A mitochondrial surveillance mechanism activated by SRSF2 mutations in hematologic malignancies.

Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.

A mitochondrial surveillance mechanism activated by SRSF2 mutations in hematologic malignancies.

Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.

MAJIQlopedia: an encyclopedia of RNA splicing variations in human tissues and cancer.

Quantification of RNA splicing variations based on RNA-Sequencing can reveal tissue- and disease-specific splicing patterns. To study such splicing variations, we introduce MAJIQlopedia, an encyclopedia of splicing variations that encompasses 86 human tissues and 41 cancer datasets. MAJIQlopedia reports annotated and unannotated splicing events for a total of 486 175 alternative splice junctions in normal tissues and 338 317 alternative splice junctions in cancer. This database, available at https://majiq.biociphers.org/majiqlopedia/, includes a user-friendly interface that provides graphical representations of junction usage quantification for each junction across all tissue or cancer types. To demonstrate case usage of MAJIQlopedia, we review splicing variations in genes WT1, MAPT and BIN1, which all have known tissue or cancer-specific splicing variations. We also use MAJIQlopedia to highlight novel splicing variations in FDX1 and MEGF9 in normal tissues, and we uncover a novel exon inclusion event in RPS6KA6 that only occurs in two cancer types. Users can download the database, request the addition of data to the webtool, or install a MAJIQlopedia server to integrate proprietary data. MAJIQlopedia can serve as a reference database for researchers seeking to understand what splicing variations exist in genes of interest, and those looking to understand tissue- or cancer-specific splice isoform usage.

Cryptic MHC-E epitope from influenza elicits a potent cytolytic T cell response.

The extent to which unconventional forms of antigen presentation drive T cell immunity is unknown. By convention, CD8 T cells recognize viral peptides, or epitopes, in association with classical major histocompatibility complex (MHC) class I, or MHC-Ia, but immune surveillance can, in some cases, be directed against peptides presented by nonclassical MHC-Ib, in particular the MHC-E proteins (Qa-1 in mice and HLA-E in humans); however, the overall importance of nonclassical responses in antiviral immunity remains unclear. Similarly uncertain is the importance of 'cryptic' viral epitopes, defined as those undetectable by conventional mapping techniques. Here we used an immunopeptidomic approach to search for unconventional epitopes that drive T cell responses in mice infected with influenza virus A/Puerto Rico/8/1934. We identified a nine amino acid epitope, termed M-SL9, that drives a co-immunodominant, cytolytic CD8 T cell response that is unconventional in two major ways: first, it is presented by Qa-1, and second, it has a cryptic origin, mapping to an unannotated alternative reading frame product of the influenza matrix gene segment. Presentation and immunogenicity of M-SL9 are dependent on the second AUG codon of the positive sense matrix RNA segment, suggesting translation initiation by leaky ribosomal scanning. During influenza virus A/Puerto Rico/8/1934 infection, M-SL9-specific T cells exhibit a low level of egress from the lungs and strong differentiation into tissue-resident memory cells. Importantly, we show that M-SL9/Qa-1-specific T cells can be strongly induced by messenger RNA vaccination and that they can mediate antigen-specific cytolysis in vivo. Our results demonstrate that noncanonical translation products can account for an important fraction of the T cell repertoire and add to a growing body of evidence that MHC-E-restricted T cells could have substantial therapeutic value.

A Bayesian model for unsupervised detection of RNA splicing based subtypes in cancers.

Identification of cancer sub-types is a pivotal step for developing personalized treatment. Specifically, sub-typing based on changes in RNA splicing has been motivated by several recent studies. We thus develop CHESSBOARD, an unsupervised algorithm tailored for RNA splicing data that captures "tiles" in the data, defined by a subset of unique splicing changes in a subset of patients. CHESSBOARD allows for a flexible number of tiles, accounts for uncertainty of splicing quantification, and is able to model missing values as additional signals. We first apply CHESSBOARD to synthetic data to assess its domain specific modeling advantages, followed by analysis of several leukemia datasets. We show detected tiles are reproducible in independent studies, investigate their possible regulatory drivers and probe their relation to known AML mutations. Finally, we demonstrate the potential clinical utility of CHESSBOARD by supplementing mutation based diagnostic assays with discovered splicing profiles to improve drug response correlation.

Identifying common transcriptome signatures of cancer by interpreting deep learning models.

