Exonic Splicing Enhancer Research Articles

Exon‐independent recruitment of SRSF1 is mediated by U1 snRNP stem‐loop 3

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5′ splice site (SS), and both factors affect 5′ SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single‐molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1‐independent process of binding to an ESE. Structural analysis and cross‐linking data show that SRSF1 contacts U1 snRNA stem‐loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5′SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals.

ATR/CHK1/WEE1 Dependency in SRSF2-Mutated MDS/AML

Background: Mutations in splicing factor gene SRSF2 are recurrent drivers in 5-15 % of patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A key property of SRSF2 mutations is that they occur early in the pathogenesis of MDS and are therefore present in all tumor cells in a patient. This property makes targetable vulnerabilities caused by SRSF2 mutations exceptionally important as they provide a way to inhibit the whole tumor. We previously demonstrated that splicing factor mutations induce R-loop-dependent activation of ATR, rendering cells sensitive to ATR inhibition. R-loops are transcription intermediates consisting of an RNA:DNA hybrid and a displaced single-stranded DNA. Accumulation of aberrant R-loops induces the ATR kinase, which activates the G2-M cell cycle checkpoint via CHK1- and WEE1-mediated signaling. In normal cells, activation of the G2-M checkpoint halts the cell cycle until R-loops have been resolved. When the ATR pathway is inhibited, checkpoint activation does not occur, causing cells with unresolved R-loops to proceed to mitosis, resulting in DNA damage and cell death. We, therefore, sought to assess primary human MDS/AML samples for sensitivity to perturbation of the ATR/CHK1/WEE1 pathway and identify mechanisms of resistance.Methods: Sensitivity of 147 AML patient samples to 515 oncology drugs was tested ex vivo. Bone marrow mononuclear cells were incubated with 5 concentrations of each drug for 72 h followed by measurement of cell viability by CTG assay. Somatic mutations were identified by exome sequencing of matched leukemic bone marrow and skin biopsy samples.Isogenic K562 cell line clones carrying SRSF2 P95H/L/R mutations were generated using CRISPR/Cas9 editing. The presence of SRSF2 mutation was confirmed by CRISPR-sequencing and expression by whole transcriptome RNA-sequencing. Drug sensitivity of the K562 clones with and without SRSF2 mutation was determined by incubating cells with 16 concentrations of prexasertib, SRA-737, adavosertib, or BAY-1895344, followed by determination of cell viability by the MTS assay.Results: Analysis of ex vivo drug sensitivities in AML patient samples identified vulnerability to CHK1 and WEE1 inhibition in SRSF2-mutated AML: SRSF2 mutation is associated with sensitivity to the CHK1 inhibitors prexasertib (p = 0.006) (Fig 1 A and B) and PF-00477736 (p = 0.002) and the WEE1 inhibitor adavosertib (p = 0.003).To establish whether the isogenic SRSF2-mutant K562 cell line models recapitulate known downstream aberrations associated with SRSF2 mutations in patients, we analyzed gene expression and splicing. SRSF2 contains an RNA binding domain with affinity to CCNG or GGNG exonic splicing enhancer sequences. Similar to what has been observed in patients, the K562 clones with SRSF2 mutation show reduced use of GGNG sequence motifs at skipped exons. These results demonstrate that isogenic K562 clones recapitulate known alterations caused by mutant SRSF2.To determine whether SRSF2 mutations induce sensitivity to inhibition of ATR, CHK1, and WEE1, we tested 10 isogenic SRSF2 mutant and 4 wild-type K562 clones. Cells with SRSF2 mutation show increased sensitivity to ATR/CHK1/WEE1 inhibition (Fig 1C). We found no significant difference in drug sensitivity between clones carrying SRSF2 P95H/L/R substitutions. Clones with higher SRSF2 mutant allele dosage are more sensitive (Fig 1D).We identified a subset of SRSF2 mutated AML samples that were resistant to CHK1 and WEE1 inhibition. All resistant AML have co-occurring RUNX1 mutations (Fig 1B). In AML, RUNX1 mutations are associated with therapy resistance, suggesting that these mutations contribute to drug resistance. To test whether RUNX1 mutations induce resistance to ATR/CHK1/WEE1 inhibition in SRSF2-mutant leukemia, we introduced RUNX1 loss-of-function mutations in isogenic K562 carrying SRSF2 mutations. Candidate resistance factors identified by ATAC and RNA-sequencing will be validated in functional assays.Conclusions: Our results indicate that SRSF2-mutated leukemia harbor a vulnerability to the inhibition of ATR, CHK1, and WEE1 kinases. Cell line models indicate that sensitivity is similar across mutant alleles and dependent on allelic copy number. Several ATR/CHK1 and WEE1 inhibitors are in development, and our results suggest that these compounds could be effective treatments for SRSF2-mutated MDS and AML. [Display omitted] DisclosuresGraubert: astrazeneca: Research Funding; Janssen: Research Funding; Calico: Research Funding.

