Pumilio1 haploinsufficiency leads to SCA1-like neurodegeneration by increasing wild-type Ataxin1 levels.

Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1, this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1(+/-) mice to SCA1 mice (Atxn1(154Q/+)) exacerbated disease progression, whereas breeding them to Atxn1(+/-) mice normalized Ataxin1 levels and largely rescued the Pum1(+/-) phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease.

Rbfox2-coordinated alternative splicing of Mef2d and Rock2 controls myoblast fusion during myogenesis.

Alternative splicing plays important regulatory roles during periods of physiological change. During development, a large number of genes coordinately express protein isoform transitions regulated by alternative splicing; however, the mechanisms that coordinate splicing and the functional integration of the resultant tissue-specific protein isoforms are typically unknown. Here we show that the conserved Rbfox2 RNA binding protein regulates 30% of the splicing transitions observed during myogenesis and is required for the specific step of myoblast fusion. Integration of Rbfox2-dependent splicing outcomes from RNA-seq with Rbfox2 iCLIP data identified Mef2d and Rock2 as Rbfox2 splicing targets. Restored activities of Mef2d and Rock2 rescued myoblast fusion in Rbfox2-depleted cultures, demonstrating functional cooperation of protein isoforms generated by coordinated alterative splicing. The results demonstrate that coordinated alternative splicing by a single RNA binding protein modulates transcription (Mef2d) and cell signaling (Rock2) programs to drive tissue-specific functions (cell fusion) to promote a developmental transition.

Mechanical Anastomotic Coupling Device versus Hand-sewn Venous Anastomosis in Head and Neck Reconstruction-An Analysis of 1694 Venous Anastomoses.

Background There is a steep learning curve to attain a consistently good result in microvascular surgery. The venous anastomosis is a critical step in free-tissue transfer. The margin of error is less and the outcome depends on the surgeon's skill and technique. Mechanical anastomotic coupling device (MACD) has been proven to be an effective alternative to hand-sewn (HS) technique for venous anastomosis, as it requires lesser skill. However, its feasibility of application in emerging economy countries is yet to be established. Material and Method We retrospectively analyzed the data of patients who underwent free-tissue transfer for head and neck reconstruction between July 2015 and October 2020. Based on the technique used for the venous anastomosis, the patients were divided into an HS technique and MACD group. Patient characteristics and outcomes were measured. Result A total of 1694 venous anastomoses were performed during the study period. There were 966 patients in the HS technique group and 719 in the MACD group. There was no statistically significant difference between the two groups in terms of age, sex, prior radiotherapy, prior surgery, and comorbidities. Venous thrombosis was noted in 62 (6.4%) patients in the HS technique group and 7 (0.97%) in the MACD group ( p = 0.000). The mean time taken for venous anastomosis in the HS group was 17 ± 4 minutes, and in the MACD group, it was 5 ± 2 minutes ( p = 0.0001). Twenty-five (2.56%) patients in the HS group and 4 (0.55%) patients in MACD group had flap loss ( p = 0.001). Conclusion MACD is an effective alternative for HS technique for venous anastomosis. There is a significant reduction in anastomosis time, flap loss, and return to operation theater due to venous thrombosis. MACD reduces the surgeon's strain, especially in a high-volume center. Prospective randomized studies including economic analysis are required to prove the cost-effectiveness of coupler devices.

Argonaute 8 (AGO8) Mediates the Elicitation of Direct Defenses against Herbivory.

