Alternative Splicing Patterns Research Articles

A new strategy to increase RNA editing at the Q/R site of GluA2 AMPA receptor subunits by targeting alternative splicing patterns of ADAR2

BackgroundThe GluA2 subunit of AMPA receptors (AMPARs) undergoes RNA editing at a specific base mediated by the enzyme ADAR2, changing the coded amino acid from a glutamine to arginine at the so-called Q/R site, which is critical for regulating calcium permeability. ADAR2 exists as multiple alternatively-spliced variants within mammalian cells with differing editing efficiency. New methodIn this study, phosphorodiamidate morpholino oligomers (PMOs) were used to increase Q/R site editing, by affecting the alternative splicing of ADAR2. ResultsPMOs targeting the ADAR2 pre-mRNA transcript successfully induced alternative splicing around the AluJ cassette leading to expression of a more active isoform with increased editing of the GluA2 subunit compared to control. Comparison with existing method(s)Previously PMOs have been used to disrupt RNA editing via steric hindrance of the GluA2 RNA duplex. In contrast we report PMOs that can increase the expression of more catalytically active variants of ADAR2, leading to enhanced GluA2 Q/R RNA editing. ConclusionsUsing PMOs to increase Q/R site editing is presented here as a validated method that would allow investigation of downstream cellular processes implicated in altered ADAR2 activity.

SplicingFactory-splicing diversity analysis for transcriptome data.

MotivationAlternative splicing contributes to the diversity of RNA found in biological samples. Current tools investigating patterns of alternative splicing check for coordinated changes in the expression or relative ratio of RNA isoforms where specific isoforms are up- or down-regulated in a condition. However, the molecular process of splicing is stochastic and changes in RNA isoform diversity for a gene might arise between samples or conditions. A specific condition can be dominated by a single isoform, while multiple isoforms with similar expression levels can be present in a different condition. These changes might be the result of mutations, drug treatments or differences in the cellular or tissue environment. Here, we present a tool for the characterization and analysis of RNA isoform diversity using isoform level expression measurements.ResultsWe developed an R package called SplicingFactory, to calculate various RNA isoform diversity metrics, and compare them across conditions. Using the package, we tested the effect of RNA-seq quantification tools, quantification uncertainty, gene expression levels and isoform numbers on the isoform diversity calculation. We analyzed a set of CD34+ hematopoietic stem cells and myelodysplastic syndrome samples and found a set of genes whose isoform diversity change is associated with SF3B1 mutations.Availability and implementationThe SplicingFactory package is freely available under the GPL-3.0 license from Bioconductor for the Windows, MacOS and Linux operating systems (https://www.bioconductor.org/packages/release/bioc/html/SplicingFactory.html).Supplementary information Supplementary data are available at Bioinformatics online.

ExOrthist: a tool to infer exon orthologies at any evolutionary distance

Several bioinformatic tools have been developed for genome-wide identification of orthologous and paralogous genes. However, no corresponding tool allows the detection of exon homology relationships. Here, we present ExOrthist, a fully reproducible Nextflow-based software enabling inference of exon homologs and orthogroups, visualization of evolution of exon-intron structures, and assessment of conservation of alternative splicing patterns. ExOrthist evaluates exon sequence conservation and considers the surrounding exon-intron context to derive genome-wide multi-species exon homologies at any evolutionary distance. We demonstrate its use in different evolutionary scenarios: whole genome duplication in frogs and convergence of Nova-regulated splicing networks (https://github.com/biocorecrg/ExOrthist).

Molecular cloning, expression, and functional features of IGF1 splice variants in sheep.

