Comprehensive database and evolutionary dynamics of U12-type introns.

During nuclear maturation of most eukaryotic pre-messenger RNAs and long non-coding RNAs, introns are removed through the process of RNA splicing. Different classes of introns are excised by the U2-type or the U12-type spliceosomes, large complexes of small nuclear ribonucleoprotein particles and associated proteins. We created intronIC, a program for assigning intron class to all introns in a given genome, and used it on 24 eukaryotic genomes to create the Intron Annotation and Orthology Database (IAOD). We then used the data in the IAOD to revisit several hypotheses concerning the evolution of the two classes of spliceosomal introns, finding support for the class conversion model explaining the low abundance of U12-type introns in modern genomes.

The hnRNP RALY regulates transcription and cell proliferation by modulating the expression of specific factors including the proliferation marker E2F1.

The heterogeneous nuclear ribonucleoproteins (hnRNP) form a large family of RNA-binding proteins that exert numerous functions in RNA metabolism. RALY is a member of the hnRNP family that binds poly-U-rich elements within several RNAs and regulates the expression of specific transcripts. RALY is up-regulated in different types of cancer, and its down-regulation impairs cell cycle progression. However, the RALY's role in regulating RNA levels remains elusive. Here, we show that numerous genes coding for factors involved in transcription and cell cycle regulation exhibit an altered expression in RALY-down-regulated HeLa cells, consequently causing impairments in transcription, cell proliferation, and cell cycle progression. Interestingly, by comparing the list of RALY targets with the list of genes affected by RALY down-regulation, we found an enrichment of RALY mRNA targets in the down-regulated genes upon RALY silencing. The affected genes include the E2F transcription factor family. Given its role as proliferation-promoting transcription factor, we focused on E2F1. We demonstrate that E2F1 mRNA stability and E2F1 protein levels are reduced in cells lacking RALY expression. Finally, we also show that RALY interacts with transcriptionally active chromatin in both an RNA-dependent and -independent manner and that this association is abolished in the absence of active transcription. Taken together, our results highlight the importance of RALY as an indirect regulator of transcription and cell cycle progression through the regulation of specific mRNA targets, thus strengthening the possibility of a direct gene expression regulation exerted by RALY.

Biased chromatin signatures around polyadenylation sites and exons.

Core RNA-processing reactions in eukaryotic cells occur cotranscriptionally in a chromatin context, but the relationship between chromatin structure and pre-mRNA processing is poorly understood. We observed strong nucleosome depletion around human polyadenylation sites (PAS) and nucleosome enrichment just downstream of PAS. In genes with multiple alternative PAS, higher downstream nucleosome affinity was associated with higher PAS usage, independently of known PAS motifs that function at the RNA level. Conversely, exons were associated with distinct peaks in nucleosome density. Exons flanked by long introns or weak splice sites exhibited stronger nucleosome enrichment, and incorporation of nucleosome density data improved splicing simulation accuracy. Certain histone modifications, including H3K36me3 and H3K27me2, were specifically enriched on exons, suggesting active marking of exon locations at the chromatin level. Together, these findings provide evidence for extensive functional connections between chromatin structure and RNA processing.

BMP signaling requires retromer-dependent recycling of the type I receptor.

The transforming growth factor ß (TGFß) superfamily of signaling pathways, including the bone morphogenetic protein (BMP) subfamily of ligands and receptors, controls a myriad of developmental processes across metazoan biology. Transport of the receptors from the plasma membrane to endosomes has been proposed to promote TGFß signal transduction and shape BMP-signaling gradients throughout development. However, how postendocytic trafficking of BMP receptors contributes to the regulation of signal transduction has remained enigmatic. Here we report that the intracellular domain of Caenorhabditis elegans BMP type I receptor SMA-6 (small-6) binds to the retromer complex, and in retromer mutants, SMA-6 is degraded because of its missorting to lysosomes. Surprisingly, we find that the type II BMP receptor, DAF-4 (dauer formation-defective-4), is retromer-independent and recycles via a distinct pathway mediated by ARF-6 (ADP-ribosylation factor-6). Importantly, we find that loss of retromer blocks BMP signaling in multiple tissues. Taken together, our results indicate a mechanism that separates the type I and type II receptors during receptor recycling, potentially terminating signaling while preserving both receptors for further rounds of activation.