MDS-Associated Splicing Factor Mutations Promote Alternative Splicing of a Differentiation-Impaired CSF3R Isoform

▪Background. Myelodysplastic syndromes (MDS) constitute the most common group of bone marrow failure disorders, characterized by ineffective hematopoiesis and a significant risk of transformation to AML. Efforts to understand the molecular basis of MDS led to the identification of acquired somatic mutations in the RNA splicing factors SF3B1, U2AF1, SRSF2, ZRSR2, and LUC7L2 in 45-85% of adult MDS patients. How they perturb hematopoiesis and promote expansion of abnormal clones remain unknown. Elevated levels of alternate isoforms of granulocyte colony stimulating factor receptor (CSF3R) patients with MDS and other myeloid neoplasias have been reported with a strong correlation reported with loss of chromosome 7. Constitutive splicing produces the canonical Class I CSF3R isoform that supports proliferation and differentiation. Alternative splicing promoting intron excision in CSF3R exon 17 results in a differentiation-defective Class IV CSF3R isoform. We hypothesized that aberrant splicing activity of MDS-associated splicing factor mutations promote Class IV CSF3R expression, resulting in dysgranulopoiesis. Methods. We constructed a CSF3R minigene that was transiently expressed in K562 cells along with eukaryotic expression vectors containing cDNAs for wild-type or mutant forms of SF3B1, U2AF1, or SRSF2. To determine role of LUC7L2, a splicing factor residing on chromosome 7, the CSF3R minigene was transiently expressed in K562 cells with knockout LUC7L2. Class IV or Class I was measure by qPCR using specific primers. Putative exonic splicing enhancer motifs (ESEs) within exon 17 were deleted in the minigene and transfected into K562 cells expressing either wildtype or mutant SRSF2. To determine impact of mutant SRSF2 on granulopoiesis, wildtype or mutant SRSF2 were overexpressed in GCSF-treated CD34+ cells and morphology was assessed on day 14. To determine impact of Class IV isoform on granulopoiesis, Lin-, Sca-1+, and c-Kit+ (LSK) cells from Csf3r null mice were transduced with Class I or Class IV and colony forming unit (CFU) assay for granulopoiesis was performed with Methocult 3534. Colony scores and cell morphology were assessed on day 8.Results. K562 cells with transient expression of mutant SRSF2 P95H and LUC7L2 knockout resulted in statistically significant increase in Class IV:I CSF3R mRNA ratios normalized to a minigene-only control. Interestingly, expression of mutant U2AF1 S34F significantly decreased Class IV:I while U2AF1 Q157P increased Class IV:I ratios. K562 cells expressing mutant SF3B1 K700E did not show significant differences in Class IV splicing. To further investigate how SRSF2 regulates CSF3R splicing, we deleted two putative SRSF2-binding ESE motifs (ESE1 and ESE2) within exon 17 of the CSF3R minigene and transfected them into K562 cells stably expressing either wildtype SRSF2 or mutant P95H, P95L, or P95R. Deletion of either ESE1 or ESE2 resulted in decreased Class IV:I in all SRSF2 P95 mutant cells compared to minigene-only and wildtype controls. Next, we assessed the effect of SRSF2 P95 mutations on neutrophil differentiation in CD34+ cells. Overexpression of SRSF2 P95H, P95L, or P95R led to increased neutrophilic precursors compared to untransduced CD34+ cells. To determine the effect of a single CSF3R isoform on granulopoiesis, we cultured LSK cells from Csf3r null mice transduced with either Class I or Class IV on MethoCult 3534 with addition of GCSF. On day 8, cells expressing Class I had more total colony numbers with significantly higher CFU-GM colonies than cells expressing Class IV compared to empty vector control.Conclusions. We demonstrated that mutated SRSF2, U2AF1, and LUC7L2 deficiency alters CSF3R splicing in its terminal exon. Interestingly, mutations on different residues of the same gene (U2AF1) had opposing effects on CSF3R splicing. Mutant SRSF2 resulted in increased intron excision to promote increased levels of Class IV isoform. Mutation of two putative SRSF2 binding sites within CSF3R exon 17 reversed the increased splicing promoted by mutant SRSF2, further support our observation that CSF3R is a target for mutant SRSF2. Our observation with overexpression of mutant SRSF2 P95 in CD34+ cells suggests that defective neutrophil differentiation is related to increased Class IV. Our findings shed insights into how aberrant splicing of CSF3R drives MDS progression and provides a new model of dysgranulopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Identification and in silico characterization of structural and functional impacts of genetic variants in milk protein genes in the Zebu breeds Guzerat and Gyr.

