Splice Failure Research Articles

Experimental and Numerical Investigations on Enhancing Ductility of Reinforced Concrete Columns with Substandard Lap Splices Using Hollow Steel Section Collars

ABSTRACT Hollow steel section (HSS) collars with significant flexural stiffness in addition to their axial stiffness are proposed to prevent brittle lap splice failures. Experimental program on five full-scale cantilever columns with and without HSS collar strengthening was conducted. The control column without strengthening failed suddenly. HSS collars at 333 mm spacing slightly improved peak capacity, at 200 mm spacing significantly improved peak capacity but showed extensive pinching, and at 100 mm spacing achieved lateral load-deflection response equal to the column without a lap splice. Nonlinear fiber-based modeling using OpenSees predicted the hysteretic response, resulting in close agreement with experimental results.

Behavior of steel-plate concrete wall-reinforced concrete slab joint with UHPC-strengthened lap-splice connection

The comprehensive integration of modular technology is the crucial pathway for advancing the upgraded Hua-long Pressurized Reactor (HPR1000). However, previous studies are limited and numerous challenges remain concerning the modular curved steel-plate concrete (SC) wall-reinforced concrete (RC) slab joint. This paper focuses on the behavior and design of the SC wall-RC slab joint through large-scale tests and numerical simulation. Two UHPC-strengthened non-contact lap splice (NS) connections, including versions with normal and headed rebars, were proposed to ensure effective load transfer and modular construction performance. The test results indicated that the four joint specimens subjected to cyclic loading did not experience lap splice failure but instead suffered interface shear failure and shear failure of the RC slab under shear-span ratios of 1.5 and 3.0, respectively. The excellent load transfer performance demonstrated by these two NS connections validates them as effective methods for this joint, with lap lengths of 21d for normal rebar and 13d for headed rebar deemed adequate. Besides, the developed three-dimensional FE model with non-linear spring element accurately simulated the test failure modes and F-Δ curves. The parametric study results recommended that the volumetric steel ratio of tie-bar should not be lower than 1.03 %. The tension of longitudinal rebars for normal and headed rebars can be fully transferred within 80 % of the designed lap length. Furthermore, a complete design method was established based on the load-transfer model, encompassing interfacial shear, lap length calculation and tie-bar configuration, and is applicable to SC-to-RC connections or joints.

Refining clinically relevant parameters for mis-splicing risk in shortened introns with donor-to-branchpoint space constraint

Intronic deletions that critically shorten donor-to-branchpoint (D-BP) distance of a precursor mRNA impose biophysical space constraint on assembly of the U1/U2 spliceosomal complex, leading to canonical splicing failure. Here we use a series of β-globin (HBB) gene constructs with intron 1 deletions to define D-BP lengths that present low/no risk of mis-splicing and lengths which are critically short and likely elicit clinically relevant mis-splicing. We extend our previous observation in EMD intron 5 of 46 nt as the minimal productive D-BP length, demonstrating spliceosome assembly constraint persists at D-BP lengths of 47-56 nt. We exploit the common HBB exon 1 β-thalassemia variant that strengthens a cryptic donor (NM_000518.5(HBB):c.79G > A) to provide a simple barometer for the earliest signs of space constraint, via cryptic donor activation. For clinical evaluation of intronic deletions, we assert D-BP lengths > 60 nt present low mis-splicing risk while space constraint increases exponentially with D-BP lengths < 55 nt, with critical risk and profound splicing abnormalities with D-BP lengths < 50 nt.

Abstract 5742: Pharmacologic modulation of RNA splicing enhances anti-tumor immunity