Biochemical defects in minor spliceosome function in the developmental disorder MOPD I.

Biallelic mutations of the human RNU4ATAC gene, which codes for the minor spliceosomal U4atac snRNA, cause the developmental disorder, MOPD I/TALS. To date, nine separate mutations in RNU4ATAC have been identified in MOPD I patients. Evidence suggests that all of these mutations lead to abrogation of U4atac snRNA function and impaired minor intron splicing. However, the molecular basis of these effects is unknown. Here, we use a variety of in vitro and in vivo assays to address this question. We find that only one mutation, 124G>A, leads to significantly reduced expression of U4atac snRNA, whereas four mutations, 30G>A, 50G>A, 50G>C and 51G>A, show impaired binding of essential protein components of the U4atac/U6atac di-snRNP in vitro and in vivo. Analysis of MOPD I patient fibroblasts and iPS cells homozygous for the most common mutation, 51G>A, shows reduced levels of the U4atac/U6atac.U5 tri-snRNP complex as determined by glycerol gradient sedimentation and immunoprecipitation. In this report, we establish a mechanistic basis for MOPD I disease and show that the inefficient splicing of genes containing U12-dependent introns in patient cells is due to defects in minor tri-snRNP formation, and the MOPD I-associated RNU4ATAC mutations can affect multiple facets of minor snRNA function.

Higher oesophageal temperature at rest and during exercise in humans with patent foramen ovale.

Respiratory system cooling occurs via convective and evaporative heat loss, so right-to-left shunted blood flow through a patent foramen ovale (PFO) would not be cooled. Accordingly, we hypothesized that PFO+ subjects would have a higher core temperature than PFO- subjects due, in part, to absence of respiratory system cooling of the shunted blood and that this effect would be dependent upon the estimated PFO size and inspired air temperature. Subjects were screened for the presence and size of a PFO using saline contrast echocardiography. Thirty well-matched males (15 PFO-, 8 large PFO+, 7 small PFO+) completed cycle ergometer exercise trials on three separate days. During Trial 1, subjects completed a V̇(O2max) test. For Trials 2 and 3, randomized, subjects completed four 2.5 min stages at 25, 50, 75 and 90% of the maximum workload achieved during Trial 1, breathing either ambient air (20.6 ± 1.0°C) or cold air (1.9 ± 3.5°C). PFO+ subjects had a higher oesophageal temperature (T(oesoph)) (P < 0.05) than PFO- subjects on Trial 1. During exercise breathing cold and dry air, PFO+ subjects achieved a higher T(oesoph) than PFO- subjects (P < 0.05). Subjects with a large PFO, but not those with a small PFO, had a higher T(oesoph) than PFO- subjects (P < 0.05) during Trial 1 and increased T(oesoph) breathing cold and dry air. These data suggest that the presence and size of a PFO are associated with T(oesoph) in healthy humans but this is explained only partially by absence of respiratory system cooling of shunted blood.

New connections between splicing and human disease.

The removal by splicing of introns from the primary transcripts of most mammalian genes is an essential step in gene expression. Splicing is performed by large, complex ribonucleoprotein particles termed spliceosomes. Mammals contain two types that splice out mutually exclusive types of introns. However, the role of the minor spliceosome has been poorly studied. Recent reports have now shown that mutations in one minor spliceosomal snRNA, U4atac, are linked to a rare autosomal recessive developmental defect. In addition, very exciting recent results of exome deep-sequencing have found that recurrent, somatic, heterozygous mutations of other splicing factors occur at high frequencies in particular cancers and pre-cancerous conditions, suggesting that alterations in the core splicing machinery can contribute to tumorigenesis. Mis-splicing of crucial genes may underlie the pathologies of all of these diseases. Identifying these genes and understanding the mechanisms involved in their mis-splicing may lead to advancements in diagnosis and treatment.