Insulin-like growth factor 1 (IGF1), also known as somatomedin C, is essential for the regulation of animal growth and development. In many species, the IGF1 gene can be alternatively spliced into multiple transcripts, encoding different pre-pro-IGF1 proteins. However, the exact alternative splicing patterns of IGF1 and the sequence information of different splice variants in sheep are still unclear. In this study, four splice variants (class 1-Ea, class 1-Eb, class 2-Ea, and class 2-Eb) were obtained, but no IGF1 Ec, similar to that found in other species, was discovered. Bioinformatics analysis showed that the four splice variants shared the same mature peptide (70 amino acids) and possessed distinct signal peptides and E peptides. Tissue expression analysis indicated that the four splice variants were broadly expressed in all tested tissues and were most abundantly expressed in the liver. In most tissues and stages, the expression of class 1-Ea was highest, and the expression of other splice variants was low. Overall, levels of the four IGF1 splice variants at the fetal and lamb stages were higher than those at the adult stage. Overexpression of the four splice variants significantly increased fibroblast proliferation and inhibited apoptosis (P < 0.05). In contrast, silencing IGF1 Ea or IGF1 Eb with siRNA significantly inhibited proliferation and promoted apoptosis (P < 0.05). Among the four splice variants, class 1-Ea had a more evident effect on cell proliferation and apoptosis. In summary, the four ovine IGF1 splice variants have different structures and expression patterns and might have different biological functions.

Nopp140-chaperoned 2'-O-methylation of small nuclear RNAs in Cajal bodies ensures splicing fidelity.

Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB)-specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at ∼80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2'-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.

PPM1G promotes the progression of hepatocellular carcinoma via phosphorylation regulation of alternative splicing protein SRSF3

Emerging evidence has demonstrated that alternative splicing has a vital role in regulating protein function, but how alternative splicing factors can be regulated remains unclear. We showed that the PPM1G, a protein phosphatase, regulated the phosphorylation of SRSF3 in hepatocellular carcinoma (HCC) and contributed to the proliferation, invasion, and metastasis of HCC. PPM1G was highly expressed in HCC tissues compared to adjacent normal tissues, and higher levels of PPM1G were observed in adverse staged HCCs. The higher levels of PPM1G were highly correlated with poor prognosis, which was further validated in the TCGA cohort. The knockdown of PPM1G inhibited the cell growth and invasion of HCC cell lines. Further studies showed that the knockdown of PPM1G inhibited tumor growth in vivo. The mechanistic analysis showed that the PPM1G interacted with proteins related to alternative splicing, including SRSF3. Overexpression of PPM1G promoted the dephosphorylation of SRSF3 and changed the alternative splicing patterns of genes related to the cell cycle, the transcriptional regulation in HCC cells. In addition, we also demonstrated that the promoter of PPM1G was activated by multiple transcription factors and co-activators, including MYC/MAX and EP300, MED1, and ELF1. Our study highlighted the essential role of PPM1G in HCC and shed new light on unveiling the regulation of alternative splicing in malignant transformation.

Human spliceosomal snRNA sequence variants generate variant spliceosomes.

Human pre-mRNA splicing is primarily catalyzed by the major spliceosome, comprising five small nuclear ribonucleoprotein complexes, U1, U2, U4, U5, and U6 snRNPs, each of which contains the corresponding U-rich snRNA. These snRNAs are encoded by large gene families exhibiting significant sequence variation, but it remains unknown if most human snRNA genes are untranscribed pseudogenes or produce variant snRNAs with the potential to differentially influence splicing. Since gene duplication and variation are powerful mechanisms of evolutionary adaptation, we sought to address this knowledge gap by systematically profiling human U1, U2, U4, and U5 snRNA variant gene transcripts. We identified 55 transcripts that are detectably expressed in human cells, 38 of which incorporate into snRNPs and spliceosomes in 293T cells. All U1 snRNA variants are more than 1000-fold less abundant in spliceosomes than the canonical U1, whereas at least 1% of spliceosomes contain a variant of U2 or U4. In contrast, eight U5 snRNA sequence variants occupy spliceosomes at levels of 1% to 46%. Furthermore, snRNA variants display distinct expression patterns across five human cell lines and adult and fetal tissues. Different RNA degradation rates contribute to the diverse steady state levels of snRNA variants. Our findings suggest that variant spliceosomes containing noncanonical snRNAs may contribute to different tissue- and cell-type–specific alternative splicing patterns.