Whole genome sequencing of bovine breeds has allowed identification of genetic variants in milk protein genes. However, functional repercussion of such variants at a molecular level has seldom been investigated. Here, the results of a multistep Bioinformatic analysis for functional characterization of recently identified genetic variants in Brazilian Gyr and Guzerat breeds is described, including predicted effects on the following: (i) evolutionary conserved nucleotide positions/regions; (ii) protein function, stability, and interactions; (iii) splicing, branching, and miRNA binding sites; (iv) promoters and transcription factor binding sites; and (v) collocation with QTL. Seventy-one genetic variants were identified in the caseins (CSN1S1, CSN2, CSN1S2, and CSN3), LALBA, LGB, and LTF genes. Eleven potentially regulatory variants and two missense mutations were identified. LALBA Ile60Val was predicted to affect protein stability and flexibility, by reducing the number the disulfide bonds established. LTF Thr546Asn is predicted to generate steric clashes, which could mildly affect iron coordination. In addition, LALBA Ile60Val and LTF Thr546Asn affect exonic splicing enhancers and silencers. Consequently, both mutations have the potential of affecting immune response at individual level, not only in the mammary gland. Although laborious, this multistep procedure for classifying variants allowed the identification of potentially functional variants for milk protein genes.

Regulation of Survival Motor Neuron Gene Expression by Calcium Signaling.

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletion, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, a defect in SMN2 splicing, involving exon 7 skipping, results in a low level of functional SMN protein. Therefore, the upregulation of SMN protein expression from the SMN2 gene is generally considered to be one of the best therapeutic strategies to treat SMA. Most of the SMA drug discovery is based on synthetic compounds, and very few natural compounds have been explored thus far. Here, we performed an unbiased mechanism-independent and image-based screen of a library of microbial metabolites in SMA fibroblasts using an SMN-specific immunoassay. In doing so, we identified brefeldin A (BFA), a well-known inhibitor of ER-Golgi protein trafficking, as a strong inducer of SMN protein. The profound increase in SMN protein was attributed to, in part, the rescue of the SMN2 pre-mRNA splicing defect. Intriguingly, BFA increased the intracellular calcium concentration, and the BFA-induced exon 7 inclusion of SMN2 splicing, was abrogated by the depletion of intracellular calcium and by the pharmacological inhibition of calcium/calmodulin-dependent kinases (CaMKs). Moreover, BFA considerably reduced the expression of Tra2-β and SRSF9 proteins in SMA fibroblasts and enhanced the binding of PSF and hnRNP M to an exonic splicing enhancer (ESE) of exon 7. Together, our results demonstrate a significant role for calcium and its signaling on the regulation of SMN splicing, probably through modulating the expression/activity of splicing factors.

Evidence in disease and non-disease contexts that nonsense mutations cause altered splicing viamotif disruption.

Transcripts containing premature termination codons (PTCs) can be subject to nonsense-associated alternative splicing (NAS). Two models have been evoked to explain this, scanning and splice motif disruption. The latter postulates that exonic cis motifs, such as exonic splice enhancers (ESEs), are disrupted by nonsense mutations. We employ genome-wide transcriptomic and k-mer enrichment methods to scrutinize this model. First, we show that ESEs are prone to disruptive nonsense mutations owing to their purine richness and paucity of TGA, TAA and TAG. The motif model correctly predicts that NAS rates should be low (we estimate 5–30%) and approximately in line with estimates for the rate at which random point mutations disrupt splicing (8–20%). Further, we find that, as expected, NAS-associated PTCs are predictable from nucleotide-based machine learning approaches to predict splice disruption and, at least for pathogenic variants, are enriched in ESEs. Finally, we find that both in and out of frame mutations to TAA, TGA or TAG are associated with exon skipping. While a higher relative frequency of such skip-inducing mutations in-frame than out of frame lends some credence to the scanning model, these results reinforce the importance of considering splice motif modulation to understand the etiology of PTC-associated disease.

Clinical and genetic analysis of six Chinese children with Poirier-Bienvenu neurodevelopmental syndrome caused by CSNK2B mutation.

Mutations in CSNK2B lead to Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), a rare neurodevelopmental disorder. Only 14 cases of POBINDS have been reported worldwide. The main manifestations are seizures, often tonic-clonic, with or without intellectual disability, growth retardation, and developmental language retardation. We conducted a comprehensive phenotypic mining and trio-whole exome sequencing on six children with POBINDS for gene diagnosis and analyzed the different variants using bioinformatics analysis software and related experiments. This paper reviews previous literature and discusses two common missense variants that lead to structural changes. Among the six patients, four, one, and one had tonic-clonic, myoclonic, and febrile seizures, respectively. Language development disorder, motor development disorder, and developmental delay/intellectual disability (DD/ID) are the main clinical features. All children had de novo mutations in CSNK2B, including three missense variants (c.410G > T/p.(Cys137Phe), c.494A > G/p.(His165Arg), and c.3G > A/p.(Met1Ile)), two splice variants (c.292-2A > T, c.558-3T > G), and one frameshift variant (c.499delC/p.(Leu167Serfs*60)). Three missense variants were predicted to be harmful by various software programs, and two splicing variants were found to produce new exonic splicing enhancers by the minigene assay. Western blot analysis showed that the frameshift variant resulted in decreased protein expression. According to a literature review, c.3G > A/p.(Met1Ile), c.292-2A > T, c.558-3T > G, and c.499delC/p.(Leu167Serfs*60) are novel variants of CSNK2B. The decrease or loss of protein function caused by CSNK2B mutations may be a pathogenic factor in this cohort. The severity of the POBINDS phenotype differs, and refractory epilepsy may be accompanied by a more serious DD/ID, language disorder, and motor retardation. At present, there is no specific treatment, and antiepileptic therapy usually requires the combination of two or more anti-epileptic drugs.