Abstract Immune checkpoint blockade therapy has revolutionized cancer care, including the treatment of advanced metastatic disease. However, most patients derive little or no clinical benefit from these therapies and many cancer types are notoriously non-responsive. Motivated by (1) the correlation between tumor neoantigen abundance and anti-tumor immunity and (2) that most cancers are characterized by widespread dysregulation of RNA processing, we reasoned that pharmacologic modulation of RNA splicing might increase cancer cell immunogenicity via the generation of splicing-derived neoantigens. We demonstrated that two compounds which modulate RNA splicing via distinct mechanisms, inhibited tumor growth and enhanced response to immune checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Critical for their clinical translatability, therapeutic doses of splicing inhibitors were non-toxic, tolerated by the host immune system, and did not affect T cell activation, proliferation, and anti-cancer killing activities. Mechanistically, splicing modulation induced stereotyped, dose-dependent “splicing failure” — dramatic intron retention, alternative exon skipping, etc. — that was consistent across multiple mouse and human tumor types. By combining RNA-seq-based peptide predictions and mass spectrometry of the MHC I-bound immunopeptidome, we identified drug-induced, splicing-derived peptides that promote the expansion of antigen-specific CD8+ T cells and trigger anti-tumor T cell responses in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic. Citation Format: James D. Thomas, Sydney X. Lu, Emma De Neef, Erich Sabio, Benoit Rousseau, Mathieu Gigoux, David A. Knorr, Benjamin Greenbaum, Yuval Elhanati, Simon J. Hogg, Andrew Chow, Arnab Ghosh, Abigail Xie, Dmitriy Zamarin, Daniel Cui, Caroline Erickson, Michael Singer, Hana Cho, Eric Wang, Bin Lu, Benjamin H. Durham, Harshal Shah, Diego Chowell, Austin M. Gabel, Yudao Shen, Jing Liu, Jian Jin, Matthew C. Rhodes, Richard E. Taylor, Henrik Molina, Jedd D. Wolchok, Taha Merghoub, Luis A. Diaz Jr, Omar Abdel-Wahab, Robert K. Bradley. Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5742.

Seismic behavior of high-performance fiber reinforced cementitious composites columns with limited reinforcement details

Many older reinforced concrete (RC) buildings were designed considering only gravity loads. The column members in such buildings could be vulnerable to earthquakes, as they have limited reinforcement details. The aim of this study was to enhance the cyclic behavior of older RC columns using high-performance fiber reinforced cementitious composites (HPFRCCs) comprising polyvinyl alcohol (PVA) fibers. Six–full scale column specimens were constructed and tested. The use of the HPFRCC improved the seismic behavior of columns and also delayed the onset of damage stares and of lap–splice failure.

Shear Behavior of FRP Connectors in Precast Sandwich Insulation Wall Panels

Glass fiber reinforced polymer (FRP) composite connectors used in precast sandwich insulation wall panels directly affect the safety of the wall. In practical applications, a precast concrete sandwich insulation wall panel is transported to the construction site for hoisting 3–5 days after steam curing, and its concrete strength typically reaches approximately 70% of the design strength (i.e., the concrete strength after natural curing for 14 days). This study investigated the natural curing of concrete for 14 days and analyzed the mechanical properties of FRP connectors with two different sections in terms of their failure mode, failure process, and load–displacement curves. Numerical analysis and finite element parametric analysis of the connectors were conducted based on experimental data. The average ultimate shear capacity of a single rectangular-section connector was 8.37 kN and that of the cross-section connector was 8.37 kN. The connectors exhibited a good shear resistance, and the rectangular-section connectors had better ductility than the cross-section connectors. The wall panel exhibited three types of failure modes: splicing failure of the fiber layer of the connector, fiber fracture in the anchorage of the connector, failure of the concrete of the anchorage, and mainly material damage of the connector itself. The error between the load simulation value and test value of a single connector was less than 10% of the numerical simulation error requirement, and the finite element simulation results were reliable. The results of the parametric simulation of the shear capacity showed that the distance between connectors, anchorage depth, and insulation layer thickness had a significant influence on the shear performance of concrete connectors.

A developmentally programmed splicing failure contributes to DNA damage response attenuation during mammalian zygotic genome activation.

Transition from maternal to embryonic transcriptional control is crucial for embryogenesis. However, alternative splicing regulation during this process remains understudied. Using transcriptomic data from human, mouse, and cow preimplantation development, we show that the stage of zygotic genome activation (ZGA) exhibits the highest levels of exon skipping diversity reported for any cell or tissue type. Much of this exon skipping is temporary, leads to disruptive noncanonical isoforms, and occurs in genes enriched for DNA damage response in the three species. Two core spliceosomal components, Snrpb and Snrpd2, regulate these patterns. These genes have low maternal expression at ZGA and increase sharply thereafter. Microinjection of Snrpb/d2 messenger RNA into mouse zygotes reduces the levels of exon skipping at ZGA and leads to increased p53-mediated DNA damage response. We propose that mammalian embryos undergo an evolutionarily conserved, developmentally programmed splicing failure at ZGA that contributes to the attenuation of cellular responses to DNA damage.