F5‐Atlanta: A novel mutation in F5 associated with enhanced East Texas splicing and FV‐short production

BackgroundElucidating the molecular pathogenesis underlying East Texas bleeding disorder (ET) led to the discovery of alternatively spliced F5 transcripts harboring large deletions within exon 13. These alternatively spliced transcripts produce a shortened form of coagulation factor V (FV) in which a large portion of its B‐domain is deleted. These FV isoforms bind tissue factor pathway inhibitor alpha (TFPIα) with high affinity, prolonging its circulatory half‐life and enhancing its anticoagulant effects. While two missense pathogenic variants highlighted this alternative splicing event, similar internally deleted FV proteins are found in healthy controls. ObjectiveWe identified a novel heterozygous 832 base pair deletion within F5 exon 13, termed F5‐Atlanta (F5‐ATL), in a patient with severe bleeding. Our objective is to investigate the effect of this deletion on F5 and FV expression. Methods & ResultsAssessment of patient plasma revealed markedly elevated levels of total and free TFPI and a FV isoform similar in size to the FV‐short described in ET. Sequencing analyses of cDNA revealed the presence of a transcript alternatively spliced using the ET splice sites, thereby removing the F5‐ATL deletion. This alternative splicing pattern was recapitulated by heterologous expression in mammalian cells. ConclusionsThese findings support a mechanistic model consisting of cis‐acting regulatory sequences encoded within F5 exon 13 that control alternative splicing at the ET splice sites and thereby regulate circulating FV‐short and TFPIα levels.

Loss of De Novo DNA Methyltransferase DNMT3A Impacts Alternative Splicing in Hematopoietic Stem Cells

DNMT3A is a de novo DNA methyltransferase that is crucial to the maintenance of normal hematopoiesis. DNMT3A is frequently mutated in hematopoietic diseases, with mutations reported in over 20% of adult acute myeloid leukemia (AML), and 10-15% of myelodysplastic syndrome (MDS) patients. Our lab has shown that loss of Dnmt3a in mice leads to enhanced self-renewal of the HSC compartment and a block in differentiation, contributing to its role in clonal hematopoiesis (Challen et al. 2011). We have also shown that loss of DNMT3A leads to a spectrum of hematologic malignancies; however, changes in DNA methylation alone are insufficient to explain the leukemic transformation, suggesting that DNMT3A could also regulate stem cells independent of methylation. The mechanisms through which DNMT3A exerts its specific effects are unclear, in part due to our relatively poor understanding of its interactome.To address this gap in knowledge, we generated a doxycycline-inducible biotinylated-DNMT3A transgene in mouse ES cells. Using co-immunoprecipitation and mass spectrometry analysis of biotinylated-DNMT3A in murine ESCs, we identified several candidate interacting proteins. Along with known partners such as DNMT3L and DNMTB, factors associated with chromatin regulation (HDACs, NuRD complex), splicing factors (SF proteins, Hnrnps, and DDXs), and ribosomes (RPSs and RPLs) were pulled down. We validated these interactions by over-expressing myc-tagged DNMT3A in HEK293T cells. Since epigenetic regulators and splicing factors are recurrently co-mutated in hematologic malignancies, we validated its interaction with splicing factors such as DDX39B, SF3B3, and SF3B1 (among others) through co-IP of DNMT3A in CD34+ HSPCs. Furthermore, we found that these interactions are RNA and DNA-independent. N-terminal deletions of DNMT3A domains revealed that the catalytic domain is crucial for its interaction with splicing factors, and AML mutant, Arg882His, disrupts this interaction. These data suggest that through its interaction with splicing factors, DNMT3A may have a crucial role in RNA splicing efficacy.To determine how the putative interaction between DNMT3A and splicing factors affects hematopoiesis, we examined whether loss of Dnmt3a impacted RNA splicing in purified HSCs after competitive transplant using our Mx1-cre-inducible Dnmt3a knockout mouse model. Using intron retention as a measure of splicing efficacy, we performed deep RNA sequencing of poly-A-enriched mRNA from HSCs (LSK CD150+ CD48-). When comparing exon-intron junctions in Dnmt3a KO versus WT HSCs, Dnmt3a KO HSCs exhibited a greater degree of intron retention. Aberrant alternative splicing was observed in genes involved in HSC differentiation and self-renewal, splicing, ribosomal biogenesis, and cell cycle regulation, suggesting that its role in RNA splicing may be an important mechanism through which DNMT3A influences hematopoiesis. In addition, we delineate a key relationship between DNMT3A, intron retention, and HSC differentiation. Inducing differentiation of WT and Dnmt3a KO HSCs with 5-Fluorouracil (5-FU) revealed key differences in alternative splicing. As WT HSCs differentiate, intron retention decreases; however, Dnmt3a KO HSCs are unable to decrease intron retention in key hematopoietic differentiation genes, leading to a block in differentiation. This was also demonstrated after inducing differentiation in WT and Dnmt3a KO mouse ESCs with Retinoic Acid (RA).To determine if loss of DNMT3A also impacts splicing in human HSPCs, we used CRISPR/Cas9 to knock out DNMT3A in human HSPCs and transplanted CD34+ cells into NSG mice. RNA sequencing of CD34+ cells 3 months after transplantation revealed aberrant alternative splicing, specifically, greater intron retention in DNMT3A KO than control cells. Together, these data suggest that DNMT3A plays a crucial role in maintaining proper splicing in HSCs.In summary, this study reveals a novel role for DNMT3A in hematopoiesis. We show that DNMT3A interacts with splicing factors and impacts global alternative splicing patterns in HSCs. In addition, we demonstrate that decreasing intron retention has a specific role in HSC differentiation. The aberrant splicing patterns seen in Dnmt3a KO HSCs may be instrumental in decrypting the crosstalk between DNA methylation and alternative splicing, and their role in HSC differentiation. DisclosuresNo relevant conflicts of interest to declare.