Identification of a Novel Splice Site Mutation in RUNX2 Gene in a Family with Rare Autosomal Dominant Cleidocranial Dysplasia.

Background:Pathogenic variants of RUNX2, a gene that encodes an osteoblast-specific transcription factor, have been shown as the cause of CCD, which is a rare hereditary skeletal and dental disorder with dominant mode of inheritance and a broad range of clinical variability. Due to the relative lack of clinical complications resulting in CCD, the medical diagnosis of this disorder is challenging, which leaves it underdiagnosed. Methods:In this study, nine healthy and affected members of an Iranian family were investigated. PCR and sequencing of all exons and exon-intron boundaries of RUNX2 (NM_001024630) gene was performed on proband. Co-segregation analysis was conducted in the other family members for the identified variant. Additionally, a cohort of 100 Iranian ethnicity-matched healthy controls was screened by ARMS-PCR method. Results:The novel splice site variant (c.860-2A>G), which was identified in the intron 6 of RUNX2 gene, co-segregated with the disease in the family, and it was absent in healthy controls. Pathogenicity of this variant was determined by several software, including HSF, which predicts the formation or disruption of splice donor sites, splice acceptor sites, exonic splicing silencer sites, and exonic splicing enhancer sites. In silico analysis predicted this novel variant to be disease causing. Conclusion:The identified variant is predicted to have an effect on splicing, which leads to exon skipping and producing a truncated protein via introducing a premature stop codon.

Identification of seven exonic variants in the SLC4A1, ATP6V1B1, and ATP6V0A4 genes that alter RNA splicing by minigene assay.

Primary distal renal tubular acidosis (dRTA) is a rare tubular disease associated with variants in SLC4A1, ATP6V0A4, ATP6V1B1, FOXⅠ1, or WDR72 genes. Currently, there is growing evidence that all types of exonic variants can alter splicing regulatory elements, affecting the precursor messenger RNA (pre-mRNA) splicing process. This study was to determine the consequences of variants associated with dRTA on pre-mRNA splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 15 candidate variants, 7 variants distributed in SLC4A1 (c.1765C>T, p.Arg589Cys), ATP6V1B1 (c.368G>T, p.Gly123Val; c.370C>T, p.Arg124Trp; c.484G>T, p.Glu162* and c.1102G>A, p.Glu368Lys) and ATP6V0A4 genes (c.322C>T, p.Gln108* and c.1572G>A, p.Pro524Pro) were identified to result in complete or incomplete exon skipping by either disruption of exonic splicing enhancers (ESEs) and generation of exonic splicing silencers, or interference with the recognition of the classic splicing site, or both. To our knowledge, this is the first study on pre-mRNA splicing of exonic variants in the dRTA-related genes. These results highlight the importance of assessing the effects of exonic variants at the mRNA level and suggest that minigene analysis is an effective tool for evaluating the effects of splicing on variants in vitro.

Targeting the Mechanism of c-Mpl Exon-Inclusion through Antisense Oligonucleotides Yields a Dominant Negative Isoform