BACKGROUND: Cancer is a set of diseases characterized by unchecked cell proliferation and invasion of surrounding tissues. The many genes that have been genetically associated with cancer or shown to directly contribute to oncogenesis vary widely between tumor types, but common gene signatures that relate to core cancer pathways have also been identified. It is not clear, however, whether there exist additional sets of genes or transcriptomic features that are less well known in cancer biology but that are also commonly deregulated across several cancer types. RESULTS: Here, we agnostically identify transcriptomic features that are commonly shared between cancer types using 13,461 RNA-seq samples from 19 normal tissue types and 18 solid tumor types to train three feed-forward neural networks, based either on protein-coding gene expression, lncRNA expression, or splice junction use, to distinguish between normal and tumor samples. All three models recognize transcriptome signatures that are consistent across tumors. Analysis of attribution values extracted from our models reveals that genes that are commonly altered in cancer by expression or splicing variations are under strong evolutionary and selective constraints. Importantly, we find that genes composing our cancer transcriptome signatures are not frequently affected by mutations or genomic alterations and that their functions differ widely from the genes genetically associated with cancer. CONCLUSIONS: Our results highlighted that deregulation of RNA-processing genes and aberrant splicing are pervasive features on which core cancer pathways might converge across a large array of solid tumor types.

Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies.

Downregulation of surface epitopes causes postimmunotherapy relapses in B-lymphoblastic leukemia (B-ALL). Here we demonstrate that mRNA encoding CD22 undergoes aberrant splicing in B-ALL. We describe the plasma membrane-bound CD22 Δex5-6 splice isoform, which is resistant to chimeric antigen receptor (CAR) T cells targeting the third immunoglobulin-like domain of CD22. We also describe splice variants skipping the AUG-containing exon 2 and failing to produce any identifiable protein, thereby defining an event that is rate limiting for epitope presentation. Indeed, forcing exon 2 skipping with morpholino oligonucleotides reduced CD22 protein expression and conferred resistance to the CD22-directed antibody-drug conjugate inotuzumab ozogamicin in vitro. Furthermore, among inotuzumab-treated pediatric patients with B-ALL, we identified one nonresponder in whose leukemic blasts Δex2 isoforms comprised the majority of CD22 transcripts. In a second patient, a sharp reduction in CD22 protein levels during relapse was driven entirely by increased CD22 exon 2 skipping. Thus, dysregulated CD22 splicing is a major mechanism of epitope downregulation and ensuing resistance to immunotherapy. SIGNIFICANCE: The mechanism(s) underlying downregulation of surface CD22 following CD22-directed immunotherapy remains underexplored. Our biochemical and correlative studies demonstrate that in B-ALL, CD22 expression levels are controlled by inclusion/skipping of CD22 exon 2. Thus, aberrant splicing of CD22 is an important driver/biomarker of de novo and acquired resistance to CD22-directed immunotherapies. See related commentary by Bourcier and Abdel-Wahab, p. 87. This article is highlighted in the In This Issue feature, p. 85.

Pharmacological activation of STING blocks SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, resulting millions of infections and deaths with few effective interventions available. Here, we demonstrate that SARS-CoV-2 evades interferon (IFN) activation in respiratory epithelial cells, resulting in a delayed response in bystander cells. Since pretreatment with IFNs can block viral infection, we reasoned that pharmacological activation of innate immune pathways could control SARS-CoV-2 infection. To identify potent antiviral innate immune agonists, we screened a panel of 75 microbial ligands that activate diverse signaling pathways and identified cyclic dinucleotides (CDNs), canonical STING agonists, as antiviral. Since CDNs have poor bioavailability, we tested the small molecule STING agonist diABZI, and found that it potently inhibits SARS-CoV-2 infection of diverse strains including variants of concern (B.1.351) by transiently stimulating IFN signaling. Importantly, diABZI restricts viral replication in primary human bronchial epithelial cells and in mice in vivo. Our study provides evidence that activation of STING may represent a promising therapeutic strategy to control SARS-CoV-2.

Alternative splicing redefines landscape of commonly mutated genes in acute myeloid leukemia.

Most genes associated with acute myeloid leukemia (AML) are mutated in less than 10% of patients, suggesting that alternative mechanisms of gene disruption contribute to this disease. Here, we find a set of splicing events that alter the expression of a subset of AML-associated genes independent of known somatic mutations. In particular, aberrant splicing triples the number of patients with reduced functional EZH2 compared with that predicted by somatic mutation alone. In addition, we unexpectedly find that the nonsense-mediated decay factor DHX34 exhibits widespread alternative splicing in sporadic AML, resulting in a premature stop codon that phenocopies the loss-of-function germline mutations observed in familial AML. Together, these results demonstrate that classical mutation analysis underestimates the burden of functional gene disruption in AML and highlight the importance of assessing the contribution of alternative splicing to gene dysregulation in human disease.