Detection of alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) in association with eCry3.1Ab resistance using RNA-seq and PacBio Iso-Seq.

Alternative splicing is a common feature in eukaryotes that not only increases the transcript diversity, but also has functional consequences. In insects, alternative splicing has been found associated with resistance to pesticides and Bt toxins. Up to date, the alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) has not been studied. To investigate its alternative splicing pattern and relation to Bt resistance, we carried out single-molecule real-time (SMRT) transcript sequencing and Iso-seq analysis on resistant, eCry3.1Ab-selected and susceptible, unselected, western corn rootworm neonate midguts which fed on seedling maize with and without eCry3.1Ab for 12 and 24 h. We present transcriptome-wide alternative splicing patterns of western corn rootworm midgut in response to feeding on eCry3.1Ab-expressing corn using a comprehensive approach that combines both RNA-seq and SMRT transcript sequencing techniques. The results showed genes in western corn rootworm are highly alternatively spliced, which happens on 67.73% of multi-exon genes. One of the alternative splicing events we identified was a novel peritrophic matrix protein with two alternative splicing isoforms. Analysis of differential exon usage between resistant and susceptible colonies showed that in eCry3.1Ab-resistant western corn rootworm, expression of one isoform was significantly higher than in the susceptible colony, while no significant differences between colonies were observed with the other isoform. Our results provide the first survey of alternative splicing in western corn rootworm and suggest that the observed alternatively spliced isoforms of peritrophic matrix protein may be associated with eCry3.1Ab resistance in western corn rootworm.

Spliceosome-regulated RSRP1-dependent NF-κB activation promotes the glioblastoma mesenchymal phenotype.

The glioblastoma (GBM) mesenchymal (MES) phenotype, induced by NF-κB activation, is characterized by aggressive tumor progression and poor clinical outcomes. Our previous analysis indicated that MES GBM has a unique alternative splicing (AS) pattern; however, the underlying mechanism remains obscure. We aimed to reveal how splicing regulation contributes to MES phenotype promotion in GBM. We screened novel candidate splicing factors that participate in NF-κB activation and MES phenotype promotion in GBM. In vitro and in vivo assays were used to explore the function of RSRP1 in MES GBM. Here, we identified that arginine/serine-rich protein 1 (RSRP1) promotes the MES phenotype by facilitating GBM cell invasion and apoptosis resistance. Proteomic, transcriptomic, and functional analyses confirmed that RSRP1 regulates AS in MES GBM through mediating spliceosome assembly. One RSRP1-regulated AS event resulted in skipping PARP6 exon 18 to form truncated, oncogenic PARP6-s. This isoform was unable to effectively suppress NF-κB. Cotreatment of cultured GBM cells and GBM tumor-bearing mice with spliceosome and NF-κB inhibitors exerted a synergistic effect on MES GBM growth. We identified a novel mechanism through which RSRP1-dependent splicing promotes the GBM MES phenotype. Targeting AS via RSRP1-related spliceosomal factors might constitute a promising treatment for GBM.