In Nicotiana attenuata, specific RNA-directed RNA polymerase (RdR1) and the Dicer-like (DCL3 and DCL4) proteins are recruited during herbivore attack to mediate the regulation of defense responses. However, the identity and role(s) of Argonautes (AGOs) involved in herbivory remain unknown. Of the 11 AGOs in the N. attenuata genome, we silenced the expression of 10. Plants silenced in NaAGO8 expression grew normally but were highly susceptible to herbivore attack. Larvae of Manduca sexta grew faster when consuming inverted-repeat stable transformants (irAGO8) plants but did not differ from the wild type when consuming plants silenced in AGO1 (a, b, and c), AGO2, AGO4 (a and b), AGO7, or AGO10 expression. irAGO8 plants were significantly compromised in herbivore-induced levels of defense metabolites such as nicotine, phenolamides, and diterpenoid glycosides. Time-course analyses revealed extensively altered microRNA profiles and the reduced accumulation of MYB8 transcripts and of the associated genes of the phenolamide and phenylpropanoid pathways as well as the nicotine biosynthetic pathway. A possible AGO8-modulated microRNA-messenger RNA target network was inferred. Furthermore, comparative analysis of domains revealed the diversity of AGO conformations, particularly in the small RNA-binding pocket, which may influence substrate recognition/binding and functional specificity. We infer that AGO8 plays a central role in the induction of direct defenses by modulating several regulatory nodes in the defense signaling network during herbivore response. Thus, our study identifies the effector AGO of the herbivore-induced small RNA machinery, which in N. attenuata now comprises RdR1, DCL3/4, and AGO8.

The RNA-binding protein Rbfox1 regulates splicing required for skeletal muscle structure and function.

The Rbfox family of RNA-binding proteins is highly conserved with established roles in alternative splicing (AS) regulation. High-throughput studies aimed at understanding transcriptome remodeling have revealed skeletal muscle as displaying one of the largest number of AS events. This finding is consistent with requirements for tissue-specific protein isoforms needed to sustain muscle-specific functions. Rbfox1 is abundant in vertebrate brain, heart and skeletal muscle. Genome-wide genetic approaches have linked the Rbfox1 gene to autism, and a brain-specific knockout mouse revealed a critical role for this splicing regulator in neuronal function. Moreover, a Caenorhabditis elegans Rbfox1 homolog regulates muscle-specific splicing. To determine the role of Rbfox1 in muscle function, we developed a conditional knockout mouse model to specifically delete Rbfox1 in adult tissue. We show that Rbfox1 is required for muscle function but a >70% loss of Rbfox1 in satellite cells does not disrupt muscle regeneration. Deep sequencing identified aberrant splicing of multiple genes including those encoding myofibrillar and cytoskeletal proteins, and proteins that regulate calcium handling. Ultrastructure analysis of Rbfox1(-/-) muscle by electron microscopy revealed abundant tubular aggregates. Immunostaining showed mislocalization of the sarcoplasmic reticulum proteins Serca1 and Ryr1 in a pattern indicative of colocalization with the tubular aggregates. Consistent with mislocalization of Serca1 and Ryr1, calcium handling was drastically altered in Rbfox1(-/-) muscle. Moreover, muscle function was significantly impaired in Rbfox1(-/-) muscle as indicated by decreased force generation. These results demonstrate that Rbfox1 regulates a network of AS events required to maintain multiple aspects of muscle physiology.

Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage.

Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utilizing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily conserved sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted exclusion of exon 11, while its siRNA-mediated knockdown prevented the stress-induced alternative splicing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. Notably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation.

The splicing factor FUBP1 is required for the efficient splicing of oncogene MDM2 pre-mRNA.

Alternative splicing of the oncogene MDM2 is a phenomenon that occurs in cells in response to genotoxic stress and is also a hallmark of several cancer types with important implications in carcinogenesis. However, the mechanisms regulating this splicing event remain unclear. Previously, we uncovered the importance of intron 11 in MDM2 that affects the splicing of a damage-responsive MDM2 minigene. Here, we have identified discrete cis regulatory elements within intron 11 and report the binding of FUBP1 (Far Upstream element-Binding Protein 1) to these elements and the role it plays in MDM2 splicing. Best known for its oncogenic role as a transcription factor in the context of c-MYC, FUBP1 was recently described as a splicing regulator with splicing repressive functions. In the case of MDM2, we describe FUBP1 as a positive splicing regulatory factor. We observed that blocking the function of FUBP1 in in vitro splicing reactions caused a decrease in splicing efficiency of the introns of the MDM2 minigene. Moreover, knockdown of FUBP1 in cells induced the formation of MDM2-ALT1, a stress-induced splice variant of MDM2, even under normal conditions. These results indicate that FUBP1 is also a strong positive splicing regulator that facilitates efficient splicing of the MDM2 pre-mRNA by binding its introns. These findings are the first report describing the regulation of alternative splicing of MDM2 mediated by the oncogenic factor FUBP1.