▪Activating mutations of the Thrombopoietin (THPO) receptor, c-MPL, have been revealed as drivers in myeloproliferative diseases (MPDs).In addition, wildtype c-MPL signaling has been shown to be required for the pathogenesis of Calreticulin-mutated MPDs and implicated in leukemic stem cell survival in CML and AML. Therefore, the ability to impair c-MPL signaling has the potential to provide a novel, targeted approach toward these difficult to treat diseases. Alternative splicing of c-MPL gives rise to a truncated dominant negative isoform, MPL-TR, which skips exons 9 and 10 and is conserved between species. Our prior work using a knockout mouse model identified the spliceosomal protein, Ott1(Rbm15), as a key regulator of alternative splicing between Mpl-TR and the full length isoform, Mpl-FL. Loss of Ott1 increased the fraction of Mpl-TR from 33% to 73% in hematopoietic stem cells (HSCs) and abolished Thpo response. Furthermore, overexpression of Mpl-TR impaired HSC function in vivo. To further delineate the global effects of Ott1 on alternative splicing within the HSC compartment, we performed RNA-seq on sorted Ott1-deleted Lin-Sca1+Kit+ (LSK) cells. DEXseq analysis of significant differential exon expression identified 1821 exon exclusion and 158 exon inclusion events in the Ott1-deleted cells compared to wildtype controls, establishing Ott1 as primarily an exon-inclusion factor. The splicing factor Srsf2 has been observed in the same complex as Ott1 and also functions primarily as a cassette exon inclusion factor, therefore we compared previously identified Srsf2 exon targets and found 15-19% overlapped with Ott1-affected exons, suggesting a functional link between the proteins in the spliceosome.Srsf2 utilizes the exon splicing enhancer (ESE) sequence to promote exon inclusion. Scanning the c-Mpl sequence revealed an ESE within exon 10 that is conserved between mice and humans. We hypothesized blocking the exon 10 ESE with an anti-sense oligonucleotide (AON) would interfere with the spliceosomal machinery responsible for c-Mpl exon-inclusion and lead to exon skipping, thus resulting in production of the dominant negative Mpl-TR isoform. An advantage of AON-targeted therapy is the ability to produce highly specific agents based on sequence complementarity. We synthesized chemically modified AONs with phosphorothioate backbones, either fully modified with 2'- O -methoxyethyl RNA (MOE) and thus designed to induce splice-switching, or designed as gapmers to elicit cleavage of their targets by RNase H. MOE-based AONs targeted towards the exon 10 ESE were the most effective at increasing the Mpl-TR isoform fraction, increasing them from 30-40% to 60-70% in murine cell lines and primary bone marrow/fetal liver cells. AONs directed toward the splice acceptors and donors of exons 9 and 10 were less effective, suggesting a key role for the exon 10 ESE in regulating c-Mpl splicing decision. To test the ability of AONs to successfully target in vivo, fluorophore-labelled AONs were used in a dose-escalation study in mice. 18 hours after tail vein injection with 1-6 mg/kg un-encapsulated AON dose, bone marrow and spleen were harvested and lineage labelled. High levels of dose-dependent uptake were observed, particularly within the myeloid compartment (granulocyte >99%; megakaryocyte 55%; macrophage 20.9%; erythroid 44.7%; B cell 5% and CD4 T 45.6% and CD8 T 77%). To assess uptake and intracellular localization of AONs within the HSC compartment, AMNIS flow cytometry was performed and showed robust nuclear localization within 64% of LSK cells and 42% of myeloid progenitors. The high levels of uptake in a wide range of hematopoietic lineages, including HSCs, confirms IV injection of naked AONs can efficiently target the hematopoietic compartment. In summary, through uncovering the pivotal cis- and trans-factors controlling splicing at the c-Mpl locus, we were able to develop a specific, effective agent for impairing the Thpo/Mpl axis. DisclosuresNo relevant conflicts of interest to declare.

RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants.

Simple SummaryIn the BRIDGES project, the breast/ovarian cancer gene RAD51D has been sequenced in >113,000 women. In the present study, we focused on the impact that 11 pre-selected RAD51D variants at the intron/exon boundaries had on the splicing process (intron removal). For this purpose, we developed a splicing reporter minigene, containing RAD51D-exons 2–9 wherein any variant could be introduced and functionally assayed for splicing alterations. All variants impaired splicing, 10 of which caused complete splicing aberrations. Moreover, we developed a minigene-based strategy to search for non-canonical, spliceogenic variants that disrupted splicing enhancers/silencers in the non-constitutive exon 3. Twenty-six BRIDGES and 16 artificial exon 3 variants were also tested. Thirty variants impaired splicing by producing variable amounts of the FL transcript. In total, up to 9 variants were classified as Likely Pathogenic, and therefore were clinically actionable. Carriers may benefit from tailored prevention protocols and therapies.RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2–9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (<3.4%) of the full-length (FL) transcript. In addition, ESE elements of the alternative exon 3 were mapped by testing four overlapping exonic microdeletions (≥30-bp), revealing an ESE-rich interval (c.202_235del) with critical sequences for exon 3 recognition that might have been affected by germline variants. Next, 26 BRIDGES variants and 16 artificial exon 3 single-nucleotide substitutions were also assayed. Thirty variants impaired splicing with variable amounts (0–65.1%) of the FL transcript, although only c.202G>A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T>C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation.

Effect of MACC1 Genetic Polymorphisms and Environmental Risk Factors in the Occurrence of Oral Squamous Cell Carcinoma.

