Hagfish genome elucidates vertebrate whole-genome duplication events and their evolutionary consequences.

Polyploidy or whole-genome duplication (WGD) is a major event that drastically reshapes genome architecture and is often assumed to be causally associated with organismal innovations and radiations. The 2R hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution. However, the timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved and whether these WGD events underlie vertebrate phenotypic diversification remains elusive. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and 2R occurred in the gnathostome stem-lineage, maximally in the late Cambrian-earliest Ordovician, after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced an additional independent genome triplication. Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only in the gnathostome but not in the cyclostome lineage, calling into question the general expectation that WGDs lead to leaps of bodyplan complexity.

SF3B1 mutation-mediated sensitization to H3B-8800 splicing inhibitor in chronic lymphocytic leukemia.

Splicing factor 3B subunit 1 (SF3B1) is involved in pre-mRNA branch site recognition and is the target of antitumor-splicing inhibitors. Mutations in SF3B1 are observed in 15% of patients with chronic lymphocytic leukemia (CLL) and are associated with poor prognosis, but their pathogenic mechanisms remain poorly understood. Using deep RNA-sequencing data from 298 CLL tumor samples and isogenic SF3B1 WT and K700E-mutated CLL cell lines, we characterize targets and pre-mRNA sequence features associated with the selection of cryptic 3' splice sites upon SF3B1 mutation, including an event in the MAP3K7 gene relevant for activation of NF-κB signaling. Using the H3B-8800 splicing modulator, we show, for the first time in CLL, cytotoxic effects in vitro in primary CLL samples and in SF3B1-mutated isogenic CLL cell lines, accompanied by major splicing changes and delayed leukemic infiltration in a CLL xenotransplant mouse model. H3B-8800 displayed preferential lethality towards SF3B1-mutated cells and synergism with the BCL2 inhibitor venetoclax, supporting the potential use of SF3B1 inhibitors as a novel therapeutic strategy in CLL.

Human EWS-FLI protein recapitulates in Drosophila the neomorphic functions that induce Ewing sarcoma tumorigenesis.

Ewing sarcoma (EwS) is a human malignant tumor typically driven by the Ewing sarcoma-Friend leukemia integration (EWS-FLI) fusion protein. A paucity of genetically modified animal models, partially owed to the high toxicity of EWS-FLI, hinders research on EwS. Here, we report a spontaneous mutant variant, EWS-FLI1FS, that circumvents the toxicity issue in Drosophila. Through proteomic and genomic analyses, we show that human EWS-FLI1FS interacts with the Drosophila homologues of EWS-FLI human protein partners, including core subunits of chromatin remodeling complexes, the transcription machinery, and the spliceosome; brings about a massive dysregulation of transcription that affects a significant fraction of known targets of EWS-FLI in human cells; and modulates splicing. We also show that EWS-FLI1FS performs in Drosophila the two major neomorphic activities that it is known to have in human cells: activation of transcription from GGAA microsatellites and out competition of ETS transcription factors. We conclude that EWS-FLI1FS reproduces in Drosophila the known oncogenic activities of EWS-FLI that drive EwS tumorigenesis in humans. These results open up an unprecedented opportunity to investigate EWS-FLI's oncogenic pathways in vivo in a genetically tractable organism.

Huntington's disease-specific mis-splicing unveils key effector genes and altered splicing factors.