Molecular evolution and diversification of the Argonaute family of proteins in plants.

BACKGROUND: Argonaute (AGO) proteins form the core of the RNA-induced silencing complex, a central component of the smRNA machinery. Although reported from several plant species, little is known about their evolution. Moreover, these genes have not yet been cloned from the ecological model plant, Nicotiana attenuata, in which the smRNA machinery is known to mediate important ecological traits. RESULTS: Here, we not only identify 11 AGOs in N. attenuata, we further annotate 133 genes in 17 plant species, previously not annotated in the Phytozome database, to increase the number of plant AGOs to 263 genes from 37 plant species. We report the phylogenetic classification, expansion, and diversification of AGOs in the plant kingdom, which resulted in the following hypothesis about their evolutionary history: an ancestral AGO underwent duplication events after the divergence of unicellular green algae, giving rise to four major classes with subsequent gains/losses during the radiation of higher plants, resulting in the large number of extant AGOs. Class-specific signatures in the RNA-binding and catalytic domains, which may contribute to the functional diversity of plant AGOs, as well as context-dependent changes in sequence and domain architecture that may have consequences for gene function were found. CONCLUSIONS: Together, the results demonstrate that the evolution of AGOs has been a dynamic process producing the signatures of functional diversification in the smRNA pathways of higher plants.

Epinephrine-induced myocardial infarction in severe anaphylaxis: is nonselective ß-blockade a contributory factor?

Epinephrine-induced myocardial ischemia in the setting of anaphylaxis is a rare event and is postulated to be due to coronary artery spasm. We report the case of a 43-year-old woman who presented to the emergency department with an anaphylactic reaction triggered by flucloxacillin. She was treated with intramuscular epinephrine, following which she developed ischemic chest pain and electrocardiographic changes, associated with troponin elevation. Subsequent coronary angiography demonstrated normal coronary arteries. In this case report, we discuss the potential role of prior nonselective ß-blockade with propranolol in predisposing such patients to ischemic cardiac events following treatment with epinephrine.

Factors predicting emergency surgery in severe mitral regurgitation following mitral balloon valvotomy.

BACKGROUND AND AIM OF THE STUDY: Acute mitral regurgitation (MR) may cause adverse hemodynamics following mitral balloon valvotomy (MBV). Some patients become severely hemodynamically unstable and require emergency mitral valve replacement (MVR), while others remain relatively stable with medical management. The study aim was to identify factors that would predict severe acute MR leading to severe hemodynamic compromise requiring emergency MVR. METHODS: Between January 2001 and July 2009, a total of 46 patients developed acute severe MR following MBV at the authors' institution. Of these patients, 11 developed severe hemodynamic compromise and required emergency MVR within 6 h of the procedure (group I), while 35 were relatively stable, improved with time, and were discharged with advice to undergo an early MVR (group II). RESULTS: The demographic profile and routine echocardiographic parameters were comparable between the two groups. In group I, the right ventricular systolic pressure (RVSP) before and after MBV was significantly higher, and a significantly higher level of calcium was present in the mitral valve leaflets. Univariate analysis of the RVSP before and after MBV predicted the occurrence of hemodynamic instability leading to emergency MVR. The receiver operating characteristic (ROC) curve for RVSP before and after MBV had a significant area under the curve (0.944, p < 0.005 and 0.940, p < 0.005, respectively). Based on the ROC data, the pre- and post-MBV RVSPs of 76 mmHg and 77 mmHg, respectively, predicted the possibility of emergency MVR, with sensitivities and specificities of 72% and 63%, and 100% and 90%, respectively. CONCLUSION: Patients undergoing MBV with an RVSP >76 mmHg and the presence of non-commissural calcium on the mitral valve leaflet, or those who develop an RVSP of 77 mmHg following the procedure will very likely require emergency MVR.

