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Ketone bodies are an alternate fuel source generated by the liver in response to low carbohydrate availability in neonates and after starvation and exhausting exercise in adulthood. However, the mechanisms regulating effective ketone body utilization in peripheral organs are poorly understood. The postnatal alternative splicing generates muscle-specific MEF2D⍺2 protein isoform of the transcription factor MEF2D. Here, we discovered that MEF2D⍺2 exon knockout (Eko) mice displayed reduced running capacity and muscle expression of all three ketolytic genes, BDH1, OXCT1, and ACAT1. Consistently, MEF2D⍺2 Eko mice showed a slower clearance of blood ketone bodies in a tolerance test. MEF2D⍺2 Eko mice displayed higher ketone body levels immediately after exercise, during the recovery period, and after feeding on a ketogenic diet. Thus, we identified MEF2D⍺2 as a crucial regulator of skeletal muscle ketone body oxidation and exercise capacity. Our results also underscore the effect of muscle-specific alternative splicing at the whole organism level.

3D rendering of 60X confocal images taken of SRSF2 (green) and SRRM2 (red) coimmunofluorescence in HeLa S3 cells after 12 hours of UVC treatment. Colocalization channel is indicated by magenta color, while the blue color represents the nucleus stained with DAPI.

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.

Supplemental Figure S1: Mutation of predicted binding sites for SRSF2 disrupt alternative splicing regulation of the MDM2 3-11-12s minigene in response to cisplatinum. Supplemental Figure S2: SRSF2 protein expression is increased after UVC treatment. Supplemental Figure S3: SRSF2 is relocalized upon UVC treatment. Supplemental Figure S4: SRSF2 has less affinity of CCNL1 exon 4 under UVC treatment. Supplemental Figure S5: Exogenous SRSF2 is expressed in the nucleus. Supplemental Table S1: Oligonucleotides

The tumor suppressor protein p53 is a transcription factor that induces G1 arrest of the cell cycle and/or apoptosis. The murine double-minute protein MDM2 and its homologue MDM4 (also known as MDMX) are critical regulators of p53. Altered transcripts of the human homologue of mdm2, MDM2, have been identified in human tumors, such as invasive carcinoma of the breast, lung carcinoma, and liposarcoma. MDM2 alternate forms act to negatively regulate the normal MDM2 gene product, thus activating p53. Although many reports have documented a plethora of tumor types characterized by MDM2 alternative transcripts, few have investigated the signals that might initiate alternative splicing. We have identified a novel role of these alternative MDM2 transcripts in the normal surveillance mechanism of the cell and in DNA damage response. We report that alternate forms of MDM2 are detected after UV irradiation. Furthermore, we show that mouse cells treated with UV are also characterized by alternative transcripts of mdm2, suggesting that this is an important and evolutionarily conserved mechanism for regulating the expression of MDM2/mdm2. An additional p53 regulator and mdm2 family member, MDM4, is likewise alternatively spliced following UV irradiation. By activating alternative splicing of both MDM2 and MDM4, yet another layer of p53 regulation is initiated by the cells in response to damage. A stepwise model for malignant conversion by which alternate forms of MDM2 and MDM4 place selective pressure on the cells to acquire additional alterations in the p53 pathway is herein proposed. (Cancer Res 2006; 66(19): 9502-8)

Supplementary Figure 1 from Genotoxic Stress Induces Coordinately Regulated Alternative Splicing of the p53 Modulators MDM2 and MDM4

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.

Regulatory small RNAs (sRNAs) play important roles in many fundamental processes in plant biology such as development, fertilization and stress responses. The AGO protein family has here a central importance in gene regulation based on their capacity to associate with sRNAs followed by mRNA targeting in a sequence-complementary manner. The present study explored Argonautes (AGOs) in the Solanaceae family, with emphasis on potato, Solanum tuberosum (St). A genome-wide monitoring was performed to provide a deeper insight into gene families, genomic localization, gene structure and expression profile against the potato late blight pathogen Phytophthora infestans. Among 15 species in the Solanaceae family we found a variation from ten AGOs in Nicotiana obtusifolia to 17 in N. tabacum. Comprehensive analyses of AGO phylogeny revealed duplication of AGO1, AGO10 and AGO4 paralogs during early radiation of Solanaceae. Fourteen AGOs were identified in potato. Orthologs of AGO8 and AGO9 were missing in the potato genome. However, AGO15 earlier annotated in tomato was identified. StAGO15 differs from the other paralogs having residues of different physico-chemical properties at functionally important amino acid positions. Upon pathogen challenge StAGO15 was significantly activated and hence may play a prominent role in sRNA-based regulation of potato defense.

Summary 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). Patients and methods 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. Results Eleven UCLP patients underwent one cleft surgery between July 2016 and June 2018 at the age of 8 to 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). Conclusion Given these preliminary results, the concept of single-stage continuous circular closure in UCLP has potential for further investigation. In particular, it remains to be proven that there are no relevant adverse effects such as inhibition of maxillary growth. Registered in clinicaltrials.gov:NCT04108416.