IsoSplitter: identification and characterization of alternative splicing sites without a reference genome.

Long-read transcriptome sequencing is designed to sequence full-length RNA molecules and advantageous for identifying alternative splice isoforms; however, in the absence of a reference genome, it is difficult to accurately locate splice sites because of the diversity of patterns of alternative splicing (AS). Based on long-read transcriptome data, we developed a versatile tool, IsoSplitter, to reverse-trace and validate AS gene “split sites” with the following features: (i) IsoSplitter initially invokes a modified SIM4 program to find transcript split sites; (ii) each split site is then quantified, to reveal transcript diversity, and putative isoforms are grouped into gene clusters; (iii) an optional step for aligning short reads is provided, to validate split sites by identifying unique junction reads, and revealing and quantifying tissue-specific alternative splice isoforms. We tested IsoSplitter AS prediction using data sets from multiple model and nonmodel plant species and showed that the IsoSplitter pipeline is efficient to handle different transcriptomes with high accuracy. Furthermore, we evaluated the IsoSplitter pipeline compared with that of the splice junction identification tools, Program to Assemble Spliced Alignments (PASA software needs a reference genome for AS identification) and AStrap, using data from the model plant Arabidopsis thaliana. We found that IsoSplitter determined more than twice as many AS events than AStrap analysis, and 94.13% of the IsoSplitter predicted AS events were also identified by the PASA analysis. Starting from a simple sequence file, IsoSplitter is an assembly-free tool for identification and characterization of AS. IsoSplitter is developed and implemented in Python 3.5 using the Linux platform and is freely available at https://github.com/Hengfu-Yin/IsoSplitter.

New insights into molecular changes in skeletal muscle aging and disease: Differential alternative splicing and senescence

Progressive loss of muscle mass and function due to muscle fiber atrophy and loss in the elderly and chronically ill is now defined as sarcopenia. It is a major contributor to loss of independence, disability, need of long-term care as well as overall mortality. Sarcopenia is a heterogenous disease and underlying mechanisms are not completely understood. Here, we newly identified and used Tmem158, alongside Cdkn1a, as relevant senescence and denervation markers (SDMs), associated with muscle fiber atrophy. Subsequent application of laser capture microdissection (LCM) and RNA analyses revealed age- and disease-associated differences in gene expression and alternative splicing patterns in a rodent sarcopenia model. Of note, genes exhibiting such differential alternative splicing (DAS) are mainly involved in the contractile function of the muscle. Many of these splicing events are also found in a mouse model for myotonic dystrophy type 1 (DM1), underscoring the premature aging phenotype of this disease. We propose to add differential alternative splicing to the hallmarks of aging.

Rbfox2 mediates exon 11 inclusion in insulin receptor pre-mRNA splicing in hepatoma cells

Insulin receptor (IR) pre-mRNA undergoes alternative splicing that produces two isoforms, IR-A and IR-B. The ratio of IR-A to IR-B varies among tissues, which strongly suggests that IR mRNA alternative splicing is regulated in a tissue-specific manner. However, the precise molecular mechanism for IR alternative splicing remains to be elucidated, especially in liver. In this study, we have analyzed IR alternative splicing mechanism by preparing a mini-gene splicing reporter with rat genomic DNA. The splicing reporter that contains exon 11 and its flanking intronic sequences could reproduce alternative splicing pattern in rat hepatoma H4IIE cells. Introducing several deletions in introns of the reporter revealed that intron 11 contains the region near exon 11 essential to promote exon 11 inclusion. This region contains an UGCAUG sequence, a specific binding site for the Rbfox splicing regulator, and mutation in this sequence results in exon 11 skipping. Furthermore, RbFox2 knockdown in H4IIE cells enhanced exon 11 skipping of endogenous IR pre-mRNA. Lastly mutations in the SRSF3 binding site of exon11 together with the Rbfox2 binding site completely abolished exon 11 inclusion with a mini-gene reporter pre-mRNA. Our results indicate that RbFox2 and SRSF3 proteins mediate exon 11 inclusion in rat hepatoma cells.

Splicing regulation in hematopoiesis.