Continuous circular closure in unilateral cleft lip and plate repair in one surgery.

The study aims at assessing wound healing and safety of single-stage two-layers continuous closure in patients with unilateral cleft lip and palate (UCLP). In this retrospective, descriptive cohort study, we assessed wound healing without fistula formation at 1, 3, and 6 months after a single-stage two-layer UCLP repair, in which the midline suture is continuously circular all along the oral and nasal sides. We examined lengths of hospital stay and the incidence of intra- and postoperative adverse events. Furthermore, we compared the cleft width at birth and on the day of surgery, after presurgical orthopaedics. Eleven UCLP patients underwent one cleft surgery between July 2016 and June 2018 at the age of 8-9 months. Full primary healing occurred in all patients without fistulas. Median length of post-operative hospital stay was 5 days (range = 4-9 days). No intra- or postoperative adverse events above Grade I (according to ClassIntra and Clavien-Dindo, respectively) occurred. Median and interquartile range (IQR) of the palatal cleft width decreased significantly from birth to surgery, i.e., from 12.0 mm (10.8-13.6 mm) to 5.0 mm (4.0-7.5 mm) anteriorly and from 14.0 mm (11.5-15.0 mm) to 7.3 mm (6.0-8.5 mm) posteriorly (p = 0.0033 in both cases). Given these preliminary results, the concept of single-stage continuous circular closure in UCLP has potential for further investigation. However, it remains to be proven that there are no relevant adverse effects such as inhibition of maxillary growth. Registered in clinicaltrials.gov:NCT04108416.

Does rheumatic valvular heart disease affect right ventricular performance?

AIM: Right ventricular (RV) function often determines clinical outcome in patients with valvular heart disease. Though difficult to assess by echocardiography, Tei index is useful in its assessment. The aims of the study were to evaluate global RV function using the Tei index in patients with rheumatic heart disease and to observe if such abnormalities in RV function were reversible post-operatively. METHOD: The study included patients with atrial septal defect (ASD, Group I, n = 15) and rheumatic valvular heart disease (RVHD, Group II, n = 18). Patients with atrial fibrillation were excluded from the study. Conventional 2-D echocardiography was performed preoperatively, immediate postoperative and in last follow-up. RESULT: ASD group had lower left LVES and LVED dimensions as compared to RHVD (p = 0.001) and better ejection fraction (EF) than RVHD group (p = 0.02). LV Tei in the ASD group was above the normal limit (> 0.5), while RV Tei was increased in the RHVD group. The median RVSP was similar in two groups (p = 0.9). The impaired LVMPI in the ASD group improved as early as 2 weeks following surgery (p = 0.09) while in patients with RHVD it deteriorated which mirrored the reduction in median LVEF (p = 0.04). Group II that had an abnormal RV Tei pre-operatively demonstrated improvement following surgery (p = 0.03). CONCLUSION: RVHD is associated with impairment of RV function. Volume overload of RV in patients of ASD is associated with normal MPI. The abnormalities in RVMPI improved as early as 2 weeks after valve surgery with sustained improvement noted at follow up.

Conserved sequences in the final intron of MDM2 are essential for the regulation of alternative splicing of MDM2 in response to stress.