Correction of mis-splicing events is a growing therapeutic approach for neurological diseases such as spinal muscular atrophy or neuronal ceroid lipofuscinosis 7, which are caused by splicing-affecting mutations. Mis-spliced effector genes that do not harbour mutations are also good candidate therapeutic targets in diseases with more complex aetiologies such as cancer, autism, muscular dystrophies or neurodegenerative diseases. Next-generation RNA sequencing (RNA-seq) has boosted investigation of global mis-splicing in diseased tissue to identify such key pathogenic mis-spliced genes. Nevertheless, while analysis of tumour or dystrophic muscle biopsies can be informative on early stage pathogenic mis-splicing, for neurodegenerative diseases, these analyses are intrinsically hampered by neuronal loss and neuroinflammation in post-mortem brains. To infer splicing alterations relevant to Huntington's disease pathogenesis, here we performed intersect-RNA-seq analyses of human post-mortem striatal tissue and of an early symptomatic mouse model in which neuronal loss and gliosis are not yet present. Together with a human/mouse parallel motif scan analysis, this approach allowed us to identify the shared mis-splicing signature triggered by the Huntington's disease-causing mutation in both species and to infer upstream deregulated splicing factors. Moreover, we identified a plethora of downstream neurodegeneration-linked mis-spliced effector genes that-together with the deregulated splicing factors-become new possible therapeutic targets. In summary, here we report pathogenic global mis-splicing in Huntington's disease striatum captured by our new intersect-RNA-seq approach that can be readily applied to other neurodegenerative diseases for which bona fide animal models are available.

Silencing of SRRM4 suppresses microexon inclusion and promotes tumor growth across cancers.

RNA splicing is widely dysregulated in cancer, frequently due to altered expression or activity of splicing factors (SFs). Microexons are extremely small exons (3-27 nucleotides long) that are highly evolutionarily conserved and play critical roles in promoting neuronal differentiation and development. Inclusion of microexons in mRNA transcripts is mediated by the SF Serine/Arginine Repetitive Matrix 4 (SRRM4), whose expression is largely restricted to neural tissues. However, microexons have been largely overlooked in prior analyses of splicing in cancer, as their small size necessitates specialized computational approaches for their detection. Here, we demonstrate that despite having low expression in normal nonneural tissues, SRRM4 is further silenced in tumors, resulting in the suppression of normal microexon inclusion. Remarkably, SRRM4 is the most consistently silenced SF across all tumor types analyzed, implying a general advantage of microexon down-regulation in cancer independent of its tissue of origin. We show that this silencing is favorable for tumor growth, as decreased SRRM4 expression in tumors is correlated with an increase in mitotic gene expression, and up-regulation of SRRM4 in cancer cell lines dose-dependently inhibits proliferation in vitro and in a mouse xenograft model. Further, this proliferation inhibition is accompanied by induction of neural-like expression and splicing patterns in cancer cells, suggesting that SRRM4 expression shifts the cell state away from proliferation and toward differentiation. We therefore conclude that SRRM4 acts as a proliferation brake, and tumors gain a selective advantage by cutting off this brake.

Cooperative epithelial phagocytosis enables error correction in the early embryo.

Errors in early embryogenesis are a cause of sporadic cell death and developmental failure1,2. Phagocytic activity has a central role in scavenging apoptotic cells in differentiated tissues3-6. However, how apoptotic cells are cleared in the blastula embryo in the absence of specialized immune cells remains unknown. Here we show that the surface epithelium of zebrafish and mouse embryos, which is the first tissue formed during vertebrate development, performs efficient phagocytic clearance of apoptotic cells through phosphatidylserine-mediated target recognition. Quantitative four-dimensional in vivo imaging analyses reveal a collective epithelial clearance mechanism that is based on mechanical cooperation by two types of Rac1-dependent basal epithelial protrusions. The first type of protrusion, phagocytic cups, mediates apoptotic target uptake. The second, a previously undescribed type of fast and extended actin-based protrusion that we call 'epithelial arms', promotes the rapid dispersal of apoptotic targets through Arp2/3-dependent mechanical pushing. On the basis of experimental data and modelling, we show that mechanical load-sharing enables the long-range cooperative uptake of apoptotic cells by multiple epithelial cells. This optimizes the efficiency of tissue clearance by extending the limited spatial exploration range and local uptake capacity of non-motile epithelial cells. Our findings show that epithelial tissue clearance facilitates error correction that is relevant to the developmental robustness and survival of the embryo, revealing the presence of an innate immune function in the earliest stages of embryonic development.

Ancient Genomic Regulatory Blocks Are a Source for Regulatory Gene Deserts in Vertebrates after Whole-Genome Duplications.