Regulatory small RNAs (sRNAs) play important roles in many fundamental processes in plant biology such as development, fertilization and stress responses. The AGO protein family has here a central importance in gene regulation based on their capacity to associate with sRNAs followed by mRNA targeting in a sequence-complementary manner. The present study explored Argonautes (AGOs) in the Solanaceae family, with emphasis on potato, Solanum tuberosum (St). A genome-wide monitoring was performed to provide a deeper insight into gene families, genomic localization, gene structure and expression profile against the potato late blight pathogen Phytophthora infestans. Among 15 species in the Solanaceae family we found a variation from ten AGOs in Nicotiana obtusifolia to 17 in N. tabacum. Comprehensive analyses of AGO phylogeny revealed duplication of AGO1, AGO10 and AGO4 paralogs during early radiation of Solanaceae. Fourteen AGOs were identified in potato. Orthologs of AGO8 and AGO9 were missing in the potato genome. However, AGO15 earlier annotated in tomato was identified. StAGO15 differs from the other paralogs having residues of different physico-chemical properties at functionally important amino acid positions. Upon pathogen challenge StAGO15 was significantly activated and hence may play a prominent role in sRNA-based regulation of potato defense.

Nicotiana attenuata (coyote tobacco) has been developed into a model plant system to understand the molecular basis of traits important for the Darwinian fitness of plants in nature. Twenty years of releases of transgenic N. attenuata plants into a nature preserve in the plant’s native habitat have greatly advanced our understanding of the traits related to herbivore resistance. We provide an account of the genome sequencing of this wild tobacco species, as well as of another sympatric diploid tobacco, N. obtusifolia, which has different responses to herbivore attack. N. attenuata is a post-fire annual with a long-lived seed bank and exhibits an impressive array of plastic responses to the environmental challenges it faces, including unpredictable herbivore and pathogen attacks, by reprogramming the expression of thousands of genes. Regulatory small RNAs, including microRNAs, along with their biogenesis and regulating machinery, are thought to tailor the much needed specificity in modulating these large-scale changes in gene expression. Here, we provide a detailed genome-informed account of the small RNA machinery and its evolution in N. attenuata.

Plant small RNAs are largely non-coding, regulatory RNAs that act with a sequence-specific binding capacity to regulate target gene expression, either before transcription, a process called transcriptional gene silencing (TGS), or after transcription, a process called post-transcriptional gene silencing (PTGS) or RNA interference (RNAi), in general. Emerging evidence from high-throughput sequencing technologies leads us in characterizing several categories and groups of small RNAs. They play essential roles in complex biological processes under various physiological conditions. Small RNA-mediated gene silencing has been proven as a promising and powerful genetic tool for the improvement of crop plants and agriculture. It has emerged as a means to probe the function of a gene of interest on a whole-genome scale. Parallel to the enormous potential and applications, this technology has met with several new challenges, such as the off-target effects, delivery of optimum and sufficient small RNAs at the targeted cells, its measure for persistence in the environment, and several ethical issues.

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 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 in genotoxic stress condition, whereas knockdown induces the expression of MDM2-ALT1 in absence of genotoxic stress. Consistently, blocking the exon 11 SRSF2 binding sites using oligonucleotides promotes MDM2-ALT1. The regulation of MDM2 splicing by SRSF2 is also conserved in mouse as mutation of one SRSF2 binding site in Mdm2 exon 11, using CRISPR-Cas9, increases the expression MDM2-ALT1 homolog Mdm2-MS2 and proliferation of NIH 3T3 cells. Taken together, these findings underscore the relevance of MDM2 alternative splicing in cancer and suggest that p53 levels can be modulated by artificially regulating MDM2 splicing.

Anaplastic large cell lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma (NHL) characterized by T or null phenotype pleomorphic cells that express CD30. Systemic ALCL is sub-classified into two distinct entities, ALK-positive (ALK+ ALCL) and ALK-negative (ALK− ALCL), according to whether it

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.

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.

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.

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.

Novel ALK fusion in anaplastic large cell lymphoma involving EEF1G, a subunit of the eukaryotic elongation factor-1 complex

Argonautes (AGOs) are the effector proteins of the RNA-induced silencing (RISC) complex, formed during the phenomena of small-RNA mediated post-transcriptional gene silencing. AGOs are a large family of proteins; their number varies from a few (4 in Chlamydomonas reinhardtii) to many (18 in Oryza sativa) in plants. Genetics-guided analysis have demonstrated the roles of some of the AGOs during growth and development of plants. Biochemical studies have further revealed differences in functional specificities among AGOs. How the AGO family expanded in different plant species during the course of evolution is starting to emerge. We hypothesized that 4 classes of AGOs evolved after divergence of unicellular green algae when an ancestral AGO underwent duplication events. Evolution of multicellularity may have coincided with the diversification of AGOs. A comparative sequence and structure analysis of the plant AGOs, including those from the mosses and the unicellular algae, show not only conformational differences between those from lower and higher plants, but also functional divergence of important sites.