Alternative splicing plays a fundamental role in generating proteome diversity and is critical in regulation of eukaryotic gene expression. It is estimated that 50% of disease-causing mutations alter splicing efficiency and/or patterns of splicing. An alternatively spliced form of murine double-minute 2, MDM2-ALT1, is associated with pediatric rhabdomyosarcoma (RMS) at high frequency in primary human tumors and RMS cell lines. We have identified that this isoform can be induced in response to specific types of stress (UV and cisplatin). However, the mechanism of alternative splicing of MDM2 in human cancer is unknown. Using UV and cisplatin to model alternative splicing of the MDM2 gene, we have developed a damage-inducible in vitro splicing system. This system employs an MDM2 minigene that mimics the damage-induced alternative splicing observed in vivo. Using this in vitro splicing system, we have shown that conserved intronic sequences in intron 11 of MDM2 are required for normal splicing. Furthermore, we showed that these intronic elements are also required for the regulated damage-induced alternative splicing of MDM2. The use of this novel damage-inducible system will allow for the systematic identification of regulatory elements and factors involved in the splicing regulation of the MDM2 gene in response to stress. This study has implications for identification of novel intervention points for development of future therapeutics for rhabdomyosarcoma.

SRSF2 Regulation of MDM2 Reveals Splicing as a Therapeutic Vulnerability of the p53 Pathway.

MDM2 is an oncogene and critical negative regulator of tumor suppressor p53. Genotoxic stress causes alternative splicing of MDM2 transcripts, which leads to alterations in p53 activity and contributes to tumorigenesis. MDM2-ALT1 is one of the alternatively spliced transcripts predominantly produced in response to genotoxic stress, and is comprised of terminal coding exons 3 and 12. Previously, we found that SRSF1 induces MDM2-ALT1 by promoting MDM2 exon 11 skipping. Here we report that splicing regulator SRSF2 antagonizes the regulation of SRSF1 by facilitating the inclusion of exon 11 through binding at two conserved exonic splicing enhancers. Overexpression of SRSF2 reduced the generation of MDM2-ALT1 under genotoxic stress, whereas SRSF2 knockdown induced the expression of MDM2-ALT1 in the absence of genotoxic stress. Blocking the exon 11 SRSF2-binding sites using oligonucleotides promoted MDM2-ALT1 splicing and induced p53 protein expression, and apoptosis in p53 wild-type cells. The regulation of MDM2 splicing by SRSF2 is also conserved in mice, as mutation of one SRSF2-binding site in Mdm2 exon 11, using CRISPR-Cas9, increased the expression of the MDM2-ALT1 homolog Mdm2-MS2. IMPLICATIONS: Taken together, the data indicate that modulating MDM2 splicing may be a useful tool for fine-tuning p53 activity in response to genotoxic stress.

Rbfox-Splicing Factors Maintain Skeletal Muscle Mass by Regulating Calpain3 and Proteostasis.

Maintenance of skeletal muscle mass requires a dynamic balance between protein synthesis and tightly controlled protein degradation by the calpain, autophagy-lysosome, and ubiquitin-proteasome systems (proteostasis). Several sensing and gene-regulatory mechanisms act together to maintain this balance in response to changing conditions. Here, we show that deletion of the highly conserved Rbfox1 and Rbfox2 alternative splicing regulators in adult mouse skeletal muscle causes rapid, severe loss of muscle mass. Rbfox deletion did not cause a reduction in global protein synthesis, but it led to altered splicing of hundreds of gene transcripts, including capn3, which produced an active form of calpain3 protease. Rbfox knockout also led to a reduction in autophagy flux, likely producing a compensatory increase in general protein degradation by the proteasome. Our results indicate that the Rbfox-splicing factors are essential for the maintenance of skeletal muscle mass and proteostasis.

Phylogenetic and Evolutionary Analysis of Plant ARGONAUTES.