We investigated how the two rounds of whole-genome duplication that occurred at the base of the vertebrate lineage have impacted ancient microsyntenic associations involving developmental regulators (known as genomic regulatory blocks, GRBs). We showed that the majority of GRBs identified in the last common ancestor of chordates have been maintained as a single copy in humans. We found evidence that dismantling of the duplicated GRB copies occurred early in vertebrate evolution often through the differential retention of the regulatory gene but loss of the bystander gene's exonic sequences. Despite the large evolutionary scale, the presence of duplicated highly conserved noncoding regions provided unambiguous proof for this scenario for multiple ancient GRBs. Remarkably, the dismantling of ancient GRB duplicates has contributed to the creation of large gene deserts associated with regulatory genes in vertebrates, providing a potentially widespread mechanism for the origin of these enigmatic genomic traits.

Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.

Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.

Retention of paternal DNA methylome in the developing zebrafish germline.

Two waves of DNA methylation reprogramming occur during mammalian embryogenesis; during preimplantation development and during primordial germ cell (PGC) formation. However, it is currently unclear how evolutionarily conserved these processes are. Here we characterise the DNA methylomes of zebrafish PGCs at four developmental stages and identify retention of paternal epigenetic memory, in stark contrast to the findings in mammals. Gene expression profiling of zebrafish PGCs at the same developmental stages revealed that the embryonic germline is defined by a small number of markers that display strong developmental stage-specificity and that are independent of DNA methylation-mediated regulation. We identified promoters that are specifically targeted by DNA methylation in somatic and germline tissues during vertebrate embryogenesis and that are frequently misregulated in human cancers. Together, these detailed methylome and transcriptome maps of the zebrafish germline provide insight into vertebrate DNA methylation reprogramming and enhance our understanding of the relationships between germline fate acquisition and oncogenesis.

A novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing.

The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies.

Molecular basis of differential 3' splice site sensitivity to anti-tumor drugs targeting U2 snRNP.

Several splicing-modulating compounds, including Sudemycins and Spliceostatin A, display anti-tumor properties. Combining transcriptome, bioinformatic and mutagenesis analyses, we delineate sequence determinants of the differential sensitivity of 3' splice sites to these drugs. Sequences 5' from the branch point (BP) region strongly influence drug sensitivity, with additional functional BPs reducing, and BP-like sequences allowing, drug responses. Drug-induced retained introns are typically shorter, displaying higher GC content and weaker polypyrimidine-tracts and BPs. Drug-induced exon skipping preferentially affects shorter alternatively spliced regions with weaker BPs. Remarkably, structurally similar drugs display both common and differential effects on splicing regulation, SSA generally displaying stronger effects on intron retention, and Sudemycins more acute effects on exon skipping. Collectively, our results illustrate how splicing modulation is exquisitely sensitive to the sequence context of 3' splice sites and to small structural differences between drugs.

The alternative splicing factor Nova2 regulates vascular development and lumen formation.

Vascular lumen formation is a fundamental step during angiogenesis; yet, the molecular mechanisms underlying this process are poorly understood. Recent studies have shown that neural and vascular systems share common anatomical, functional and molecular similarities. Here we show that the organization of endothelial lumen is controlled at the post-transcriptional level by the alternative splicing (AS) regulator Nova2, which was previously considered to be neural cell-specific. Nova2 is expressed during angiogenesis and its depletion disrupts vascular lumen formation in vivo. Similarly, Nova2 depletion in cultured endothelial cells (ECs) impairs the apical distribution and the downstream signalling of the Par polarity complex, resulting in altered EC polarity, a process required for vascular lumen formation. These defects are linked to AS changes of Nova2 target exons affecting the Par complex and its regulators. Collectively, our results reveal that Nova2 functions as an AS regulator in angiogenesis and is a novel member of the 'angioneurins' family.

An alternative splicing event amplifies evolutionary differences between vertebrates.

