Mis-spliced Transcripts Research Articles

Steering research on mRNA splicing in cancer towards clinical translation.

Splicing factors are affected by recurrent somatic mutations and copy number variations in several types of haematologic and solid malignancies, which is often seen as prima facie evidence that splicing aberrations can drive cancer initiation and progression. However, numerous spliceosome components also 'moonlight' in DNA repair and other cellular processes, making their precise role in cancer difficult to pinpoint. Still, few would deny that dysregulated mRNA splicing is a pervasive feature of most cancers. Correctly interpreting these molecular fingerprints can reveal novel tumour vulnerabilities and untapped therapeutic opportunities. Yet multiple technological challenges, lingering misconceptions, and outstanding questions hinder clinical translation. To start with, the general landscape of splicing aberrations in cancer is not well defined, due to limitations of short-read RNA sequencing not adept at resolving complete mRNA isoforms, as well as the shallow read depth inherent in long-read RNA-sequencing, especially at single-cell level. Although individual cancer-associated isoforms are known to contribute to cancer progression, widespread splicing alterations could be an equally important and, perhaps, more readily actionable feature of human cancers. This is to say that in addition to 'repairing' mis-spliced transcripts, possible therapeutic avenues include exacerbating splicing aberration with small-molecule spliceosome inhibitors, targeting recurrent splicing aberrations withsynthetic lethal approaches, and training the immune system to recognize splicing-derived neoantigens.

Genome sequencing enables diagnosis and treatment of SLC5A6 neuropathy.

The sodium-dependent multivitamin transporter encoded by SLC5A6 is responsible for uptake of biotin, pantothenic acid, and α-lipoic acid. Thirteen individuals from eight families are reported with pathogenic biallelic SLC5A6 variants. Phenotype ranges from multisystem metabolic disorder to childhood-onset peripheral motor neuropathy. We report three additional affected individuals with biallelic SLC5A6 variants. In Family A, a male proband (AII:1) presenting in early childhood with gross motor regression, motor axonal neuropathy, recurrent cytopenia and infections, and failure to thrive was diagnosed at 12 years of age via genome sequencing (GS) with a paternal NM_021095.4:c.393+2T>C variant and a maternal c.1285A>G p.(Ser429Gly) variant. An uncle with recurrent cytopenia and peripheral neuropathy was subsequently found to have the same genotype. We also report an unrelated female with peripheral neuropathy homozygous for the c.1285A>G p.(Ser429Gly) recurrent variant identified in seven reported cases, including this study. RT-PCR studies on blood mRNA from AII:1 showed c.393+2T>C caused mis-splicing with all canonically spliced transcripts in AII:1 containing the c.1285A>G variant. SLC5A6 mRNA expression in AII:1 fibroblasts was ~50% of control levels, indicative of nonsense-mediated decay of mis-spliced transcripts. Biotin uptake studies on AII:1 fibroblasts, expressing the p.(Ser429Gly) variant, showed an ~90% reduction in uptake compared to controls. Targeted treatment of AII:1 with biotin, pantothenic acid, and lipoic acid resulted in clinical improvement. Health Economic analyses showed implementation of GS as an early investigation could have saved $ AUD 105,988 and shortened diagnostic odyssey and initiation of treatment by up to 7 years.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43-depleted human iPSC-derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43-depleted human iPSC-derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

Unleashing the potential of catalytic RNAs to combat mis-spliced transcripts.

Human transcriptome can undergo RNA mis-splicing due to spliceopathies contributing to the increasing number of genetic diseases including muscular dystrophy (MD), Alzheimer disease (AD), Huntington disease (HD), myelodysplastic syndromes (MDS). Intron retention (IR) is a major inducer of spliceopathies where two or more introns remain in the final mature mRNA and account for many intronic expansion diseases. Potential removal of such introns for therapeutic purposes can be feasible when utilizing bioinformatics, catalytic RNAs, and nano-drug delivery systems. Overcoming delivery challenges of catalytic RNAs was discussed in this review as a future perspective highlighting the significance of utilizing synthetic biology in addition to high throughput deep sequencing and computational approaches for the treatment of mis-spliced transcripts.

Patient-specific MDS-RS iPSCs define the mis-spliced transcript repertoire and chromatin landscape of SF3B1-mutant HSPCs

AbstractSF3B1K700E is the most frequent mutation in myelodysplastic syndrome (MDS), but the mechanisms by which it drives MDS pathogenesis remain unclear. We derived a panel of 18 genetically matched SF3B1K700E- and SF3B1WT-induced pluripotent stem cell (iPSC) lines from patients with MDS with ring sideroblasts (MDS-RS) harboring isolated SF3B1K700E mutations and performed RNA and ATAC sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from them. We developed a novel computational framework integrating splicing with transcript usage and gene expression analyses and derived a SF3B1K700E splicing signature consisting of 59 splicing events linked to 34 genes, which associates with the SF3B1 mutational status of primary MDS patient cells. The chromatin landscape of SF3B1K700E HSPCs showed increased priming toward the megakaryocyte- erythroid lineage. Transcription factor motifs enriched in chromatin regions more accessible in SF3B1K700E cells included, unexpectedly, motifs of the TEA domain (TEAD) transcription factor family. TEAD expression and transcriptional activity were upregulated in SF3B1-mutant iPSC-HSPCs, in support of a Hippo pathway-independent role of TEAD as a potential novel transcriptional regulator of SF3B1K700E cells. This study provides a comprehensive characterization of the transcriptional and chromatin landscape of SF3B1K700E HSPCs and nominates novel mis-spliced genes and transcriptional programs with putative roles in MDS-RS disease biology.