Viral-induced alternative splicing of host genes promotes influenza replication.

Viral infection induces the expression of numerous host genes that impact the outcome of infection. Here, we show that infection of human lung epithelial cells with influenza A virus (IAV) also induces a broad program of alternative splicing of host genes. Although these splicing-regulated genes are not enriched for canonical regulators of viral infection, we find that many of these genes do impact replication of IAV. Moreover, in several cases, specific inhibition of the IAV-induced splicing pattern also attenuates viral infection. We further show that approximately a quarter of the IAV-induced splicing events are regulated by hnRNP K, a host protein required for efficient splicing of the IAV M transcript in nuclear speckles. Finally, we find an increase in hnRNP K in nuclear speckles upon IAV infection, which may alter accessibility of hnRNP K for host transcripts thereby leading to a program of host splicing changes that promote IAV replication.

Alternative splicing and cancer: insights, opportunities, and challenges from an expanding view of the transcriptome.

Over the past decade there has been increased awareness of the potential role of alternative splicing in the etiology of cancer. In particular, advances in RNA-Sequencing technology and analysis has led to a wave of discoveries in the last few years regarding the causes and functional relevance of alternative splicing in cancer. Here we discuss the current understanding of the connections between splicing and cancer, with a focus on the most recent findings. We also discuss remaining questions and challenges that must be addressed in order to use our knowledge of splicing to guide the diagnosis and treatment of cancer.

Deep profiling and custom databases improve detection of proteoforms generated by alternative splicing.

Alternative pre-mRNA splicing has long been proposed to contribute greatly to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)-based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom peptide database trained on analysis of RNA-seq data with MAJIQ, an algorithm optimized to detect and quantify differential and unannotated splice junction usage. We matched tandem mass spectra acquired by data-dependent acquisition (DDA) against our custom RNA-seq based database, as well as SWISS-PROT and RefSeq databases to improve identification of splicing-derived proteoforms by 28% compared with use of the SWISS-PROT database alone. Altogether, we identified peptide evidence for 554 alternate proteoforms corresponding to 274 genes. Our increased depth and detection of proteins also allowed us to track changes in the transcriptome and proteome induced by T-cell stimulation, as well as fluctuations in protein subcellular localization. In sum, our data here confirm that use of generic databases in proteomic studies underestimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.

RNA Binding Protein CELF2 Regulates Signal-Induced Alternative Polyadenylation by Competing with Enhancers of the Polyadenylation Machinery.

The 3' UTR (UTR) of human mRNAs plays a critical role in controlling protein expression and function. Importantly, 3' UTRs of human messages are not invariant for each gene but rather are shaped by alternative polyadenylation (APA) in a cell state-dependent manner, including in response to T cell activation. However, the proteins and mechanisms driving APA regulation remain poorly understood. Here we show that the RNA-binding protein CELF2 controls APA of its own message in a signal-dependent manner by competing with core enhancers of the polyadenylation machinery for binding to RNA. We further show that CELF2 binding overlaps with APA enhancers transcriptome-wide, and almost half of 3' UTRs that undergo T cell signaling-induced APA are regulated in a CELF2-dependent manner. These studies thus reveal CELF2 to be a critical regulator of 3' UTR identity in T cells and demonstrate an additional mechanism for CELF2 in regulating polyadenylation site choice.

Spontaneous visual search during the first two years: Improvement with age but no evidence of efficient search.

Efficient visual search, wherein reaction times to acquire targets are largely independent of array size, is commonly observed in adults. Evidence for efficient search in infants may imply that selective attention to visual features is similar across development. In the current cross-sectional eye-tracking study, we examined spontaneous visual search at 6, 12, 18, and 24 months. Infants were presented with Random arrays (one target among 7, 13, or 26 pseudorandomly distributed elements) and Circle arrays (one target among 4, 7, or 13 elements arranged in a circle). Contrary to predictions, we did not find evidence of efficient search among infants. With increasing array size, time-to-target increased, the proportion of targets fixated (analogous to accuracy) decreased, and the proportion of first looks to the target decreased for both types of array (ps < .001). For Random arrays, the proportion of first looks to the target was similar to chance for all ages and array sizes; for Circle arrays, it exceeded chance for some ages and array sizes. The proportion of targets fixated and first looks to target increased with age across display types (ps < .05). We also tested adults with the same stimuli under similar conditions; the adults showed evidence of efficient visual search. Possible explanations and implications are discussed.