Alternative splicing (AS) generates extensive transcriptomic and proteomic complexity. However, the functions of species- and lineage-specific splice variants are largely unknown. Here we show that mammalian-specific skipping of polypyrimidine tract-binding protein 1 (PTBP1) exon 9 alters the splicing regulatory activities of PTBP1 and affects the inclusion levels of numerous exons. During neurogenesis, skipping of exon 9 reduces PTBP1 repressive activity so as to facilitate activation of a brain-specific AS program. Engineered skipping of the orthologous exon in chicken cells induces a large number of mammalian-like AS changes in PTBP1 target exons. These results thus reveal that a single exon-skipping event in an RNA binding regulator directs numerous AS changes between species. Our results further suggest that these changes contributed to evolutionary differences in the formation of vertebrate nervous systems.

Pneumatosis intestinalis and volvulus: a rare association / Pneumatose intestinal e volvo: uma associação rara

Pneumatosis intestinalis (PI) is a rare condition, especially when associated with volvulus; it is often misdiagnosed and inappropriately treated. We present the case of a 27 year-old woman suffering from an acute abdomen. An abdominal tomography was performed revealing Pneumatosis intestinalis. Once in the operating theatre sigmoid volvulus was diagnosed and Hartmann surgery performed. Histology showed intestinal ischemia. During the hospital stay, evolution was favourable. The authors present this case and a brief theoretical review, due to its rarity and clinical interest.

A pneumatose intestinal (PI) é uma condição pouco frequente, sendo ainda mais rara em associação com volvo; sendo muitas vezes mal diagnosticada e tratada inapropriadamente. Apresentamos o caso de uma mulher de 27 anos com um quadro de abdómen agudo. Realizou TAC abdominal que demonstrou pneumatose intestinal. Intra-operatoriamente foi diagnosticado volvo da sigmoideia e optado por cirurgia de Hartmann. O resultado anatomo-patológico da peça foi compatível com isquémia intestinal. Durante o internamento hospital, a doente evoluiu favoravelmente. Os autores apresentam este caso e uma breve revisão teórica, pela sua raridade e interesse clínico.

Metástase óssea como forma de apresentação de carcinoma hepatocelular / Bone metastasis as the presenting symptom of hepatocellular carcinoma

O carcinoma hepatocelular (CHC) é o tumor maligno primário do fígado mais frequente, apresentando na maioria das vezes em doentes cirróticos. O espectro de apresentação é muito variado e as manifestações clínicas dependem da fase evolutiva da doença. A progressão local e sistémica do carcinoma hepatocelular é frequente e as metástases ósseas não são incomuns. Apesar de as metástases ósseas serem uma forma de apresentação rara de carcinoma hepatocelular, por vezes estas precedem as manifestações hepáticas pelo que o carcinoma hepatocelular deve ser incluído no diagnóstico diferencial de lesões ósseas osteolíticas. Os autores apresentam um caso clínico e uma breve revisão teórica, pela sua raridade e importância clínica, sublinhando a importância do diagnóstico diferencial de carcinoma hepatocelular um doente previamente assintomático, sem doença hepática conhecida anteriormente, com um fractura patológica...

Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver, presenting most often in cirrhotic patients. The spectrum of presentation is very varied and clinical manifestations depend on the phase of the disease. The local and systemic progression of hepatocellular carcinoma is frequent and bone metastases are not uncommon. Although bone metastases are a rare form of presentation of hepatocellular carcinoma, sometimes they precede hepatic manifestations and that's way hepatocellular carcinoma should be included in the differential diagnosis of osteolytic bone lesions. The authors present a case report and a brief literature review, due to its rarity and clinical importance, stressing the importance of the differential diagnosis of hepatocellular carcinoma in a previously healthy patient without previously known liver disease, with a pathological fracture...

Widespread intron retention in mammals functionally tunes transcriptomes.