Isogenic MDS-RS Patient-Derived iPSCs Define the Mis-Spliced Transcript Repertoire and Chromatin Landscape of SF3B1-Mutant Hematopoietic Stem/Progenitor Cells

▪The somatic hotspot mutation SF3B1K700E is characteristically found in myelodysplastic syndrome with ring sideroblasts (MDS-RS) and frequently occurs as an isolated mutation. However, our understanding of how this mutation drives MDS pathogenesis remains limited. To explore the downstream consequences of the SF3B1K700E mutation and its role in disease pathogenesis, we generated a panel of isogenic SF3B1K700E and SF3B1WT induced pluripotent stem cell (iPSC) lines from 3 MDS-RS patients with isolated SF3B1K700E mutation (3 SF3B1K700E and 3 SF3B1WT lines per patient, total 18). Upon hematopoietic differentiation, SF3B1K700E cells exhibited lower growth and colony-forming ability, compared to SF3B1WT cells, recapitulating hallmark phenotypes of MDS cells.To investigate the effects of the SF3B1K700E mutation on the transcriptome and chromatin landscape, we performed RNA- and ATAC- sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from the panel of the 18 isogenic SF3B1K700E and SF3B1WT iPSC lines. Principal component analysis (PCA) and hierarchical clustering based on gene expression grouped the iPSC lines primarily by genotype (SF3B1K700E vs SF3B1WT) and secondarily by genetic background. To assess the impact of the SF3B1K700E mutation at the exon, transcript and gene level, we developed an analytical framework integrating differential splicing with differential transcript usage and differential gene expression analyses. We thus discovered 59 splicing events linked to 34 genes (most statistically significant events that also mapped to differentially used transcripts and differentially expressed genes). This SF3B1K700E splicing signature includes genes previously reported as mis-spliced in SF3B1K700E cells (e.g BRD9, ABCB7), as well as novel genes. We tested this signature against a published dataset of primary MDS patient samples (Pellagatti et al.). PCA based on the inclusion level of the splicing events of our signature separated SF3B1-mutated MDS patients from patients without splicing factor mutations (SF-WT) or healthy individuals. Furthermore, it identified one patient erroneously annotated as SF-WT that clustered together with the SF3B1-mutated patients, who had a, previously overlooked, 6bp in-frame deletion spanning the K700E hotspot.By comparing the chromatin accessibility profiles of SF3B1K700E and SF3B1WT iPSC-HSPCs to those defined in primary human cell types along the hematopoietic hierarchy (Corces et al.), we found that the chromatin landscape of SF3B1K700E HSPCs resembled more this of megakaryocyte-erythroid progenitor cells (MEPs) and erythroid cells, whereas that of SF3B1WT HSPCs resembled more granulocyte-monocyte progenitors (GMPs) and monocytes. This finding may underlie the more prominent involvement of the erythroid lineage in the pathology and clinical presentation of MDS-RS. To interrogate transcriptional programs in SF3B1K700E mutant cells, we performed transcription factor (TF) motif enrichment analysis. Motifs enriched in ATAC-Seq peaks more accessible in SF3B1K700E cells that were linked to genes upregulated in SF3B1K700E cells, included motifs of several TFs with known roles in hematopoiesis (GATA, ETS, STAT, AP-1). Unexpectedly, motifs of the TEAD TFs were also enriched. The TEAD family of TFs are best known as effectors of the Hippo signaling pathway, with important roles in various biological processes and malignancies, albeit no clear links to adult hematopoiesis or hematologic disease. TEAD2 and TEAD4 were upregulated in SF3B1-mutant, compared to the WT, iPSC-HSPCs and TEAD transcriptional activity, measured with a luciferase reporter construct, was higher in SF3B1K700E, compared to SF3B1WT iPSC-HSPCs. We did not find expression or activation of YAP or TAZ, which bind to DNA as a complex with TEAD upon Hippo pathway activation. These results support a Hippo-independent increase of TEAD expression and activity in SF3B1K700E cells.In summary, we generated a panel of isogenic patient-derived iPSCs that allowed us to comprehensively characterize the transcriptome and chromatin landscape of SF3B1K700E HSPCs in an isogenic system, derive a splicing signature of SF3B1K700E and identify the TEAD TF as a new transcriptional regulator of SF3B1K700E mutant HSPCs.G. Asimomitis, A.G. Deslauriers: shared 1 st authorshipE. Papaemmanuil, E.P. Papapetrou: shared senior authorship DisclosuresDeslauriers: Novo Nordisk A/S: Current Employment. Hellström-Lindberg: Celgene: Research Funding. Papaemmanuil: Isabl Technologies: Divested equity in a private or publicly-traded company in the past 24 months; Kyowa Hakko Kirin Pharma: Consultancy.