Structural-functional interactions of NS1-BP protein with the splicing and mRNA export machineries for viral and host gene expression.

The influenza virulence factor NS1 protein interacts with the cellular NS1-BP protein to promote splicing and nuclear export of the viral M mRNAs. The viral M1 mRNA encodes the M1 matrix protein and is alternatively spliced into the M2 mRNA, which is translated into the M2 ion channel. These proteins have key functions in viral trafficking and budding. To uncover the NS1-BP structural and functional activities in splicing and nuclear export, we performed proteomics analysis of nuclear NS1-BP binding partners and showed its interaction with constituents of the splicing and mRNA export machineries. NS1-BP BTB domains form dimers in the crystal. Full-length NS1-BP is a dimer in solution and forms at least a dimer in cells. Mutations suggest that dimerization is important for splicing. The central BACK domain of NS1-BP interacts directly with splicing factors such as hnRNP K and PTBP1 and with the viral NS1 protein. The BACK domain is also the site for interactions with mRNA export factor Aly/REF and is required for viral M mRNA nuclear export. The crystal structure of the C-terminal Kelch domain shows that it forms a ß-propeller fold, which is required for the splicing function of NS1-BP. This domain interacts with the polymerase II C-terminal domain and SART1, which are involved in recruitment of splicing factors and spliceosome assembly, respectively. NS1-BP functions are not only critical for processing a subset of viral mRNAs but also impact levels and nuclear export of a subset of cellular mRNAs encoding factors involved in metastasis and immunity.

Aberrant splicing in B-cell acute lymphoblastic leukemia.

Aberrant splicing is a hallmark of leukemias with mutations in splicing factor (SF)-encoding genes. Here we investigated its prevalence in pediatric B-cell acute lymphoblastic leukemias (B-ALL), where SFs are not mutated. By comparing these samples to normal pro-B cells, we found thousands of aberrant local splice variations (LSVs) per sample, with 279 LSVs in 241 genes present in every comparison. These genes were enriched in RNA processing pathways and encoded ∼100 SFs, e.g. hnRNPA1. HNRNPA1 3'UTR was most pervasively mis-spliced, yielding the transcript subject to nonsense-mediated decay. To mimic this event, we knocked it down in B-lymphoblastoid cells and identified 213 hnRNPA1-regulated exon usage events comprising the hnRNPA1 splicing signature in pediatric leukemia. Some of its elements were LSVs in DICER1 and NT5C2, known cancer drivers. We searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 additional genes. Seventy-seven LSVs out of 81 were confirmed using two large independent B-ALL RNA-seq datasets, and the twenty most common B-ALL drivers, including NT5C2, showed higher prevalence of aberrant splicing than of somatic mutations. Thus, post-transcriptional deregulation of SF can drive widespread changes in B-ALL splicing and likely contributes to disease pathogenesis.

Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing.

Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3-dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3-dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3-dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3ß phosphorylated these proteins in vitro RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing.

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy.

UNLABELLED: The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. SIGNIFICANCE: CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

TRAP150 interacts with the RNA-binding domain of PSF and antagonizes splicing of numerous PSF-target genes in T cells.

PSF (a.k.a. SFPQ) is a ubiquitously expressed, essential nuclear protein with important roles in DNA damage repair and RNA biogenesis. In stimulated T cells, PSF binds to and suppresses the inclusion of CD45 exon 4 in the final mRNA; however, in resting cells, TRAP150 binds PSF and prevents access to the CD45 RNA, though the mechanism for this inhibition has remained unclear. Here, we show that TRAP150 binds a region encompassing the RNA recognition motifs (RRMs) of PSF using a previously uncharacterized, 70 residue region we have termed the PSF-interacting domain (PID). TRAP150's PID directly inhibits the interaction of PSF RRMs with RNA, which is mediated through RRM2. However, interaction of PSF with TRAP150 does not appear to inhibit the dimerization of PSF with other Drosophila Behavior, Human Splicing (DBHS) proteins, which is also dependent on RRM2. Finally, we use RASL-Seq to identify ∼40 T cell splicing events sensitive to PSF knockdown, and show that for the majority of these, PSF's effect is antagonized by TRAP150. Together these data suggest a model in which TRAP150 interacts with dimeric PSF to block access of RNA to RRM2, thereby regulating the activity of PSF toward a broad set of splicing events in T cells.