Patterns of missplicing due to somatic U2AF1 mutations in myeloid neoplasms.

Recently, recurrent mutations of spliceosomal genes were frequently identified in myeloid malignancies, as well as other types of cancers. One of these spliceosomal genes, U2AF1, was affected by canonical somatic mutations in aggressive type of myeloid malignancies. We hypothesized that U2AF1 mutations causes defects of splicing (missplicing) in specific genes and that such misspliced genes might be important in leukemogenesis. We analyzed RNA deep sequencing to compare splicing patterns of 201 837 exons between the cases with U2AF1 mutations (n = 6) and wild type (n = 14). We identified different alternative splicing patterns in 35 genes comparing cells with mutant and wild-type U2AF1. U2AF1 mutations are associated with abnormal splicing of genes involved in functionally important pathways, such as cell cycle progression and RNA processing. In addition, many of these genes are somatically mutated or deleted in various cancers. Of note is that the alternative splicing patterns associated with U2AF1 mutations were associated with specific sequence signals at the affected splice sites. These novel observations support the hypothesis that U2AF1 mutations play a significant role in myeloid leukemogenesis due to selective missplicing of tumor-associated genes.

Mutations in the spliceosome machinery, a novel and ubiquitous pathway in leukemogenesis.

Myelodysplastic syndromes (MDSs) are chronic and often progressive myeloid neoplasms associated with remarkable heterogeneity in the histomorphology and clinical course. Various somatic mutations are involved in the pathogenesis of MDS. Recently, mutations in a gene encoding a spliceosomal protein, SF3B1, were discovered in a distinct form of MDS with ring sideroblasts. Whole exome sequencing of 15 patients with myeloid neoplasms was performed, and somatic mutations in spliceosomal genes were identified. Sanger sequencing of 310 patients was performed to assess phenotype/genotype associations. To determine the functional effect of spliceosomal mutations, we evaluated pre-mRNA splicing profiles by RNA deep sequencing. We identified additional somatic mutations in spliceosomal genes, including SF3B1, U2AF1, and SRSF2. These mutations alter pre-mRNA splicing patterns. SF3B1 mutations are prevalent in low-risk MDS with ring sideroblasts, whereas U2AF1 and SRSF2 mutations are frequent in chronic myelomonocytic leukemia and advanced forms of MDS. SF3B1 mutations are associated with a favorable prognosis, whereas U2AF1 and SRSF2 mutations are predictive for shorter survival. Mutations affecting spliceosomal genes that result in defective splicing are a new leukemogenic pathway. Spliceosomal genes are probably tumor suppressors, and their mutations may constitute diagnostic biomarkers that could potentially serve as therapeutic targets.

SF3B1 haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic syndromes.

Whole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

Caenorhabditis elegans SMA-10/LRIG is a conserved transmembrane protein that enhances bone morphogenetic protein signaling.

Bone morphogenetic protein (BMP) pathways control an array of developmental and homeostatic events, and must themselves be exquisitely controlled. Here, we identify Caenorhabditis elegans SMA-10 as a positive extracellular regulator of BMP-like receptor signaling. SMA-10 acts genetically in a BMP-like (Sma/Mab) pathway between the ligand DBL-1 and its receptors SMA-6 and DAF-4. We cloned sma-10 and show that it has fifteen leucine-rich repeats and three immunoglobulin-like domains, hallmarks of an LRIG subfamily of transmembrane proteins. SMA-10 is required in the hypodermis, where the core Sma/Mab signaling components function. We demonstrate functional conservation of LRIGs by rescuing sma-10(lf) animals with the Drosophila ortholog lambik, showing that SMA-10 physically binds the DBL-1 receptors SMA-6 and DAF-4 and enhances signaling in vitro. This interaction is evolutionarily conserved, evidenced by LRIG1 binding to vertebrate receptors. We propose a new role for LRIG family members: the positive regulation of BMP signaling by binding both Type I and Type II receptors.