Pathogenic Intronic Splice-Affecting Variants in MYBPC3 in Three Patients with Hypertrophic Cardiomyopathy

Genetic variants in MYBPC3 are one of the most common causes of hypertrophic cardiomyopathy (HCM). While variants in MYBPC3 affecting canonical splice site dinucleotides are a well-characterised cause of HCM, only recently has work begun to investigate the pathogenicity of more deeply intronic variants. Here, we present three patients with HCM and intronic splice-affecting MYBPC3 variants and analyse the impact of variants on splicing using in vitro minigene assays. We show that the three variants, a novel c.927-8G>A variant and the previously reported c.1624+4A>T and c.3815-10T>G variants, result in MYBPC3 splicing errors. Analysis of blood-derived patient RNA for the c.3815-10T>G variant revealed only wild type spliced product, indicating that mis-spliced transcripts from the mutant allele are degraded. These data indicate that the c.927-8G>A variant of uncertain significance and likely benign c.3815-10T>G should be reclassified as likely pathogenic. Furthermore, we find shortcomings in commonly applied bioinformatics strategies to prioritise variants impacting MYBPC3 splicing and re-emphasise the need for functional assessment of variants of uncertain significance in diagnostic testing.

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness.

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and backsplicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM-dependent linear-splicing events using splice-switching-antisense-oligonucleotides was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.

β-Amyrin Synthase1 Controls the Accumulation of the Major Saponins Present in Pea (Pisum sativum).

The use of pulses as ingredients for the production of food products rich in plant proteins is increasing. However, protein fractions prepared from pea or other pulses contain significant amounts of saponins, glycosylated triterpenes that can impart an undesirable bitter taste when used as an ingredient in foodstuffs. In this article, we describe the identification and characterization of a gene involved in saponin biosynthesis during pea seed development, by screening mutants obtained from two Pisum sativum TILLING (Targeting Induced Local Lesions INGenomes) populations in two different genetic backgrounds. The mutations studied are located in a gene designated PsBAS1 (β-amyrin synthase1), which is highly expressed in maturing pea seeds and which encodes a protein previously shown to correspond to an active β-amyrin synthase. The first allele is a nonsense mutation, while the second mutation is located in a splice site and gives rise to a mis-spliced transcript encoding a truncated, nonfunctional protein. The homozygous mutant seeds accumulated virtually nosaponin without affecting the seed nutritional or physiological quality. Interestingly, BAS1 appears to control saponin accumulation in all other tissues of the plant examined. These lines represent a first step in the development of pea varieties lacking bitterness off-flavors in their seeds. Our work also shows that TILLING populations in different genetic backgrounds represent valuable genetic resources for both crop improvement and functional genomics.

A cryptic splice-altering KCNQ1 variant in trans with R259L leading to Jervell and Lange-Nielsen syndrome

Here we report an infant with clinical findings suggestive of Jervell and Lange-Nielsen syndrome (JLNS), including a prolonged QT interval (LQTS) and chronic bilateral sensorineural deafness. NGS analysis revealed one known heterozygous pathogenic missense variant, KCNQ1 p.R259L, previously associated with LQTS but insufficient to explain the cardioauditory disorder. In a screening of proximal intronic regions, we found a heterozygous variant, KCNQ1 c.1686−9 T > C, absent from controls and previously undescribed. Several splicing prediction tools returned low scores for this intronic variant. Driven by the proband’s phenotype rather than the neutral predictions, we have characterized this rare intronic variant. Family analysis has shown that the proband inherited the missense and the intronic variants from his mother and father, respectively. A minigene splicing assay revealed that the intronic variant induced an additional transcript, arising from skipping of exon 14, which was translated into a truncated protein in transfected cells. The splice-out of exon 14 creates a frameshift in exon 15 and a stop codon in exon 16, which is the last exon of KCNQ1. This mis-spliced transcript is expected to escape nonsense-mediated decay and predicted to encode a truncated loss-of-function protein, KCNQ1 p.L563Kfs*73. The analysis of endogenous KCNQ1 expression in the blood of the proband’s parents detected the aberrant transcript only in the patient’s father. Taken together, these analyses confirmed the proband’s diagnosis of JLNS1 and indicated that c.1686−9 T > C is a cryptic splice-altering variant, expanding the known genetic spectrum of biallelic KCNQ1 variant combinations leading to JLNS1.

Complex landscape of alternative splicing in myeloid neoplasms.