Splicing mutations are among the most recurrent genetic perturbations in hematological malignancies, highlighting an important impact of splicing regulation in hematopoietic development. However, compared to our understanding of splicing factor mutations in hematological malignancies, studies of splicing components and alternative splicing in normal hematopoiesis have been less well investigated. Here, we outline the most recent findings on splicing regulation in normal hematopoiesis and discuss the important questions in the field. Recent studies have highlighted the critical role of splicing regulation in hematopoiesis, including characterization of splicing components in normal hematopoiesis, investigation of transcriptional alterations on splicing, and identification of stage-specific alternative splicing events during hematopoietic development. These interesting findings provide insights on hematopoietic regulation at a co-transcriptional level. More high-throughput RNA ribonucleic acid (RNA) sequencing and functional genomic screens are needed to advance our knowledge of critical alternative splicing patterns in shaping hematopoiesis.

The Vesicle‐Mediated Transport Genes, Snap23 , Tmed2 , and Trip10 , are Alternatively Spliced During Striated Muscle Development

The reprogramming of alternative splicing patterns during development is a hallmark of tissue maturation and identity. Heart and skeletal (striated) muscle tissues exhibit the most highly conserved splicing programs, which substantially change during postnatal development. Notably, alterations of these splicing events are associated with severe cardiac and muscular diseases. In striated muscle, a collection of genes encoding membrane trafficking proteins are spliced in a developmental stage-specific manner. However, few splice isoforms have been characterized, and little is known about their functional roles during heart and skeletal muscle development. We hypothesize that coordinated splicing of membrane trafficking genes represents a splicing network, which functions to regulate the transport of cargo that is critical for muscle development and homeostasis. Here, we characterize three proteins involved in vesicle-mediated transport that are alternatively spliced in striated muscle: SNAP23, TMED2, and TRIP10. The synaptosome-associated protein 23 (SNAP23) is a SNARE protein that mediates vesicle fusion with the plasma membrane during exocytosis, the transmembrane emp23 domain-containing protein 2 (TMED2) regulates vesicle budding during secretion, and the thyroid hormone receptor interactor 10 (TRIP10) is involved in endocytosis and membrane tubulation. First, we found that during striated muscle development, the Snap23, Tmed2, and Trip10 pre-mRNAs undergo splicing changes of a single exon. Second, these three splicing transitions are characterized by a shift from short to long isoform expression that are regulated by the RBPs quaking (QKI) and polypyrimidine tract binding protein 1 (PTBP1). Third, depletion of SNAP23 in muscle cells results in reduced cell viability and differentiation, which can be rescued with conditioned media from differentiated cells or by seeding cells on collagen-coated plates. Finally, exclusive expression of the short SNAP23 isoform leads to increased fusion of myoblasts into myotubes that exhibit greater myotube area and a higher nuclei-per-myotube ratio. Taken together, our data suggest that splicing of Snap23 is an important regulator of myogenesis, where SNAP23 isoform expression controls secretion of cargo that is necessary for muscle cell fusion and myotube size. Preliminary studies show that disruption of TMED2 and TRIP10 isoform levels in muscle cells likewise affects differentiation. Elucidating the mechanisms of regulation and the functional consequences of splicing networks will be fundamental to our understanding of muscle development and disease.

Combined Transcriptome Analysis Reveals the Ovule Abortion Regulatory Mechanisms in the Female Sterile Line of Pinus tabuliformis Carr.

Ovule abortion is a common phenomenon in plants that has an impact on seed production. Previous studies of ovule and female gametophyte (FG) development have mainly focused on angiosperms, especially in Arabidopsis thaliana. However, because it is difficult to acquire information about ovule development in gymnosperms, this remains unclear. Here, we investigated the transcriptomic data of natural ovule abortion mutants (female sterile line, STE) and the wild type (female fertile line, FER) of Pinus tabuliformis Carr. to evaluate the mechanism of ovule abortion during the process of free nuclear mitosis (FNM). Using single-molecule real-time (SMRT) sequencing and next-generation sequencing (NGS), 18 cDNA libraries via Illumina and two normalized libraries via PacBio, with a total of almost 400,000 reads, were obtained. Our analysis showed that the numbers of isoforms and alternative splicing (AS) patterns were significantly variable between FER and STE. The functional annotation results demonstrate that genes involved in the auxin response, energy metabolism, signal transduction, cell division, and stress response were differentially expressed in different lines. In particular, AUX/IAA, ARF2, SUS, and CYCB had significantly lower expression in STE, showing that auxin might be insufficient in STE, thus hindering nuclear division and influencing metabolism. Apoptosis in STE might also have affected the expression levels of these genes. To confirm the transcriptomic analysis results, nine pairs were confirmed by quantitative real-time PCR. Taken together, these results provide new insights into ovule abortion in gymnosperms and further reveal the regulatory mechanisms of ovule development.