Alternative splicing (AS) of precursor RNAs is responsible for greatly expanding the regulatory and functional capacity of eukaryotic genomes. Of the different classes of AS, intron retention (IR) is the least well understood. In plants and unicellular eukaryotes, IR is the most common form of AS, whereas in animals, it is thought to represent the least prevalent form. Using high-coverage poly(A)(+) RNA-seq data, we observe that IR is surprisingly frequent in mammals, affecting transcripts from as many as three-quarters of multiexonic genes. A highly correlated set of cis features comprising an "IR code" reliably discriminates retained from constitutively spliced introns. We show that IR acts widely to reduce the levels of transcripts that are less or not required for the physiology of the cell or tissue type in which they are detected. This "transcriptome tuning" function of IR acts through both nonsense-mediated mRNA decay and nuclear sequestration and turnover of IR transcripts. We further show that IR is linked to a cross-talk mechanism involving localized stalling of RNA polymerase II (Pol II) and reduced availability of spliceosomal components. Collectively, the results implicate a global checkpoint-type mechanism whereby reduced recruitment of splicing components coupled to Pol II pausing underlies widespread IR-mediated suppression of inappropriately expressed transcripts.

A global regulatory mechanism for activating an exon network required for neurogenesis.

The vertebrate and neural-specific Ser/Arg (SR)-related protein nSR100/SRRM4 regulates an extensive program of alternative splicing with critical roles in nervous system development. However, the mechanism by which nSR100 controls its target exons is poorly understood. We demonstrate that nSR100-dependent neural exons are associated with a unique configuration of intronic cis-elements that promote rapid switch-like regulation during neurogenesis. A key feature of this configuration is the insertion of specialized intronic enhancers between polypyrimidine tracts and acceptor sites that bind nSR100 to potently activate exon inclusion in neural cells while weakening 3' splice site recognition and contributing to exon skipping in nonneural cells. nSR100 further operates by forming multiple interactions with early spliceosome components bound proximal to 3' splice sites. These multifaceted interactions achieve dominance over neural exon silencing mediated by the splicing regulator PTBP1. The results thus illuminate a widespread mechanism by which a critical neural exon network is activated during neurogenesis.

Distinct types of disorder in the human proteome: functional implications for alternative splicing.

Intrinsically disordered regions have been associated with various cellular processes and are implicated in several human diseases, but their exact roles remain unclear. We previously defined two classes of conserved disordered regions in budding yeast, referred to as "flexible" and "constrained" conserved disorder. In flexible disorder, the property of disorder has been positionally conserved during evolution, whereas in constrained disorder, both the amino acid sequence and the property of disorder have been conserved. Here, we show that flexible and constrained disorder are widespread in the human proteome, and are particularly common in proteins with regulatory functions. Both classes of disordered sequences are highly enriched in regions of proteins that undergo tissue-specific (TS) alternative splicing (AS), but not in regions of proteins that undergo general (i.e., not tissue-regulated) AS. Flexible disorder is more highly enriched in TS alternative exons, whereas constrained disorder is more highly enriched in exons that flank TS alternative exons. These latter regions are also significantly more enriched in potential phosphosites and other short linear motifs associated with cell signaling. We further show that cancer driver mutations are significantly enriched in regions of proteins associated with TS and general AS. Collectively, our results point to distinct roles for TS alternative exons and flanking exons in the dynamic regulation of protein interaction networks in response to signaling activity, and they further suggest that alternatively spliced regions of proteins are often functionally altered by mutations responsible for cancer.

Tissue-specific alternative splicing remodels protein-protein interaction networks.

Alternative splicing plays a key role in the expansion of proteomic and regulatory complexity, yet the functions of the vast majority of differentially spliced exons are not known. In this study, we observe that brain and other tissue-regulated exons are significantly enriched in flexible regions of proteins that likely form conserved interaction surfaces. These proteins participate in significantly more interactions in protein-protein interaction (PPI) networks than other proteins. Using LUMIER, an automated PPI assay, we observe that approximately one-third of analyzed neural-regulated exons affect PPIs. Inclusion of these exons stimulated and repressed different partner interactions at comparable frequencies. This assay further revealed functions of individual exons, including a role for a neural-specific exon in promoting an interaction between Bridging Integrator 1 (Bin1)/Amphiphysin II and Dynamin 2 (Dnm2) that facilitates endocytosis. Collectively, our results provide evidence that regulated alternative exons frequently remodel interactions to establish tissue-dependent PPI networks.