Myeloid neoplasms are characterized by frequent mutations in at least seven components of the spliceosome that have distinct roles in the process of pre-mRNA splicing. Hotspot mutations in SF3B1, SRSF2, U2AF1 and loss of function mutations in ZRSR2 have revealed widely different aberrant splicing signatures with little overlap. However, previous studies lacked the power necessary to identify commonly mis-spliced transcripts in heterogeneous patient cohorts. By performing RNA-Seq on bone marrow samples from 1,258 myeloid neoplasm patients and 63 healthy bone marrow donors, we identified transcripts frequently mis-spliced by mutated splicing factors (SF), rare SF mutations with common alternative splicing (AS) signatures, and SF-dependent neojunctions. We characterized 17,300 dysregulated AS events using a pipeline designed to predict the impact of mis-splicing on protein function. Meta-splicing analysis revealed a pattern of reduced levels of retained introns among disease samples that was exacerbated in patients with splicing factor mutations. These introns share characteristics with “detained introns,” a class of introns that have been shown to promote differentiation by detaining pro-proliferative transcripts in the nucleus. In this study, we have functionally characterized 17,300 targets of mis-splicing by the SF mutations, identifying a common pathway by which AS may promote maintenance of a proliferative state.

A non-invasive diagnostic assay for rapid detection and characterization of aberrant mRNA-splicing by nonsense mediated decay inhibition

BackgroundInterpretation of genetic variants detected by sequencing of genomic DNA, which may cause splicing defects, regularly requires mRNA analysis. Usually, only bioinformatic testing is provided, because simple and non-invasive assay protocols are lacking. Furthermore, the detection of mis-splicing is often hampered by nonsense mediated mRNA decay (NMD). MethodsStarting from a case of Pompe disease with two potential splicing variants an assay for the analysis of splice defects in general was developed. We analyzed the transcripts from the gene of interest by standard methods after short-term culture of the patient's lymphocytes in the presence and absence of a NMD inhibitor. Variant and wild type transcript expression were quantified by allele specific PCR in the patient and both parents and the expression ratio with/without NMD inhibition was calculated for each transcript. ResultsNMD detection in lymphocytes was optimized and evaluated by analyzing a naturally occurring NMD transcript. Several compounds inhibited NMD successfully, including potential therapeutic agents. Sample storage for up to 4 days at room temperature prior to lymphocyte isolation did not affect results. In a proof of concept we identified two candidate variants as severe splicing variants in a patient with Pompe disease, but the strategy can also be used to screen for any mis-spliced transcripts prone to NMD. ConclusionsWe developed a simple, non-invasive assay for the detection and characterization of potential splicing variants. This is essential, because early and near-term diagnosis and disease classification is required to facilitate therapy in many genetic diseases.

Identification of Recurrent Alternative RNA Splicing in Adverse-Risk Acute Myeloid Leukemia

RNA splicing is a fundamental biological process that generates protein diversity from a finite set of genes. Recurrent somatic mutations of genes involved in RNA splicing are present at high frequency in Myelodysplasia (up to 70%) but less so in Acute Myeloid Leukemia (AML; less than 20%). To investigate whether there were aberrant and recurrent RNA splicing events in the AML transcriptome that were associated with poor prognosis in the absence of splicing factor mutations, we developed a bioinformatics pipeline to systematically annotate and quantify alternative splicing events from RNA-sequencing data (Fig A). We first analysed publicly available RNA-seq data from The Cancer Genome Atlas (TCGA, n=170). We focussed on non-M3 AML patients with no splicing factor mutations (based on reported genomic sequencing and verified by re-analysis of RNA-seq data from all patients) who had received intensive chemotherapy. We segregated these patients based on their European Leukaemia Net (ELN) risk classification and identified 1290 alternatively spliced events impacting 910 genes that were significantly different (FDR<0.05) between all ELNAdv (n=41) versus all ELNFav patients (n=21, Fig B). The majority were exon skipping events (716 events, 62%, Fig B-C), followed by intron retention (201 events, 15.6%, Fig B). We next used RNA-seq data from a second non-M3 AML patient cohort (ClinSeq- Sweden; ELNAdv, n=75 and ELNFav, n=47), detecting 2507 events mapping to 1566 genes. Comparing across the two cohorts, 222 shared genes were detected to be affected by alternative splicing (Fig D). Ingenuity pathway analysis associated these genes with pathways related to protein translation. In order to prioritise those alternatively spliced events most likely to have a deleterious function, we developed an analytical framework to predict their impact on protein structure (Fig E). 87 alternatively spliced events, 25.81% of the commonly shared splicing events, relating to 78 genes (35.13% of all genes) were predicted to directly alter highly conserved protein domains within the affected genes, leading to either a complete (~25%, Fig E) or a partial loss of a domain (20%, Fig E). These in silico predictions are likely to be an underestimate of the true impact, as splicing alterations mapping to poorly annotated domains or affecting the tertiary structure of proteins would be missed. A number of splicing factors themselves were differentially spliced, with the alternative splicing predicted to have functional consequences. This was exemplified by hnRNPA1, a factor with well-established roles in splicing, is itself alternatively spliced in patients and predicted to be deleterious. Consistent with this, motif scanning analyses indicated that a number of mis-spliced transcripts had hnRNPA1 binding motifs (Fig F). To assess the impact of these alternatively spliced events (that were predicted to also disrupt highly conserved protein domains) on the transcriptome, we simultaneously quantified differential gene expression. IPA analysis of the 602 genes that were differentially expressed between ELNAdv and ELNFav patients and shared between both TCGA and ClinSeq cohorts indicated that they were associated with pathways (Fig G) that were distinct from those associated with aberrantly spliced genes (Fig D). A number of pathways related to inflammation were enriched amongst the genes observed to be upregulated in ELNAdv patients (Fig G). Network analyses integrating the alternatively spliced genes with differentially expressed genes revealed strong interactions (Fig H), indicating functional associations between these biological events. Given these strong network interactions, we investigated the potential prognostic significance of these alternatively spliced events. To this end, we utilised machine-learning methods to derive a "splicing signature" of four mis-spliced genes with a predictive capacity equivalent to the ELN (Fig I). The splicing signature further refined existing risk prediction algorithms to improve the classification of patients (Fig J). Taken together, we report the presence of extensive deregulation of RNA splicing in AML patients even in the absence of splicing factor mutations. Many of these events were shared in patients with adverse outcomes and their impact on the AML transcriptome points towards vulnerabilities that could be targeted. Figure Disclosures Unnikrishnan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lehmann:TEVA: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Abbive: Membership on an entity's Board of Directors or advisory committees. Pimanda:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Transposon insertion causes cadherin mis-splicing and confers resistance to Bt cotton in pink bollworm from China