A multimodal analysis of genomic and RNA splicing features in myeloid malignancies.

RNA splicing dysfunctions are more widespread than what is believed by only estimating the effects resulting by splicing factor mutations (SFMT) in myeloid neoplasia (MN). The genetic complexity of MN is amenable to machine learning (ML) strategies. We applied an integrative ML approach to identify co-varying features by combining genomic lesions (mutations, deletions, and copy number), exon-inclusion ratio as measure of RNA splicing (percent spliced in, PSI), and gene expression (GE) of 1,258 MN and 63 normal controls. We identified 15 clusters based on mutations, GE, and PSI. Different PSI levels were present at various extents regardless of SFMT suggesting that changes in RNA splicing were not strictly related to SFMT. Combination of PSI and GE further distinguished the features and identified PSI similarities and differences, common pathways, and expression signatures across clusters. Thus, multimodal features can resolve the complex architecture of MN and help identifying convergent molecular and transcriptomic pathways amenable to therapies.

Defects in spliceosomal machinery: a new pathway of leukaemogenesis.

Proper splicing of pre-mRNA is required for protein synthesis and therefore is a fundamental cellular function. The discovery of a variety of somatic spliceosomal mutations in haematological malignancies, including myeloid neoplasms and chronic lymphocytic leukaemia has pointed to a new leukaemogenic pathway involving spliceosomal dysfunction. Theoretically, spliceosomal mutations can lead to activation of incorrect splice sites, intron retention or aberrant alternative splicing occurring in patterns generated by mutations of individual spliceosomal proteins. Such events can produce a defective balance between protein isoforms leading to functional consequences including defective regulation of proliferation and differentiation. The observed pattern of occurrence of highly specific missense mutations, coupled with the lack of nonsense mutations and deletions, implies a gain-of-function or better gain-of-dysfunction mechanism. Incorrect splicing of downstream genes, such as tumour suppressor genes, may result in haploinsufficient expression through nonsense-mediated mRNA decay. Thus, spliceosomal mutations may, depending on the pattern of affected proteins, lead to similar functional effects on tumour suppressor genes as chromosomal deletions, epigenetic silencing or inactivating/hypomorphic mutations. The prognostic value of the most common mutations and their phenotypic association in the clinical setting is currently under investigation. It is likely that spliceosomal mutations may indicate sensitivity to spliceosome inhibitors applied in the form of a synthetic lethal approach. This review discusses the most current aspects of spliceosomal research in the context of haematological malignancies.

Antiplatelet Therapy in Acute Myocardial Infarction and Cardiogenic Shock: Insights From the National Cardiogenic Shock Initiative.

BACKGROUND: Patients presenting with acute myocardial infarction complicated by cardiogenic shock (AMI-CS) are at high risk for impaired antiplatelet activity secondary to malabsorption, systemic hypoperfusion, hypothermia, need for mechanical ventilation, and high use of analgesics. The use of antiplatelet therapy in these high-risk patients is not well studied. METHODS: Using the National Cardiogenic Shock Initiative database, we analyzed patients who presented with AMI-CS at 60 hospitals from March 2018 to December 2020. All patients were treated using a standard shock protocol. Herein, the patterns of antiplatelet use are described. RESULTS: A total of 204 patients were included in the analysis, of which 174 (85.3%) presented with ST-segment elevation myocardial infarction (STEMI). The majority (84.3%) received antiplatelet therapy before percutaneous coronary intervention (PCI); of those who received antiplatelets, 77.9% received aspirin, 55.2% received an oral P2Y12 inhibitor, and 19.2% received intravenous (IV) antiplatelet therapy. Ticagrelor was the most common P2Y12 inhibitor administered (41.9%), followed by clopidogrel (12.2%) and prasugrel (1.2%). Only 18.6% of oral antiplatelet agents were crushed. Baseline characteristics of patients who received IV vs non-IV antiplatelet agents were similar. Thrombolysis in Myocardial Infarction (TIMI) 0 flow was present in 69.6% of patients before PCI and aspiration thrombectomy was performed in 24.5% of patients. The presence of STEMI, cardiac arrest, cardiopulmonary resuscitation, hypothermia, vasopressor use, elevated lactate levels, or number of vessels treated did not influence the use of IV antiplatelet agents. CONCLUSIONS: The use of crushed and IV antiplatelet agents in AMI-CS is low. Further studies are needed in this high-risk population to assess whether more potent antiplatelet inhibition will improve outcomes.