SF3B14 is involved in the centrosome regulation through splicing of TUBGCP6 pre-mRNA

Splicing precursor messenger RNA (pre-mRNA) is a critical step to produce physiologically functional protein. Splicing failure not only gives rise to dysfunctional proteins but also generates abnormal protein function, which causes several diseases. Several pre-mRNA splicing factors are reported to regulate mitosis directly at mitotic structures and/or indirectly through controlling the pre-mRNA splicing for mitotic proteins. In this study, we described the mitotic functions of SF3B14, a component of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), which we identified as a candidate involved in mitosis based on the large-scale RNA interference (RNAi) screen of the nucleolar proteome database. We observed that SF3B14 depletion caused prolonged mitosis and several mitotic defects, such as monopolar spindle and chromosome misalignment during metaphase. Although SF3B14 was found in the nucleolar proteome database, our immunofluorescent stainings demonstrated that SF3B14 was predominantly localized in the nucleoplasm and excluded from the nucleolus during interphase. In addition, SF3B14 did not colocalize with specific mitotic structures during mitosis, which is not in line with its direct mitotic function. Notably, we found that the SF3B14 depletion reduced protein levels of TUBGCP6, required for centrosome regulation, and increased the unspliced/spliced ratio of its mRNA. Taken together, we propose that the pre-mRNA of TUBGCP6 is one of the targets for SF3B14 splicing through which SF3B14 controls mitotic chromosome behavior.

CFM6 is an Essential CRM Protein Required for the Splicing of nad5 Transcript in Arabidopsis Mitochondria.

Plant chloroplast RNA splicing and ribosome maturation (CRM)-domain-containing proteins are capable of binding RNA to facilitate the splicing of group I or II introns in chloroplasts, but their functions in mitochondria are less clear. In the present study, Arabidopsis thaliana CFM6, a protein with a single CRM domain, was expressed in most plant tissues, particularly in flower tissues, and restricted to mitochondria. Mutation of CFM6 causes severe growth defects, including stunted growth, curled leaves, delayed embryogenesis and pollen development. CFM6 functions specifically in the splicing of group II intron 4 of nad5, which encodes a subunit of mitochondrial complex I, as evidenced by the loss of nad5 intron 4 splicing and high accumulation of its pretranscripts in cfm6 mutants. The phenotypic and splicing defects of cfm6 were rescued in transgenic plants overexpressing 35S::CFM6-YFP. Splicing failure in cfm6 also led to the loss of complex I activity and to its improper assembly. Moreover, dysfunction of complex I induced the expression of proteins or genes involved in alternative respiratory pathways in cfm6. Collectively, CFM6, a previously uncharacterized CRM domain-containing protein, is specifically involved in the cis-splicing of nad5 intron 4 and plays a pivotal role in mitochondrial complex I biogenesis and normal plant growth.

RNA components of the spliceosome regulate tissue- and cancer-specific alternative splicing.

Alternative splicing of pre-mRNAs plays a pivotal role during the establishment and maintenance of human cell types. Characterizing the trans-acting regulatory proteins that control alternative splicing has therefore been the focus of much research. Recent work has established that even core protein components of the spliceosome, which are required for splicing to proceed, can nonetheless contribute to splicing regulation by modulating splice site choice. We here show that the RNA components of the spliceosome likewise influence alternative splicing decisions. Although these small nuclear RNAs (snRNAs), termed U1, U2, U4, U5, and U6 snRNA, are present in equal stoichiometry within the spliceosome, we found that their relative levels vary by an order of magnitude during development, across tissues, and across cancer samples. Physiologically relevant perturbation of individual snRNAs drove widespread gene-specific differences in alternative splicing but not transcriptome-wide splicing failure. Genes that were particularly sensitive to variations in snRNA abundance in a breast cancer cell line model were likewise preferentially misspliced within a clinically diverse cohort of invasive breast ductal carcinomas. As aberrant mRNA splicing is prevalent in many cancers, we propose that a full understanding of such dysregulated pre-mRNA processing requires study of snRNAs, as well as protein splicing factors. Together, our data show that the RNA components of the spliceosome are not merely basal factors, as has long been assumed. Instead, these noncoding RNAs constitute a previously uncharacterized layer of regulation of alternative splicing, and contribute to the establishment of global splicing programs in both healthy and malignant cells.