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) are cultivated extensively, but rapid evolution of resistance by pests reduces their efficacy. We report a 3,370-bp insertion in a cadherin gene associated with resistance to Bt toxin Cry1Ac in the pink bollworm (Pectinophora gossypiella), a devastating global cotton pest. We found the allele (r15) harboring this insertion in a field population from China. The insertion is a miniature inverted repeat transposable element (MITE) that contains two additional transposons and produces two mis-spliced transcript variants (r15A and r15B). A strain homozygous for r15 had 290-fold resistance to Cry1Ac, little or no cross-resistance to Cry2Ab, and completed its life cycle on Bt cotton producing Cry1Ac. Inheritance of resistance was recessive and tightly linked with r15. For transformed insect cells, susceptibility to Cry1Ac was greater for cells producing the wild-type cadherin than for cells producing the r15 mutant proteins. Recombinant cadherin protein occurred on the cell surface in cells transformed with the wild-type or r15A sequences, but not in cells transformed with the r15B sequence. The similar resistance of pink bollworm to Cry1Ac in laboratory- and field-selected insects from China, India and the U.S. provides a basis for developing international resistance management practices.

Pink Bollworm Resistance to Bt Toxin Cry1Ac Associated with an Insertion in Cadherin Exon 20.

Insecticidal proteins from Bacillus thuringiensis (Bt) are widely used to control insect pests, but their efficacy is reduced when pests evolve resistance. We report on a novel allele (r16) of the cadherin gene (PgCad1) in pink bollworm (Pectinophora gossypiella) associated with resistance to Bt toxin Cry1Ac, which is produced by transgenic cotton. The r16 allele isolated from a field population in China has 1545 base pairs of a degenerate transposon inserted in exon 20 of PgCad1, which generates a mis-spliced transcript containing a premature stop codon. A strain homozygous for r16 had 300-fold resistance to Cry1Ac, 2.6-fold cross-resistance to Cry2Ab, and completed its life cycle on transgenic Bt cotton producing Cry1Ac. Inheritance of Cry1Ac resistance was recessive and tightly linked with r16. Compared with transfected insect cells expressing wild-type PgCad1, cells expressing r16 were less susceptible to Cry1Ac. Recombinant cadherin protein was transported to the cell membrane in cells transfected with the wild-type PgCad1 allele, but not in cells transfected with r16. Cadherin occurred on brush border membrane vesicles (BBMVs) in the midgut of susceptible larvae, but not resistant larvae. These results imply that the r16 allele mediates Cry1Ac resistance in pink bollworm by interfering with the localization of cadherin.

