NA activity as marker for airborne virus detection

Viruses offer a limited range of targets for their detection. To date, PCR and RT-PCR have been widely used for detection of viruses. In the case of environmental air sampling, the ability to detect a broad range of viruses would constitute a significant advantage for preventing outbreaks of airborne-transmitted viral infections. Given that neuraminidase is found on some respiratory virus species of medical or agricultural importance, this enzyme could theoretically be used to detect several different airborne viruses in a single assay. The aim of the present study was to evaluate the potential of neuraminidase activity as a marker for rapid detection of airborne viruses. We first validated the use of a low-pathogenic strain of Newcastle disease virus (NDV) as a model airborne virus. Our findings revealed that neuraminidase activity-based assays are almost as sensitive as RT-PCR assays currently used for detection of NDV. We also validated the utilization of a neuraminidase substrate specific to viral neuraminidase. Experiments conducted in a controlled chamber demonstrated that the neuraminidase activity is preserved after aerosolization, air sampling using impingement and handling. Finally, we tested our method with swine barn air samples. Our results demonstrate that neuraminidase activity-based assays are suitable for detection of viruses in air samples

The SRSF4–GAS5-Glucocorticoid Receptor Axis Regulates Ventricular Hypertrophy.

RATIONALE: RBPs (RNA-binding proteins) play critical roles in human biology and disease. Aberrant RBP expression affects various steps in RNA processing, altering the function of the target RNAs. The RBP SRSF4 (serine/arginine-rich splicing factor 4) has been linked to neuropathies and cancer. However, its role in the heart is completely unknown. OBJECTIVE: To investigate the role of SRSF4 in the heart. METHODS AND RESULTS: Echocardiography of mice specifically lacking SRSF4 in the heart (SRSF4 KO) revealed left ventricular hypertrophy and increased cardiomyocyte area, which led to progressive diastolic dysfunction with age. SRSF4 KO mice showed altered electrophysiological activity under isoproterenol-induced cardiac stress, with a post-QRS depression and a longer QT interval, indicating an elevated risk of sudden cardiac death. RNA-Seq analysis revealed expression changes in several long noncoding RNAs, including GAS5 (growth arrest-specific 5), which we identified as a direct SRSF4 target in cardiomyocytes by individual-nucleotideresolution cross-linking and immuno-precipitation. GAS5 is a repressor of the GR (glucocorticoid receptor) and was downregulated in SRSF4 KO hearts. This corresponded with elevated GR transcriptional activity in cardiomyocytes, leading to increases in hypertrophy markers and cell size. Furthermore, hypertrophy in SRSF4 KO cardiomyocytes was reduced by overexpressing GAS5. CONCLUSIONS: Loss of SRSF4 expression results in cardiac hypertrophy, diastolic dysfunction, and abnormal repolarization. The molecular mechanism underlying this effect involves GAS5 downregulation and consequent elevation of GR transcriptional activity. Our findings may help to develop new therapeutic tools for the treatment of cardiac hypertrophy and myocardial pathology in patients with Cushing syndrome.post-print2695 K

The SRSF4–GAS5-Glucocorticoid Receptor Axis Regulates Ventricular Hypertrophy.

RATIONALE: RBPs (RNA-binding proteins) play critical roles in human biology and disease. Aberrant RBP expression affects various steps in RNA processing, altering the function of the target RNAs. The RBP SRSF4 (serine/arginine-rich splicing factor 4) has been linked to neuropathies and cancer. However, its role in the heart is completely unknown. OBJECTIVE: To investigate the role of SRSF4 in the heart. METHODS AND RESULTS: Echocardiography of mice specifically lacking SRSF4 in the heart (SRSF4 KO) revealed left ventricular hypertrophy and increased cardiomyocyte area, which led to progressive diastolic dysfunction with age. SRSF4 KO mice showed altered electrophysiological activity under isoproterenol-induced cardiac stress, with a post-QRS depression and a longer QT interval, indicating an elevated risk of sudden cardiac death. RNA-Seq analysis revealed expression changes in several long noncoding RNAs, including GAS5 (growth arrest-specific 5), which we identified as a direct SRSF4 target in cardiomyocytes by individual-nucleotideresolution cross-linking and immuno-precipitation. GAS5 is a repressor of the GR (glucocorticoid receptor) and was downregulated in SRSF4 KO hearts. This corresponded with elevated GR transcriptional activity in cardiomyocytes, leading to increases in hypertrophy markers and cell size. Furthermore, hypertrophy in SRSF4 KO cardiomyocytes was reduced by overexpressing GAS5. CONCLUSIONS: Loss of SRSF4 expression results in cardiac hypertrophy, diastolic dysfunction, and abnormal repolarization. The molecular mechanism underlying this effect involves GAS5 downregulation and consequent elevation of GR transcriptional activity. Our findings may help to develop new therapeutic tools for the treatment of cardiac hypertrophy and myocardial pathology in patients with Cushing syndrome.post-print2695 K

Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage

NMR-derived secondary structure of the full-length Ox40 mRNA 3′UTR and its multivalent binding to the immunoregulatory RBP Roquin