Experimental investigation on the deformation capacity of lap splices under cyclic loading

Correct detailing and positioning of lap splices is essential in order to prevent premature failure of reinforced concrete structural members. Especially before the introduction of capacity design guidelines, lap splices were often placed in member regions that undergo inelastic deformations under earthquake loading. When assessing the seismic performance of such members, not only the lap splice strength, which was assessed in previous studies, but also information on the deformation capacity of lap splices is required. This paper analyses the results of a recently concluded experimental programme on spliced RC wall boundary elements tested under uniaxial tension–compression cyclic loading. The study aimed at investigating the influence of lap splice length, confining reinforcement and loading history on the deformation capacity of lap splices. The latter is defined as the average strain, at the onset of splice failure, ascribed to deformations originating from the lap splice zone. Analysis of the test results showed that the deformation capacity of lap splices: (1) increases with lap splice length; (2) increases with confining reinforcement but the effectiveness of the confining reinforcement is dependent on the lap splice length; (3) decreases with larger imposed compression levels; (4) is larger for bottom-casted with respect to top-casted lap splices. Finally, an empirical model is proposed to estimate the strain capacity of lap splices, which provides a good fit with the experimental results.

Spt5 modulates cotranscriptional spliceosome assembly in Saccharomyces cerevisiae.

There is increasing evidence from yeast to humans that pre-mRNA splicing occurs mainly cotranscriptionally, such that splicing and transcription are functionally coupled. Currently, there is little insight into the contribution of the core transcription elongation machinery to cotranscriptional spliceosome assembly and pre-mRNA splicing. Spt5 is a member of the core transcription elongation machinery and an essential protein, whose absence in budding yeast causes defects in pre-mRNA splicing. To determine how Spt5 affects pre-mRNA splicing, we used the auxin-inducible degron system to conditionally deplete Spt5 in Saccharomyces cerevisiae and assayed effects on cotranscriptional spliceosome assembly and splicing. We show that Spt5 is needed for efficient splicing and for the accumulation of U5 snRNPs at intron-containing genes, and therefore for stable cotranscriptional assembly of spliceosomes. The defect in cotranscriptional spliceosome assembly can explain the relatively mild splicing defect as being a consequence of the failure of cotranscriptional splicing. Coimmunoprecipitation of Spt5 with core spliceosomal proteins and all spliceosomal snRNAs suggests a model whereby Spt5 promotes cotranscriptional pre-mRNA splicing by stabilizing the association of U5 snRNP with spliceosome complexes as they assemble on the nascent transcript. If this phenomenon is conserved in higher eukaryotes, it has the potential to be important for cotranscriptional regulation of alternative splicing.

CYCLIC BEHAVIOUR OF LIGHTLY REINFORCED CONCRETE COLUMNS WITH NON-DUCTILE LAP SPLICES

Experimental testing of lightly reinforced concrete column was conducted to investigate the collapse behavior of such column under cyclic lateral loading. Six column specimens, which have low longitudinal reinforcement and lack of confinement, were detailed with no lap splice, and non-ductile lap splice within or outside critical region. Placing the short, unconfined column's lap splice within critical region caused peak moment to fall short under its nominal moment capacity. In contrast, moment capacity of the specimen containing non-ductile lap splice outside critical region was in close agreement with those of specimen without lap splice. However, its inelastic damage region was moving away from the beam-column interface, resulted in degradation of drift capacity and rapid degradation of lateral strength. The presence of non-ductile lap splice outside critical region also potentially shift column's collapse mechanism from flexure to flexure-shear critical. The ability of lightly reinforced concrete columns to maintain its axial load carrying capacity to large drift ratios despite heavy damage and significant loss of lateral load carrying capacity indicates that lap splice failure does not create sudden collapse hazard.