LUC7L2 Is a Novel RNA-Splicing Regulatory Factor Mutated in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are unique among cancers because of the frequent occurrence of somatic mutations impacting spliceosome machinery. At least 65% of MDS patients harbor a mutation in one of several splicing factors including U2AF1, SF3B1 and SRSF2. Whole exome sequencing of MDS bone marrow uncovered somatic frameshift mutations in LUC7L2, the mammalian ortholog of a yeast splicing factor. LUC7L2 is located in the most commonly deleted region of chromosome 7. Deletions and frameshifts lead to haploinsufficient expression and therefore it can be approximated that a combined 14% of MDS patients have low expression of LUC7L2. Restoring expression of LUC7L2 in del(7q)-iPSCs partially rescues the differentiation of iPSCs into CD45+ myeloid progenitors. Although perhaps partly due to associated losses of other genes on chromosome 7, low expression of LUC7L2 correlates with a poorer patient prognosis, so its haploinsufficiency may play an important role in bone marrow failure. While U2AF1, SF3B1, and SRSF2 are well-characterized splicing factors, the function of LUC7L2 in pre-mRNA splicing is unexamined and its role in the MDS pathogenesis is undefined. We hypothesize that low expression of LUC7L2 results in the aberrant splicing of oncogenes and tumor suppressor gene transcripts thus reducing expression or altering function and contributing to the pathogenesis of MDS.We have characterized LUC7L2 as an alternative splicing regulatory protein that plays a repressive role in the regulation of alternative RNA splicing. We generated HEK-293 cells overexpressing V5-tagged LUC7L2 for immunoprecipitation-mass spectrometry, to ascertain protein interactions with LUC7L2. LUC7L2 co-immunoprecipitated with splicing regulators which are involved in splice site recognition. We performed cross-linking-IP-high-throughput-sequencing (CLIP-seq) to identify LUC7L2 binding sites on RNA. We identified 301 LUC7L2 RNA-binding sites as well as binding sites on U1 and U2 which is common for splicing regulatory proteins. Metagene analysis of these binding sites showed that LUC7L2 bound near splice sites in exonic sequences.We knocked down LUC7L2 expression in HEK293 and K562 cells to phenocopy the frameshifts and deletions observed in MDS patients. We used a PCR-based assay to measure the splicing efficiency of introns near LUC7L2-binding sites. Knockdown of LUC7L2 increased the splicing efficiency of 8/13 selected introns; this suggests that LUC7L2 represses selective splice site usage. We also performed RNA-seq to characterize global mis-splicing events. Analysis of RNA transcripts revealed a multitude of splicing changes, including enhanced exclusion of alternative introns. Knockdown LUC7L2 cells exhibited-altered expression of other splicing factors; this could have further contributed to the vast number of splicing changes observed.To identify specific splicing changes that could contribute to the pathogenesis of MDS, we compared the splicing profiles of LUC7L2-knockdown in K562 cells with RNA-seq data from K562 cells expressing U2AF1S34F, SRSF2P95H or SF3B1K700E. This analysis yielded several exon-skipping splicing patterns in cancer-relevant transcripts, such as oncogene PRC1, splicing factor PTBP1 and MRPL33. Additionally, we noticed commonly mis-spliced transcripts among the four datasets in which the missplicing events occurred in the functional domain, potentially conferring a functional change. Surprisingly, we observed missplicing of U2AF1 in LUC7L2-knockdown, SRSF2P95H, and SF3B1K700E K562 cells, which altered the length of the RNA-recognition UHM domain by inclusion of a mutually exclusive exon or retention of an intron. In this way, low expression of LUC7L2, or point mutants U2AF1S34F, SRSF2P95H, and SF3B1K700E,could alter U2AF1 function as a distal convergence point.In summary, we identified a novel splicing factor implicated in the pathogenesis of MDS. We characterized LUC7L2 as a splicing repressor and discovered many splicing changes caused by low expression of LUC7L2. Several genes were also mis-spliced in U2AF1S34F, SRSF2P95H and SF3B1K700E K562 cells targeting these for further study. Commonly mis-spliced targets such as U2AF1 may indicate that some of the novel therapeutics may have spliceosome mutation agnostic effects. If this applies to the LUC7L2 mutations, then they may also be effective in del7/del7q cases. DisclosuresCarraway:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; FibroGen: Consultancy; Jazz: Speakers Bureau; Novartis: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Consultancy, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Saunthararajah:Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy.

Mutations in the RNA Splicing Factor SF3B1 Promote Transformation through MYC Stabilization

