene polymorphism influences susceptibility to colorectal cancer. The mutant allele of CCND1 in IBD-associated neoplasia leads to a greater frequency of alternate splicing during transcription, resulting in a more stable CCND1 protein. This creates a higher concentration of CCND1, facilitating easier passage through the G1/S checkpoint, abnormal cell cycle progression, and possibly carcinogenesis. We conducted a case-control study involving 396 individuals with IBD. IBD subgroups included CD, UC, and indeterminate colitis (IC). We studied patients with sporadic colorectal cancer (n=75) and patients without gastrointestinal disease as a control group (n=93). We extracted DNA from blood and performed polymerase chain reaction followed by high-performance liquid chromatography to screen for mutations. We confirmed the polymorphism at nucleotide A870G in exon 4. For statistical analysis, we used exact analyses of two-way contingency tables. Power calculations were done and correction for multiple testing was performed by computing the false discovery rate (FDR). Our study had a power of 75% at a 0.05 significance level. A870G SNP allele frequency in the IBD group was 44.8%, compared to 51.6% in the control population. Only the IC group showed a significant association with CCND1 splice site after correction for multiple testing (FDR=0.042). There were no differences between the other IBD groups and controls. We found an association between CCND1 A870G SNP and IC group only (p=0.014, FDR=0.042). However, our data do not show an association between CCND1 A870G SNP and CD-associated or UC-associated neoplasia.

Use of alternate splicing to identify subgroups in uniformly treated newly-diagnosed myeloma

8602 Gene function is modulated at multiple levels. Besides expression level changes, post-transcriptional changes such as alternate splicing alter specificity of gene function. Misregulation of alternate splicing is associated with various disease processes including cancer. We have analyzed alternate splicing in myeloma (MM) using high throughput exon array analysis. Affymetrix Human Exon 1.0 ST Arrays used in this investigation, besides expression levels, also provides information on presence of each exon and identifies recurrent alternately spliced genes. We evaluated 170 newly-diagnosed patients with MM treated homogeneously in tandem transplantation IFM trials using CD138 purified MM cells. Xray array tool (Biotique systems) and dChip software was modified to analyze exon array data. We first normalized gene-level expression values across samples, and then identified differentially expressed exons to identify alternate splicing events. We observed over 100 genes with alternate splicing in myeloma with frequency in more than 20% patients. Unsupervised hierarchical clustering showed two groups within myeloma patients which differ in overall survival (OS). Dividing the group based on response, 52 alternately spliced exons were identified as influencing response to therapy including CD79A exon 4, XRCC exon 3 and 17, PRKDC exon 4, NIT 2 exon 4, Calmin exon2, MADH1 exon 7, TBX5 exon 2, Amyloid beta exon 14 with the highest frequency of alternate splicing. Amongst these, 32 genes had the most influence on response with over 50% differential frequency in patients achieving CR. Similarly 85 alternately spliced genes are influencing OS between groups at 48 month survival cut-off. 49 genes within this group had the most influence on OS. Shared spliced variants between both response and survival groups suggesting their biological and functional significance. Further validation of these alternate splicing is under investigations. This study highlights that alternate splicing is observed with significant frequency and demand need for evaluation of not only the expression level of genes but also post translational modifications. The spliced genes identified here are important target for therapy as well as possible immune modulation. No significant financial relationships to disclose.

Genome-wide analyses of alternative splicing in plants: Opportunities and challenges

Alternative splicing (AS) creates multiple mRNA transcripts from a single gene. While AS is known to contribute to gene regulation and proteome diversity in animals, the study of its importance in plants is in its early stages. However, recently available plant genome and transcript sequence data sets are enabling a global analysis of AS in many plant species. Results of genome analysis have revealed differences between animals and plants in the frequency of alternative splicing. The proportion of plant genes that have one or more alternative transcript isoforms is approximately 20%, indicating that AS in plants is not rare, although this rate is approximately one-third of that observed in human. The majority of plant AS events have not been functionally characterized, but evidence suggests that AS participates in important plant functions, including stress response, and may impact domestication and trait selection. The increasing availability of plant genome sequence data will enable larger comparative analyses that will identify functionally important plant AS events based on their evolutionary conservation, determine the influence of genome duplication on the evolution of AS, and discover plant-specific cis-elements that regulate AS. This review summarizes recent analyses of AS in plants, discusses the importance of further analysis, and suggests directions for future efforts.

Alternative splicing and protein function

BackgroundAlternative splicing is a major mechanism of generating protein diversity in higher eukaryotes. Although at least half, and probably more, of mammalian genes are alternatively spliced, it was not clear, whether the frequency of alternative splicing is the same in different functional categories. The problem is obscured by uneven coverage of genes by ESTs and a large number of artifacts in the EST data.ResultsWe have developed a method that generates possible mRNA isoforms for human genes contained in the EDAS database, taking into account the effects of nonsense-mediated decay and translation initiation rules, and a procedure for offsetting the effects of uneven EST coverage. Then we computed the number of mRNA isoforms for genes from different functional categories. Genes encoding ribosomal proteins and genes in the category "Small GTPase-mediated signal transduction" tend to have fewer isoforms than the average, whereas the genes in the category "DNA replication and chromosome cycle" have more isoforms than the average. Genes encoding proteins involved in protein-protein interactions tend to be alternatively spliced more often than genes encoding non-interacting proteins, although there is no significant difference in the number of isoforms of alternatively spliced genes.ConclusionFiltering for functional isoforms satisfying biological constraints and accountung for uneven EST coverage allowed us to describe differences in alternative splicing of genes from different functional categories. The observations seem to be consistent with expectations based on current biological knowledge: less isoforms for ribosomal and signal transduction proteins, and more alternative splicing of interacting and cell cycle proteins.