Control of posttranscriptional mRNA decay is a crucial determinant of cell homeostasis and differentiation. mRNA lifetime is governed by cis-regulatory elements in their 3′ untranslated regions (UTR). Despite ongoing progress in the identification of cis elements we have little knowledge about the functional and structural integration of multiple elements in 3′UTR regulatory hubs and their recognition by mRNA-binding proteins (RBPs). Structural analyses are complicated by inconsistent mapping and prediction of RNA fold, by dynamics, and size. We here, for the first time, provide the secondary structure of a complete mRNA 3′UTR. We use NMR spectroscopy in a divide-and-conquer strategy complemented with SAXS, In-line probing and SHAPE-seq applied to the 3′UTR of Ox40 mRNA, which encodes a T-cell co-receptor repressed by the protein Roquin. We provide contributions of RNA elements to Roquin-binding. The protein uses its extended bi-modal ROQ domain to sequentially engage in a 2:1 stoichiometry with a 3′UTR core motif. We observe differential binding of Roquin to decay elements depending on their structural embedment. Our data underpins the importance of studying RNA regulation in a full sequence and structural context. This study serves as a paradigm for an approach in analysing structured RNA-regulatory hubs and their binding by RBPs

The ribosomal RNA gene promoter and adjacent cis-acting DNA sequences govern plasmid DNA partitioning and stable inheritance in the parasitic protozoan Leishmania

Detailed analysis of the Leishmania donovani ribosomal RNA (rRNA) gene promoter region has allowed the identification of cis-acting sequences involved in plasmid DNA partitioning and stable plasmid inheritance. We report that plasmids bearing the 350 bp rRNA promoter along with the 200 bp region immediately 3′ to the promoter exhibited a 6.5-fold increase in transformation frequency and were transmitted to daughter cells as single-copy molecules. This is in contrast to what has been observed for plasmid molecules in this organism so far. Moreover, we show that these low-copy-number plasmids displayed a remarkable mitotic stability in the absence of selective pressure. The region in the vicinity of the RNA pol I transcription initiation site, and also in the adjacent 200 nt, displays a complex structural organization and shares sequence similarity to the yeast autonomously replicating consensus sequence and centromere DNA elements. Deletion analyses indicated that these elements were necessary but not sufficient for plasmid DNA partitioning and stable inheritance, and that the rRNA promoter region was required for optimal function. These results suggest an interplay between RNA pol I transcription, DNA replication, DNA partitioning and mitotic stability in trypanosomatids. This is the first example of defined DNA elements for plasmid partitioning and stable inheritance in the protozoan parasite Leishmania

SRSF3 and SRSF7 modulate 3′UTR length through suppression or activation of proximal polyadenylation sites and regulation of CFIm levels

Background: Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3′ untranslated regions (3′UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown. Results: Here we combine iCLIP and 3′-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3′UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3′UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3′UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3′UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3′UTRs. Conclusions; We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation

SRSF3 and SRSF7 modulate 3'UTR length through suppression or activation of proximal polyadenylation sites and regulation of CFIm levels.

BACKGROUND Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3' untranslated regions (3'UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown. RESULTS Here we combine iCLIP and 3'-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3'UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3'UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3'UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3'UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3'UTRs. CONCLUSIONS We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation

The SRSF4–GAS5-Glucocorticoid Receptor Axis Regulates Ventricular Hypertrophy

Objective: To investigate the role of SRSF4 in the heart. Methods and Results: Echocardiography of mice specifically lacking SRSF4 in the heart (SRSF4 KO) revealed left ventricular hypertrophy and increased cardiomyocyte area, which led to progressive diastolic dysfunction with age. SRSF4 KO mice showed altered electrophysiological activity under isoproterenol-induced cardiac stress, with a post-QRS depression and a longer QT interval, indicating an elevated risk of sudden cardiac death. RNA-Seq analysis revealed expression changes in several long noncoding RNAs, including GAS5 (growth arrest-specific 5), which we identified as a direct SRSF4 target in cardiomyocytes by individual-nucleotide-resolution cross-linking and immuno-precipitation. GAS5 is a repressor of the GR (glucocorticoid receptor) and was downregulated in SRSF4 KO hearts. This corresponded with elevated GR transcriptional activity in cardiomyocytes, leading to increases in hypertrophy markers and cell size. Furthermore, hypertrophy in SRSF4 KO cardiomyocytes was reduced by overexpressing GAS5. Conclusions: Loss of SRSF4 expression results in cardiac hypertrophy, diastolic dysfunction, and abnormal repolarization. The molecular mechanism underlying this effect involves GAS5 downregulation and consequent elevation of GR transcriptional activity. Our findings may help to develop new therapeutic tools for the treatment of cardiac hypertrophy and myocardial pathology in patients with Cushing syndrome.European UnionMinisterio de Economía y CompetitividadInstituto de Salud Carlos IIIComunidad de MadridPlan Estatal I+D+I 2013-2016Ministerio de Ciencia, Innovación y UniversidadesFundación Pro-CNICSevero Ochoa Center de ExcellenceDepto. de Medicina y Cirugía AnimalFac. de VeterinariaTRUEpu