Mutations in the RNA splicing factor SF3B1 are recurrent in CLL and myeloid neoplasms but their functional role in promoting tumorigenesis remain poorly understood. While SF3B1 mutations have been identified as promoting use of aberrant 3' splice sites (3'ss), consistent identification of mis-spliced transcripts and pathways that functionally link mutant SF3B1 to transformation remains elusive. Moreover, large-scale analyses of the impact of mutant SF3B1 on gene expression and gene regulatory networks, which may be distinct from aberrant splicing changes, remain to be performed. We therefore sought to elucidate the effects of SF3B1 mutations across hematopoietic malignancies and cancer lineages at the level of both mRNA splicing and expression. To this end, we collected RNA-seq data from 79 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations. The most frequent hotspot, K700E, was the most common mutation in CLL and breast cancers while mutations at position R625 were restricted to melanomas (Figure A, B).Regulatory network analysis of differentially expressed genes in SF3B1 mutated CLL identified MYC as the top master regulator (Figure C). MYC activation in SF3B1 mutated CLL was also verified by differential expression analyses (Figure D) and was common to SF3B1K700E mutant cancers while absent in cancers with mutations affecting R625. Taken together, these observations suggested that tumors harboring SF3B1K700E mutations activate the MYC transcriptional program.We next sought to verify the effects of c-Myc activation by mutant Sf3b1 in the B-cell lineage in vivo. We crossed Cd19-cre Sf3b1K700E/+ mice with Eμ-Myc transgenic mice to generate Cd19-cre+ control, Sf3b1K700E/+, Eμ-MycTg/+, and Sf3b1K700E/+Eμ-MycTg/+ double-mutant mice. While control or single mutant primary mice did not develop disease over one year, double-mutant mice developed a lethal B-cell malignancy. This effect was consistent in serial transplantation, where mice transplanted with double-mutant cells had shorter survival compared to single-mutant controls (Figure E). These data provide the first evidence that SF3B1 mutations contribute to tumorigenesis in vivo.To understand the molecular mechanism for MYC activation across SF3B1 mutant human and mouse cells, we analyzed RNA-seq data from CLL patients, isogenic Nalm-6 cells, and splenic B-cells from the mouse models. This revealed a significant overlap in aberrant (3'ss) events across SF3B1 mutant samples. Interestingly, mis-spliced events across mouse and human SF3B1K700E mutant samples identified aberrant 3'ss usage and decay of PPP2R5A (Figure F), a gene whose product has previously been shown to regulate c-MYC protein stability and the only gene whose aberrant splicing was most prominent in K700E compared with R625 mutant SF3B1. PPP2R5A is a subunit of the PP2A phosphatase complex that dephosphorylates Serine 62 (S62) of c-MYC, resulting in an unstable form of c-MYC that is a substrate for proteasomal degradation. Consistent with this, SF3B1K700E mutant cells exhibited dramatic increase in S62-phosphorylated c-MYC and increased stability of c-MYC protein. MYC expression, stability, and S62 phosphorylation could be abrogated in SF3B1 mutant cells by restoring PPP25RA expression. In addition to c-MYC S62 phosphorylation, PPP2R5A-containing PP2A reduced S70 phosphorylation of BCL2 (a modification important for apoptosis induction) in SF3B1 mutant cells. To functionally evaluate the importance of impaired PP2A enzymatic activity in SF3B1 mutant cells further, we assessed the therapeutic potential of the FDA-approved oral PP2A activator, FTY-720. SF3B1 mutant cells were more sensitive to FTY-720 treatment than SF3B1 WT counterparts, experiencing growth arrest at lower concentration (Figure G). Moreover, both S62-phosphorylated c-MYC and S70-phosphorylated BCL2 decreased in a dose-dependent manner upon treatment with FTY-720 (Figure H).Here through combined evaluation of the effects of the SF3B1 mutation on splicing, gene expression, and transcriptional networks across cancer types, we identify a novel mechanism by which mutant SF3B1-mediated alterations in RNA splicing contribute to activation of oncogenic MYC through effects on MYC proteolysis. Moreover, these data highlight a novel therapeutic approach targeting the impact of mutant SF3B1 on post-translational modification of MYC. DisclosuresMato:Janssen: Consultancy, Honoraria; Celgene: Consultancy; Prime Oncology: Speakers Bureau; TG Therapeutics: Research Funding; Regeneron: Research Funding; Abbvie: Consultancy; Sunesis: Honoraria, Research Funding; Acerta: Research Funding; AstraZeneca: Consultancy; Pharmacyclics: Consultancy, Honoraria, Research Funding.

Human aniridia limbal epithelial cells lack expression of keratins K3 and K12

Aniridia is a rare disease of the eye that affects the iris, lens and the cornea. In about 90% of the cases, patients showed a loss of PAX6 function. Patients with aniridia often develop aniridia-related keratopathy (ARK), due to limbal stem cell insufficiency.The aim of this study was to determine the differentiation status of limbal epithelial cells (LECs) in patients with ARK.Epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in KSFM medium supplemented with EGF and BPE. Normal cells were obtained from limbus region of cadaveric control patients. Cells were analyzed with RT-PCR, qPCR and Western blot to evaluate expression of the developmental transcription factor, PAX6, potential stem cell markers, ΔNp63α and ABCG2, and corneal differentiation markers, keratin 12 (K12) and K3. Conjunctival differentiation markers, keratin 13 (K13) and K19 were also investigated. Cells were immunostained to evaluate K3, PAX6, and p63α protein expression. Protein coding sequence of PAX6 from patient LEC-cDNA was cloned and sequenced.RT-PCR showed that K3 and K12 transcripts were absent from patient cells, but present in healthy control preparations. Transcription levels of PAX6, ABCG2, and p63α of aniridia patients show no differences compared to normal control cells. Western blot showed reduced PAX6, protein levels in aniridia-LECs compared to control-LECs. Immunostaining also showed reduced PAX6 and K3 expression in aniridia-LECs compared to control-LECs. One aniridia patient showed a loss of stop codon in half of the cloned transcripts. In the second aniridia patient mRNA degradation through nonsense mediated decay seems to be very likely since we could not identify the mutation c.174C > T (Refseq. NM_000280), or misspliced transcripts in cDNA.We identified decreased PAX6 protein levels in aniridia patients in addition to decreased K12 mRNA levels compared to control cells. This result indicates an altered differentiation of limbal epithelial cells of aniridia patients. Further studies are necessary to evaluate the mechanism of differentiation of limbal epithelial cells in aniridia.

Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity.