Comparative sequence analysis is widely used for the reconstruction of phylogeny and for understanding the evolutionary history of gene families. Here, we describe the methodologies to reconstruct the phylogenetic and evolutionary history of a gene family across genomes with a focus on the ARGONAUTE (AGO) family of proteins in plants. The method described here may easily be adapted for studying molecular evolution of a wide variety of gene families. We enlist methods as well as parameters for the collection of molecular data (nucleic acids and peptides), preparation of datasets, and selection of evolutionary models and various methods for the phylogenetic and evolutionary analysis, such as maximum likelihood and Bayesian inference.

Genome-wide characterization of cellulases from the hemi-biotrophic plant pathogen, Bipolaris sorokiniana, reveals the presence of a highly stable GH7 endoglucanase.

BACKGROUND: Bipolaris sorokiniana is a filamentous fungus that causes spot blotch disease in cereals like wheat and has severe economic consequences. However, information on the identities and role of the cell wall-degrading enzymes (CWDE) in B. sorokiniana is very limited. Several fungi produce CWDE like glycosyl hydrolases (GHs) that help in host cell invasion. To understand the role of these CWDE in B. sorokiniana, the first step is to identify and annotate all possible genes of the GH families like GH3, GH6, GH7, GH45 and AA9 and then characterize them biochemically. RESULTS: We confirmed and annotated the homologs of GH3, GH6, GH7, GH45 and AA9 enzymes in the B. sorokiniana genome using the sequence and domain features of these families. Quantitative real-time PCR analyses of these homologs revealed that the transcripts of the BsGH7-3 (3rd homolog of the GH 7 family in B. sorokiniana) were most abundant. BsGH7-3, the gene of BsGH7-3, was thus cloned into pPICZαC Pichia pastoris vector and expressed in X33 P. pastoris host to be characterized. BsGH7-3 enzyme showed a temperature optimum of 60 °C and a pHopt of 8.1. BsGH7-3 was identified to be an endoglucanase based on its broad substrate specificity and structural comparisons with other such endoglucanases. BsGH7-3 has a very long half-life and retains 100% activity even in the presence of 4 M NaCl, 4 M KCl and 20% (v/v) ionic liquids. The enzyme activity is stimulated up to fivefold in the presence of Mn+2 and Fe+2 without any deleterious effects on enzyme thermostability. CONCLUSIONS: Here we reanalysed the B. sorokiniana genome and selected one GH7 enzyme for further characterization. The present work demonstrates that BsGH7-3 is an endoglucanase with a long half-life and no loss in activity in the presence of denaturants like salt and ionic liquids, and lays the foundation towards exploring the Bipolaris genome for other cell wall-degrading enzymes.

Dimorphism in the P2Y1 ADP receptor gene is associated with increased platelet activation response to ADP.

OBJECTIVE: The platelet ADP receptors P2Y1 and P2Y12 play a pivotal role in platelet aggregation. There is marked interindividual variation in platelet response to ADP. We studied whether genetic variants in the P2Y1 or P2Y12 genes affect platelet response to ADP. METHODS AND RESULTS: The P2Y1 and P2Y12 genes were screened for polymorphisms. Associations between selected polymorphisms and the platelet response to ADP (0.1, 1.0, and 10 micromol/L), assessed by whole blood flow cytometric measurement of fibrinogen binding to activated glycoprotein IIb-IIIa, were then determined in 200 subjects. Five polymorphisms were found in the P2Y1 gene and 11 in the P2Y12 gene. All polymorphisms were silent. A P2Y1 gene dimorphism, 1622AG, was associated with a significant (P=0.007) effect on platelet ADP response, with a greater response in carriers of the G allele (frequency 0.15). The effect was seen at all concentrations of ADP but greatest at 0.1 mumol/L ADP, where the response in GG homozygotes was on average 130% higher than that seen in AA homozygotes (P=0.006). CONCLUSIONS: A common genetic variant at the P2Y1 locus is associated with platelet reactivity to ADP. This genotype effect partly explains the interindividual variation in platelet response to ADP and may have clinical implications with regard to thrombotic risk.