MACC1 (Metastasis Associated in Colon Cancer 1) is found to regulate the hepatocyte growth factor (HGF)/Met signal pathway, and plays an important role in tumor proliferation, angiogenesis, and metastasis. However, the relationships between MACC1 SNPs (single nucleotide polymorphisms) and oral cancer are still blurred. In this study, five SNPs (rs3095007, rs1990172, rs4721888, rs975263, and rs3735615) were genotyped in 911 oral cancer patients and 1200 healthy individuals by real-time polymerase chain reaction (PCR), and the associations of oral cancer with the SNP genotypes, environmental risk factors, and clinicopathological characteristics were further analyzed. Our results showed that individuals who had GC genotype or C-allele (GC + CC) in rs4721888 would have a higher risk for oral cancer incidence than GG genotype after adjustment for betel quid chewing, cigarette smoking, and alcohol drinking. Moreover, the 715 oral cancer patients with a betel quid chewing habit, who had C-allele (TC + CC) in rs975263, would have a higher risk for lymph node metastasis. Further analyses of the sequences of rs4721888 revealed that the C-allele of rs4721888 would be a putative exonic splicing enhancer. In conclusion, MACC1 SNP rs4721888 would elevate the susceptibility for oral cancer, and SNP rs975263 would increase the metastasis risk for oral cancer patients with a betel quid chewing habit. Our data suggest that SNP rs4721888 could be a putative genetic marker for oral cancer, and SNP rs975362 may have the potential to be a prognostic marker of metastasis in an oral cancer patient.

Daily temperature cycles promote alternative splicing of RNAs encoding SR45a, a splicing regulator in maize.

Elevated temperatures enhance alternative RNA splicing in maize (Zea mays) with the potential to expand the repertoire of plant responses to heat stress. Alternative RNA splicing generates multiple RNA isoforms for many maize genes, and here we observed changes in the pattern of RNA isoforms with temperature changes. Increases in maximum daily temperature elevated the frequency of the major modes of alternative splices (AS), in particular retained introns and skipped exons. The genes most frequently targeted by increased AS with temperature encode factors involved in RNA processing and plant development. Genes encoding regulators of alternative RNA splicing were themselves among the principal AS targets in maize. Under controlled environmental conditions, daily changes in temperature comparable to field conditions altered the abundance of different RNA isoforms, including the RNAs encoding the splicing regulator SR45a, a member of the SR45 gene family. We established an “in protoplast” RNA splicing assay to show that during the afternoon on simulated hot summer days, SR45a RNA isoforms were produced with the potential to encode proteins efficient in splicing model substrates. With the RNA splicing assay, we also defined the exonic splicing enhancers that the splicing-efficient SR45a forms utilize to aid in the splicing of model substrates. Hence, with rising temperatures on hot summer days, SR45a RNA isoforms in maize are produced with the capability to encode proteins with greater RNA splicing potential.

Targeting novel human transient receptor potential ankyrin 1 splice variation with splice-switching antisense oligonucleotides.

Activation of transient receptor potential ankyrin 1 (TRPA1) channels by both environmental irritants and endogenous inflammatory mediators leads to excitation of the nerve endings, resulting in acute sensation of pain, itch, or chronic neurogenic inflammation. As such, TRPA1 channels are actively pursued as therapeutic targets for various pathological nociception and pain disorders. We uncovered that exon 27 of human TRPA1 (hTRPA1) could be alternatively spliced into hTRPA1_27A and hTRPA1_27B splice variants. The resulting channel variants displayed reduced expression, weakened affinity to interact with WT, and suffered from complete loss of function because of disruption of the C-terminal coiled-coil domain. Using a human minigene construct, we revealed that binding of splicing factor serine/arginine-rich splicing factor 1 (SRSF1) to the exonic splicing enhancer was critical for the inclusion of intact exon 27. Knockdown of SRSF1, mutation within exonic splicing enhancer, or masking SRSF1 binding with antisense oligonucleotides promoted alternative splicing within exon 27. Finally, antisense oligonucleotides-induced alternative splicing produced transcript and protein variants that could be functionally determined as diminished endogenous TRPA1 activity in human Schwann cell-line SNF96.2 and hiPSCs-derived sensory neurons. The outcome of the work could potentially offer a novel therapeutic strategy for treating pain by targeting alternative splicing of hTRPA1.

Synonymous variants of the ATP7B gene may cause abnormal splicing of mRNA by affecting the exonic splicing enhancers

To explore the effect of rare synonymous variants of the ATP7B gene on the splicing of its precursor mRNA. A total of 248 rare synonymous variants with allelic frequency of <0.005 were retrieved from the ExAc database. Human Splicing Finder (HSF) was used to predict their effect on the splicing of precursor mRNA. And ESE Finder 3.0 was used to predict the effect of such variants on the binding ability of SR protein family. Rare synonymous variants affecting the binding of two or more SR proteins were selected and verified with an in vitro mini gene splicing report system. HSF analysis indicated that 136 of the 248 rare synonymous variants may destroy the exonic splicing enhancer (ESE) motif. Analysis using ESE Finder 3.0 indicated that 19 of them may affect the binding of two or more SR proteins at the same time. In vitro mini gene experiment confirmed that the c.1620C>T (p.L540L) and c.3888C>T (p.A1296A) variants could lead to abnormal splicing of the corresponding exons, resulting in complete skipping of exon 4 and 25% increase in the skipping of exon 18, respectively. Synonymous variants may affect the splicing of precursor mRNA in various ways, particularly the destruction of ESE motif. This study confirmed that the c.1620C>T (p.L540L) and c.3888C>T (p.A1296A) variants can affect the mRNA splicing of the ATP7B gene, resulting in skipping of corresponding exons, which may provide a basis for genetic diagnosis and consultation of carriers.