Cyclin D1 gene (CCND1) polymorphism and the risk of squamous cell carcinoma of the larynx

Cyclin D1 is one of the key proteins involved in cell cycle control, and it is believed that its overexpression may be connected with tumorigenesis. A reason for cyclin D1 deregulation may be connected to a common G870A polymorphism at codon 242 in exon 4 of the CCND1 gene. This single nucleotide substitution, localized in the conserved splice donor site between exon 4 and the intron 4 boundary, might modulate the frequency of alternative splicing. It has been postulated that the A allele results in a higher level of mRNA (transcript b) encoding a protein with an altered C-terminal domain. The influence of CCND1 G-->A polymorphism for the risk of cancer and the prognosis of patients with different types of solid tumors has already been suggested. This study was conducted to investigate the association between the cyclin D1 gene polymorphism and laryngeal cancer risk, as well as the clinical outcome. We also examined the relationship between genotype/allele distributions and the cyclin D1 expression profile. The genotyping study was done using the PCR-RFLP method in 63 patients with larynx cancer and 102 healthy controls. The heterozygotic genotype GA as well as a combination of GA and AA genotypes were associated with an increased risk of larynx cancer compared to the GG genotype (OR =3.02; P =0.004 and OR =2.52; P =0.013, respectively). The A allele frequency was higher in cancer cases (0.484) than in controls (0.416) that were connected with a slightly increased risk of cancer development (OR =1.34); however, the difference was not significant. The AA genotype was associated with an early cancer onset compared to the GG genotype (median age: 51.5 and 63.0 years, respectively). We also demonstrated that the AA genotype was associated with the occurrence of lymph node metastases (OR =3.26) and a higher level of cyclin D1 overexpression. These results suggest that the CCND1 A allele may be a genetic factor that modulates the risk of larynx cancer development, and it may also have an effect on tumor biology and disease prognosis.

Alternative splicing ofMDM2 mRNA in lung carcinomas and lung cell lines

The MDM2 gene is overexpressed in several human tumors and its product may be processed into various isoforms. Recently, alternative splicing forms of MDM2 mRNA have been detected in various types of tumors. In this study, lung tissue from human non small cell lung cancers was examined for MDM2 mRNA splicing variants by nested RT-PCR. Of the 117 lung cancer tissue samples analyzed, a total of 31 (26.5%) had splice variants for the MDM2 gene, while 59 (50.4%) had undetectable levels of MDM2 transcript. Further analysis indicated that the predominant variant for 26 of the 31 samples with alternative MDM2 splicing products was MDM2-657, a splice variant lacking exons 3-11. Significant associations were found between the frequency of alternative splicing and the gender and smoking habits of the patients. Approximately 36% of male patients had alternative splicing of MDM2 compared with only 9.5% of female patients (P = 0.008); 44.2% of the smoker patients had alternative MDM2 splice forms versus 16.2% of nonsmokers (P = 0.003). Furthermore, most normal lung cell lines examined possessed only full-length MDM2 mRNA, while among several lung cancer cell lines, only H1355 and CaLu-1 cells lacked alternatively spliced MDM2 transcripts. When H1355 cells were treated in vitro with the cigarette smoke carcinogen benzo[a]pyrene (B[a]P) or the B[a]P metabolite benzo[a]pyrene diolepoxide (BPDE), three MDM2 splicing products were detected by nested RT-PCR. Finally, with the use of several specific inhibitors, we found that BPDE-induced MDM2 mRNA alternative splicing in H1355 cells may occur through the PI3K or MAPK pathway. Overall, our results suggest that carcinogens present in cigarette smoke increase the risk of alternative MDM2 splicing, which is highly associated with lung cancer.

High frequency of alternative splicing of human genes participating in the HIV-1 life cycle: a model using TSG101, betaTrCP, PPIA, INI1, NAF1, and PML.

Alternative splicing may generate splice forms with different biologic roles or missing protein domains implicated in the interaction with HIV-1. To address this issue, 6 human genes were investigated-tumor suppressor gene 101 (TSG101), beta-transducin repeats containing protein (betaTrCP), peptidyl-proly cis-trans isomerase, cyclophilin A (PPIA), integrase interactor 1 protein (INI1), Nef-associated factor 1 (NAF1), and promyelacytic leukemia (PML)-involved in the viral life cycle and HIV-1 pathogenesis. All 6 genes presented alternative splicing, and a combined bioinformatic and reverse transcription polymerase chain reaction (RT-PCR) analysis identified 27 new variants for a total of 53 splice forms (an average of 9 variants per gene). The predicted frequency of the various splice forms based on expressed sequence tags (EST) analysis corresponded to the semiquantitative findings on RT-PCR analysis for the cell culture systems and for native CD4 cells investigated. Interindividual variation in the frequencies of various splice forms in CD4 T cells from blood donors was observed for INI1. Cell type-specific variation of splice pattern was observed for NAF1. Eight splice forms lacked or modified motifs implicated in the interaction with HIV-1, underscoring the potential interest of assessing alternative splicing when investigating viral cell biology and pathogenesis.