Forensic Engineering Analysis of an Electrical Substation Fire in a Manufacturing Plant in Brazil

A cable splice failure in one of the cables associated with one of the 6 MVAR capacitor banks in an electrical substation at a manufacturing plant in South America caused a fire in the 88/4.16kV electrical substation. The fire caused the plant to stop production for approximately 29 days while temporary repairs were made. Operating two shifts per day/seven days a week, and stopping for maintenance once a year, the manufacturing production generates approximately $750,000 in revenue per day. The cable splice failure caused an electrical short circuit in the substation 4.16kV distribution system for approximately 120 seconds. The cable splice failure ignited the adjacent cables in the cable tray, causing damage to various sections of the 4.16kV cables, three 88kV disconnect switches, and four 88kV – 4.16kV transformers. The cable fire in the electrical substation resulted in property damages and business interruption losses with an estimated value of $20 million. The four 88kV – 4.16kV transformers that were in service at the time of the substation fire were exposed to voltage transients and electromagnetic forces produced by the short-circuit currents for approximately120 seconds.

Experimental Research on Tension Lap Splice in Reactive Powder Concrete Beams Exposed to Repeated Loading

This research is a part of an experimental study to examine the effect of lap splicing tension steel bars reactive powder concrete (RPC) beams under repeated loads. Eight RPCbeams whose tension steel bars were spliced at mid-span for a length equals 20 times the bar diameter and one RPC beam without lap splice were casted and tested. These beams were simply supported and tested up to failure under the action of two point repeated loads. The studied parameters were: the steel fiber volumetric ratio (1.5%,1.75% and2%), diameter of tension steel bars (12mm, 16mm and 20mm) and the repeated loading regime in which three types of loading were used depending on the minimum to maximum ratio of the applied load. The first loading regime with ratio of 0% with 0 kN for the minimum load while the maximum was the load beyond that causes yielding of steel bars and this is determined from the previous monotonic load test. The second type with 27% ratio (30 kN for the minimum and 105-110 kN for the maximum). The last type was with 20% ratio (the minimum12 kN and the maximum 60 kN).It should be mentioned that 10mm bar diameter was used to the top reinforcement and stirrups for all beams. The mid-span deflection as well as cracks propagation were recorded for each beam throughout the test. The main results showed that the adopted spliced length of tension steel bars was sufficient in monotonic load but insufficient under the action of high number of cycles of the repeated load. In addition, there were beams of splice failure that having low steel fiber ratio or larger diameter of tension steel bars.

Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome.

Many COL4A5 splice region variants have been described in patients with X-linked Alport syndrome, but few have been confirmed by functional analysis to actually cause defective splicing. We sought to demonstrate that a novel COL4A5 splice region variant in a family with Alport syndrome is pathogenic using functional studies. We also describe an alternative method of diagnosis. Targeted next-generation sequencing results of an individual with Alport syndrome were analyzed and the results confirmed by Sanger sequencing in family members. A splicing reporter minigene assay was used to examine the variant's effect on splicing in transfected cells. Plucked hair follicles from patients and controls were examined for collagen IV proteins using immunofluorescence microscopy. A novel splice region mutation in COL4A5, c.1780-6T>G, was identified and segregated with disease in this family. This variant caused frequent skipping of exon 25, resulting in a frameshift and truncation of collagen α5(IV) protein. We also developed and validated a new approach to characterize the expression of collagen α5(IV) protein in the basement membranes of plucked hair follicles. Using this approach we demonstrated reduced collagen α5(IV) protein in affected male and female individuals in this family, supporting frequent failure of normal splicing. Differing normal to abnormal transcript ratios in affected individuals carrying splice region variants may contribute to variable disease severity observed in Alport families. Examination of plucked hair follicles in suspected X-linked Alport syndrome patients may offer a less invasive alternative method of diagnosis and serve as a pathogenicity test for COL4A5 variants of uncertain significance.