GATA2 Mutation in Twins with Transient Aplastic Anemia after Chloramphenicol Exposure; A Follow up to Nagao and Mauer 1969

A case of identical twins who developed transient aplastic anemia after chloramphenicol exposure was reported by Nagao and Mauer in the New England Journal of Medicine in 1969. Bone marrow (BM) morphology and tritiated thymidine labeling indices were suggestive of a failure of delivery cells from the undifferentiated stem cell compartment. They concluded that an underlying genetic factor which predisposed to the development of drug induced aplastic anemia was highly likely. We now report long term follow up on the twins from that article and have identified that they both harbour a mutation in GATA2. Twin 1 presented with severe pancytopenia at age 8 months, 3 months after chloramphenicol exposure. His leukocytes and erythrocytes had normalized with supportive care, but platelet count (PC) remained low at 92,000/mm3. Later in life, he was diagnosed with transfusion acquired hepatitis C, treated with Sofosbuvir/Velpatasvir. He did not develop recurrent infections, warts, lymphedema, or pulmonary symptoms. At age 51, he was referred to Hematology for asymptomatic thrombocytopenia with a PC of 17,000/mm3. BM aspirate and biopsy showed a variably cellular marrow with relative erythroid hyperplasia and megaloblastoid features. Cytogenetics, FISH for myelodysplastic syndrome (MDS) related abnormalities and a 33 gene panel for MDS/chronic myelomonocytic leukemia related mutations by next generation sequencing were all normal. A next generation sequencing panel of 59 genes associated with hereditary BM failure syndromes was remarkable only for a previously unreported heterozygous mutation in GATA2 (c1040C&amp;gt;A, pThr347Asn). Twin 2 was also exposed to chloramphenicol at 5 months of age and was found to be pancytopenic when evaluated for BM donation for his brother. His pancytopenia spontaneously resolved at the end of observation. Subsequent medical problems included partial complex seizures, dysautonomia, vertigo, chronic pain syndrome and recurrent Clostridium difficile colitis. He had no history of other recurrent infections, warts, lymphedema, or pulmonary symptoms. He had mild asymptomatic cytopenias noted with a white blood cell count of 3600/mm3, a hemoglobin of 12.9 g/dl and a PC of 97,000/mm3 at the age of 50 years. At the age of 52, he was referred to hematology and was asymptomatic with a normal hemogram. DNA sequencing confirmed an identical GATA2 mutation. GATA2, a DNA binding transcription factor with two zinc finger domains, plays a significant role in gene regulation, cell fate decisions and maintenance of the hematopoietic stem cell pool. Excessive GATA2 activity is reported to prevent stem cells from leaving the pool, while insufficient GATA2 activity causes stem cells to differentiate at increased rates thereby depleting the pool. Homozygous GATA deletions/mutations are lethal. Reported mutations include deletions, frame shift mutations, nonsense mutations and point mutations. Many point mutations appear to act by causing haplo-insufficiency. The point mutation in these patients maps in proximity to the C-zinc finger domain and could potentially disrupt an exonic splicing enhancer causing an in-frame deletion of exon six. Clinical syndromes associated with GATA2 mutations include familial BM failure and cytopenia; familial myelodysplastic syndromes and acute myeloid leukemia; immunodeficiency with monocytopenia and recurrent viral, fungal and mycobacterial infections; immunodeficiency with decreased natural killer, dendritic, B cells, and monocytes; Emberger syndrome presenting with myelodysplastic syndrome, warts and lymphedema; and pulmonary alveolar proteinosis. Cytopenia has been the only clinically obvious manifestation in these twins. Chloramphenicol has been associated with two forms of hematological toxicity. The first is a dose related reversible bone marrow suppression presenting as anemia, and the second is idiosyncratic, often fatal, aplastic anemia. It was proposed that these may be opposite ends of a spectrum with an underlying genetic predisposition leading to the more severe forms. The finding of a GATA2 mutation in these patients supports Nagao and Maurer's speculation that an underlying genetic factor affecting stem cell regulation predisposed to the development of chloramphenicol induced aplastic anemia in these twins. Figure Disclosures No relevant conflicts of interest to declare.

Engineered U7 Small Nuclear RNA Restores Correct β-Globin Pre-mRNA Splicing in Mouse βIVS2-654-Thalassemic Erythroid Progenitor Cells

Restoration of correct splicing of βIVS2-654-globin pre-mRNA was previously accomplished in erythroid cells from β-thalassemia/HbE patients by an engineered U7 small nuclear RNA (snRNA) that carried a sequence targeted to the cryptic branch point and an exonic splicing enhancer, U7.BP+623 snRNA. In this study, this approach was tested in thalassemic mice carrying the βIVS2-654 mutation. While correction of βIVS2-654 pre-mRNA splicing was achieved in erythroid progenitors transduced with a lentiviral vector carrying the U7.BP+623 snRNA, a high level of truncated U7.BP+623 snRNA was also observed. The discrepancy of processing of the modified U7 snRNA in human and mouse constructs hamper the evaluation of pathologic improvement in mouse model.