BackgroundApproximately 75% of clinically definite long QT syndrome (LQTS) cases are caused by mutations in the KCNQ1, KCNH2 and SCN5A genes. Of these mutations, a small proportion (3.2-9.2%) are predicted to affect splicing. These mutations present a particular challenge in ascribing pathogenicity.MethodsHere we report an analysis of the transcriptional consequences of two mutations, one in the KCNQ1 gene (c.781_782delinsTC) and one in the SCN5A gene (c.2437-5C>A), which are predicted to affect splicing. We isolated RNA from lymphocytes and used a directed PCR amplification strategy of cDNA to show mis-spliced transcripts in mutation-positive patients.ResultsThe loss of an exon in each mis-spliced transcript had no deduced effect on the translational reading frame. The clinical phenotype corresponded closely with genotypic status in family members carrying the KCNQ1 splice variant, but not in family members with the SCN5A splice variant. These results are put in the context of a literature review, where only 20% of all splice variants reported in the KCNQ1, KCNH2 and SCN5A gene entries in the HGMDPro 2015.4 database have been evaluated using transcriptional assays.ConclusionsPrediction programmes play a strong role in most diagnostic laboratories in classifying variants located at splice sites; however, transcriptional analysis should be considered critical to confirm mis-splicing. Critically, this study shows that genuine mis- splicing may not always imply clinical significance, and genotype/phenotype cosegregation remains important even when mis-splicing is confirmed.

The Role of LUC7L2 in Splicing and MDS

Chromosomal abnormalities are common in myelodysplastic syndrome (MDS) and other myeloid malignancies. -7/del7q is found in 15% of MDS patients, but the pathological consequences of this deletion are unknown. To identify genes in the -7/del7q region that may contribute to pathogenesis of MDS, whole exome sequencing of MDS patient samples was used to detect novel somatic nonsense/frameshift mutations. Novel somatic mutations in the gene LUC7L2 were found in 27 malignant cases.Cases of mutant LUC7L2 hemizygosity, heterozygosity and homozygosity were observed in this cohort. The mutations were frameshift or nonsense, resulting in a premature stop codon and decreased expression of the LUC7L2 mRNA. LUC7L2 is located in the most commonly deleted region of -7/del7q; 7q34, which is deleted in 85% of -7/del7q patients. Patients with LUC7L2 mutations and those harboring -7/del7q have similar and statistically shorter overall survival than those with normal LUC7L2 expression levels.LUC7L2 is a poorly characterized, splicing-related protein. The function of LUC7L2 is largely unknown, but it is the ortholog of yeast protein LUC7p, which is involved in recruitment of early splicing factors. Recent studies have shown that over 65% of MDS patients harbor mutations in one of several proteins involved in pre-mRNA splicing. Therefore, we hypothesize that LUC7L2 acts as a mammalian splicing factor and deficiency of LUC7L2 results in aberrant splicing of transcripts that contribute to the pathogenesis of MDS.We characterized the role LUC7L2 in splicing by showing that LUC7L2 interacts with splicing factors using immunoprecipitation followed by mass spectrometry. Immunoprecipitation of LUC7L2 and interacting proteins revealed that LUC7L2 interacts with many proteins in the SR and HnRNP families of splicing regulators suggesting that LUC7L2 may also play a role as a splicing regulator. Splicing regulatory proteins are a diverse group that can influence constitutive splicing as well as alternative splicing. Therefore, to understand the mRNA targets of LUC7L2, we performed RNA CLIP-Seq. This revealed that LUC7L2 binds at least 301 pre-mRNA transcripts as well as spliceosomal snRNAs. LUC7L2 binding is enriched near splice junctions, with 117 of binding sites lying within 100bp of a splice site.To understand how LUC7L2 regulates splicing, we knocked down LUC7L2 in HEK293 cells and subjected the cells to a qRT-PCR based intron splicing efficiency assay as well as a splicing sensitive microarray. The intron splicing efficiency assay was performed on a subset of LUC7L2 targets identified by CLIP-Seq and showed that LUC7L2-deficiency often promotes the splicing of introns, suggesting that LUC7L2 acts as a splicing repressor. To understand the effect of LUC7L2 deficiency on all LUC7L2-targets, the splicing sensitive microarray identified splicing changes near LUC7L2-binding sites. LUC7L2-deficiency significantly alters the splicing of 151 transcripts that contain LUC7L2 binding sites.Missplicing of transcripts can alter protein function, leading to profound repercussions in the cell. The microarray analysis of LUC7L2-deficient cells identified several misspliced transcripts that are involved in the processes of proliferation, differentiation, and apoptosis, which are disrupted in MDS hematopoietic stem cells. Exons 2, 7 and 8 of the RUNX1 transcript are frequently skipped in the LUC7L2 knockdown cells. Although the mRNA expression of RUNX1 remains the same, several downstream transcripts that are regulated by RUNX1 have significantly increased or decreased expression, suggesting altered function of the RUNX1 protein. RUNX1 is a transcription factor that is required for HSC differentiation into myeloid progenitor cells. Altered splicing of RUNX1 and changes in the RUNX1 pathway could cause defects in the HSC differentiation process.In conclusion, novel somatic mutations in have been observed in LUC7L2 in patients with MDS. LUC7L2 is a protein that acts as a splicing regulator and deficiency of this protein results in missplicing of transcripts, including factors involved in differentiation, which may contribute to the pathogenesis of MDS. DisclosuresMakishima:The Yasuda Medical Foundation: Research Funding.