Genetic Drivers of Head and Neck Squamous Cell Carcinoma: Aberrant Splicing Events, Mutational Burden, HPV Infection and Future Targets.

Head and neck cancers include cancers that originate from a variety of locations. These include the mouth, nasal cavity, throat, sinuses, and salivary glands. These cancers are the sixth most diagnosed cancers worldwide. Due to the tissues they arise from, they are collectively named head and neck squamous cell carcinomas (HNSCC). The most important risk factors for head and neck cancers are infection with human papillomavirus (HPV), tobacco use and alcohol consumption. The genetic basis behind the development and progression of HNSCC includes aberrant non-coding RNA levels. However, one of the most important differences between healthy tissue and HNSCC tissue is changes in the alternative splicing of genes that play a vital role in processes that can be described as the hallmarks of cancer. These changes in the expression profile of alternately spliced mRNA give rise to various protein isoforms. These protein isoforms, alternate methylation of proteins, and changes in the transcription of non-coding RNAs (ncRNA) can be used as diagnostic or prognostic markers and as targets for the development of new therapeutic agents. This review aims to describe changes in alternative splicing and ncRNA patterns that contribute to the development and progression of HNSCC. It will also review the use of the changes in gene expression as biomarkers or as the basis for the development of new therapies.

Dissecting the impact of regional identity and the oncogenic role of human-specific NOTCH2NL in an hESC model of H3.3G34R-mutant glioma

H3.3G34R-mutant gliomas are lethal tumors of the cerebral hemispheres with unknown mechanisms of regional specificity and tumorigenicity. We developed a human embryonic stem cell (hESC)-based model of H3.3G34R-mutant glioma that recapitulates the key features of the tumors with cell-type specificity to forebrain interneuronal progenitors but not hindbrain precursors. We show that H3.3G34R, ATRX, and TP53 mutations cooperatively impact alternative RNA splicing events, particularly suppression of intron retention. This leads to increased expression of components of the Notch pathway, notably NOTCH2NL, a human-specific gene family. We also uncover a parallel mechanism of enhanced NOTCH2NL expression via genomic amplification of its locus in some H3.3G34R-mutant tumors. These findings demonstrate a novel mechanism whereby evolutionary pathways that lead to larger brain size in humans are co-opted to drive tumor growth.

Altered Expression of MBNL Family of Alternative Splicing Factors in Colorectal Cancer.

Colorectal cancer is currently the third leading cause of cancer-related deaths and recently, alternative splicing has risen as its important regulator and potential treatment target. In the present study, we analyzed gene expression of the MBNL family of regulators of alternative splicing in various stages of colorectal cancer development, together with the MBNL-target splicing events in FOXP1 and EPB41L3 genes and tumor-related CD44 variants. Samples of tumor tissue and non-malignant mucosa from 108 patients were collected. After RNA isolation and reverse transcription, the relative gene expression of a selected gene panel was tested by quantitative real-time PCR, followed by statistical analysis. MBNL expression was decreased in tumor tissue compared to non-tumor mucosa. In addition, lower expression was observed for the variants of FOXP1 and EPB41L3, while higher expression in tumor tissue was detected both for total CD44 and its cancer-related variants 3 and 6. Transcript levels of the MBNL genes were not found to be related to any of the studied clinicopathological characteristics. Multiple significant associations were identified in the target gene panel, including higher transcript levels of FOXP1 and CD44v3 in patients with distant metastases and connections between recurrence-free survival and altered levels of FOXP1 and CD44v3. Our results identified for the first-time deregulation of MBNL genes in colorectal cancer. Down-regulation of their transcripts in tumor tissue compared to matched non-tumor mucosa can lead to transition of alternative splicing patterns towards a less differentiated phenotype, which highlights the importance of alternative splicing regulation for tumor growth and propagation.