A new mechanism for a familiar mutation \u2013 bovine DGAT1 K232A modulates gene expression through multi-junction exon splice enhancement

BackgroundThe DGAT1 gene encodes an enzyme responsible for catalysing the terminal reaction in mammary triglyceride synthesis, and underpins a well-known pleiotropic quantitative trait locus (QTL) with a large influence on milk composition phenotypes. Since first described over 15 years ago, a protein-coding variant K232A has been assumed as the causative variant underlying these effects, following in-vitro studies that demonstrated differing levels of triglyceride synthesis between the two protein isoforms.ResultsWe used a large RNAseq dataset to re-examine the underlying mechanisms of this large milk production QTL, and hereby report novel expression-based functions of the chr14 g.1802265AA > GC variant that encodes the DGAT1 K232A substitution. Using expression QTL (eQTL) mapping, we demonstrate a highly-significant mammary eQTL for DGAT1, where the K232A mutation appears as one of the top associated variants for this effect. By conducting in vitro expression and splicing experiments in bovine mammary cell culture, we further show modulation of splicing efficiency by this mutation, likely through disruption of an exon splice enhancer as a consequence of the allele encoding the 232A variant.ConclusionsThe relative contributions of the enzymatic and transcription-based mechanisms now attributed to K232A remain unclear; however, these results suggest that transcriptional impacts contribute to the diversity of lactation effects observed at the DGAT1 locus.

Deep intronic F8 c.5999-27A>G variant causes exon 19 skipping and leads to moderate hemophilia A.

: Hemophilia A, an X-linked recessive bleeding disorder, is caused by mutations of F8 gene. In about 2% hemophilia A patients, no exonic mutation of F8 gene was found. We aimed to identify deep intronic mutations of F8 gene. We reanalyzed the next-generation sequencing data of six hemophilia A patients with negative F8 variant in either coding region or splice site. Deep intronic F8 c.5999-27A>G variant (NM_000132.3) was found in two unrelated moderate hemophilia A patients from different region, and one patient's mother was mild hemophilia A patient. Splice site prediction algorithms showed no impact of this variant on F8 mRNA splicing of exon 19, including Human Splicing Finder 3.1, NNSPLICE 0.9, NetGene2, and Transcript-inferred Pathogenicity score. Exonic splicing enhancer was predicted by ESEfinder, and no difference was found between the wild type and mutant sequence. The branch point predicted by SVM-BPfinder suggested that F8 c.5999-27A>G variant may disrupt the branch point in intron 18 and affect the acceptor site splicing of F8 exon 19. Sanger sequencing of F8 cDNA from peripheral blood mononuclear cells confirmed that F8 c.5999-27A>G variant caused F8 exon 19 skipping in proband and his mother. Skewed X chromosome inactivation was found in another X chromosome of this mother, combined with F8 c.5999-27A>G variant in trans. In conclusion, our study suggests that deep intronic F8 c.5999-27A>G variant may be responsible for F8 exon 19 skipping and lead to moderate hemophilia A. Systematic reanalysis of next-generation sequencing data could promote the diagnostic yields.

Mutation of CFAP57, a protein required for the asymmetric targeting of a subset of inner dynein arms in Chlamydomonas, causes primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, reduced fertility, and randomization of the left/right body axis. It is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious axonemal defect for pathogenic variants using whole exome capture, next generation sequencing, and bioinformatic analysis assuming an autosomal recessive trait. We identified one subject with an apparently homozygous nonsense variant [(c.1762C>T), p.(Arg588*)] in the uncharacterized CFAP57 gene. Interestingly, the variant results in the skipping of exon 11 (58 amino acids), which may be due to disruption of an exonic splicing enhancer. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Nasal cells from the PCD patient express a shorter, mutant version of CFAP57 and the protein is not incorporated into the axoneme. The missing 58 amino acids include portions of WD repeats that may be important for loading onto the intraflagellar transport (IFT) complexes for transport or docking onto the axoneme. A reduced beat frequency and an alteration in ciliary waveform was observed. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is highly conserved in organisms that assemble motile cilia. CFAP57 is allelic with the BOP2/IDA8/FAP57 gene identified previously in Chlamydomonas reinhardtii. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass tag (TMT) mass spectroscopy shows that FAP57 is missing, and the “g” inner dyneins (DHC7 and DHC3) and the “d” inner dynein (DHC2) are reduced, but the FAP57 paralog FBB7 is increased. Together, our data identify a homozygous variant in CFAP57 that causes PCD that is likely due to a defect in the inner dynein arm assembly process.