The conserved 3' end domain of U6atac snRNA can direct U6 snRNA to the minor spliceosome.

U6 and U6atac snRNAs play analogous critical roles in the major U2-dependent and minor U12-dependent spliceosomes, respectively. Previous results have shown that most of the functional cores of these two snRNAs are either highly similar in sequence or functionally interchangeable. Thus, a mechanism must exist to restrict each snRNA to its own spliceosome. Here we show that a chimeric U6 snRNA containing the unique and highly conserved 3' end domain of U6atac snRNA is able to function in vivo in U12-dependent spliceosomal splicing. Function of this chimera required the coexpression of a modified U4atac snRNA; U4 snRNA could not substitute. Partial deletions of this element in vivo, as well as in vitro antisense experiments, showed that the 3' end domain of U6atac snRNA is necessary to direct the U4atac/U6atac.U5 tri-snRNP to the forming U12-dependent spliceosome. In vitro experiments also uncovered a role for U4atac snRNA in this targeting.

Histamine-receptor blockade reduces blood flow but not muscle glucose uptake during postexercise recovery in humans.

Skeletal muscle vasodilatation persists following a single bout of exercise and can potentially influence glucose uptake by recovering muscle. To investigate whether blood flow is a rate-limiting component in postexercise muscle glucose uptake, we tested the hypothesis that oral ingestion of H(1)- and H(2)-receptor antagonists, known to attenuate the sustained postexercise vasodilatation, would reduce leg glucose uptake after a bout of cycling. Healthy, recreationally active subjects (n = 8) exercised for 1 h at 60% of peak oxygen consumption on each of two days, with (blockade) and without (control) histamine-receptor antagonism. For 2 h of recovery following exercise, arteriovenous glucose differences were assessed from the radial artery and femoral vein, and leg blood flow was measured using Doppler ultrasonography on the common femoral artery. Femoral blood flow following exercise was 65.4 ± 16.4 ml min(-1) lower on the blockade day compared with the control day (P < 0.05). Likewise, glucose delivery was 0.177 ± 0.045 mmol min(-1) lower with blockade (P < 0.05). However, histamine-receptor antagonism produced no consistent effect on leg glucose uptake following exercise, due to high interindividual variability. In conclusion, while oral ingestion of H(1)- and H(2)-receptor antagonists alters postexercise recovery by attenuating vasodilatation, leg glucose uptake is not universally affected in recreationally active individuals.

Functional analyses of human LUC7-like proteins involved in splicing regulation and myeloid neoplasms.

Vertebrates have evolved three paralogs, termed LUC7L, LUC7L2, and LUC7L3, of the essential yeast U1 small nuclear RNA (snRNA)-associated splicing factor Luc7p. We investigated the mechanistic and regulatory functions of these putative splicing factors, of which one (LUC7L2) is mutated or deleted in myeloid neoplasms. Protein interaction data show that all three proteins bind similar core but distinct regulatory splicing factors, probably mediated through their divergent arginine-serine-rich domains, which are not present in Luc7p. Knockdown of each factor reveals mostly unique sets of significantly dysregulated alternative splicing events dependent on their binding locations, which are largely non-overlapping. Notably, knockdown of LUC7L2 alone significantly upregulates the expression of multiple spliceosomal factors and downregulates glycolysis genes, possibly contributing to disease pathogenesis. RNA binding studies reveal that LUC7L2 and LUC7L3 crosslink to weak 5' splice sites and to the 5' end of U1 snRNA, establishing an evolutionarily conserved role in 5' splice site selection.

Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia.

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.