Association of New Mutant Allele, w4‐nw, at W4 Locus with Near‐White Flower Color in Soybean

An ethyl methanesulfonate‐induced mutant (PE1033) with near‐white flowers was isolated from the soybean [Glycine max (L.) Merr.] cultivar Pungsannamul with purple flowers that had the W1w3W4 genotype. In the present study, we aimed to investigate the genetic and molecular bases of the flower color variation displayed by the PE1033 mutant. Analyses of genomic sequences and reverse transcription–polymerase chain reaction (RT‐PCR) revealed that the color variation of PE1033 flowers originated from a recessive mutation in the W4 locus that encodes dihydroflavonol 4‐reductase (DFR) 2. The new mutant allele, named w4‐nw, harbored a single‐nucleotide substitution (G to A) at the 5′ spliceosome recognition site in the first intron of DFR2, resulting in the failure of splicing and producing two different abnormal transcripts: w4‐nw‐a and w4‐nw‐b. The former encoded a truncated DFR2 protein because of the occurrence of a premature stop codon in the first intron, which was retained in the transcript; the latter resulted from alternative splicing of the first intron and encoded a DFR2 protein that lacked 11 amino acid residues in the N‐terminus. Derived cleaved amplified polymorphism sequence (dCAPS) analysis revealed a tight cosegregation pattern between the w4‐nw mutant allele and near‐white color phenotype. In conclusion, the novel mutation in the W4 locus, w4‐nw, caused the production of near‐white flowers under the w3 allelic background of PE1033.

Collapse Assessment of the Alto Rio Building in the 2010 Chile Earthquake

The 2010 Mw = 8.8 earthquake in Chile subjected thousands of modern buildings to strong shaking. The 15-story, residential, Torre Alto Rio building was the only modern reinforced concrete building over ten stories to collapse. Since the concrete building code used in Chile is based on ACI 318-95, it is a valuable exercise to assess the performance of this building and to identify possible reasons for collapse. Potential reasons for collapse were studied using post-earthquake observed damage, structural drawings, and nonlinear static and dynamic response analyses. Analysis results indicate that collapse was likely influenced by a number of factors, including flexural-compression failure at the web boundary of T-shaped walls below discontinuities on the east side of the building, shear damage in walls across the central corridor at the first level, and tensile fracture and splice failure of the wall boundary and web vertical reinforcement at the ground line on the west side of the building.

Abstract 18382: Rna Splicing Regulated by A2bp1 is Essential for Cardiac Function in Zebrafish

Objective: Alternative splicing (AS) is one of the key mechanisms for the proteomic and functional diversity of eukaryotes. However, the complex nature of AS, its associated regulators and their targets are only partially understood. In the present study we investigated the transcriptomic diversity in the zebrafish heart using RNA-Sequencing and elucidated the functional role of the splicing regulator A2BP1 in vivo. Results: Using RNA-Sequencing we characterized the cardiac transcriptome of 48 hours post fertilization (hpf) old zebrafish embryos and compared the expression of genes and their isoforms to whole fish tissue. Besides the known cardiac genes, we found several previously described genes, highly expressed in cardiac tissue. The analysis of RNA-Seq data indicates that 14% of all genes expressed in the heart undergo AS by single exon-skipping/inclusion. To determine the effect of splicing factors on mRNA splicing we investigated the functional role of splicing regulator a2bp1 in vivo by using the zebrafish as a model organism. Morpholino-mediated a2bp1 knockdown in zebrafish embryos led to progressive cardiac contractile dysfunction, suggesting an important role of a2bp1 in maintenance of cardiac function. Splicing analysis revealed that loss of a2bp1 does not result in a completely splicing failure, but rather alters the splicing pattern of specific target genes. Here we identified novel spliceforms and potentialy novel targets of splicing factor a2bp1. Splice-junction blockage experiments showed that a balanced isoform expression of the targets actn3a, hug, ktn1, ptpla and camk2g is necessary for maintaining cardiac function in zebrafish. We assume, that the a2bp1-knockdown phenotype is not caused by missplicing of specific targets rather by the cumulative effect of many splicing abnormalities. Conclusion: Our study reveal a novel splicing regulator that is necessary for normal heart function. We showed that dysfunction of a2bp1 not only leads to heart failure, but show that a2bp1 mediates the splicing of different transcripts which might mediate the observed phenotype. Our results highlight the importance of balanced mRNA splicing in the heart and represents intriguing opportunities for novel therapeutic approaches.

U2AF1 mutations alter splice site recognition in hematological malignancies.

Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3′ splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3′ splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1’s zinc finger domains.