Splice-switching of the insulin receptor pre-mRNA alleviates tumorigenic hallmarks in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is an aggressive pediatric tumor with a poor prognosis for metastasis and recurrent disease. Large-scale sequencing endeavors demonstrate that Rhabdomyosarcomas have a dearth of precisely targetable driver mutations. However, IGF-2 signaling is known to be grossly altered in RMS. The insulin receptor (IR) exists in two alternatively spliced isoforms, IR-A and IR-B. The IGF-2 signaling molecule binds both its innate IGF-1 receptor as well as the insulin receptor variant A (IR-A) with high affinity. Mitogenic and proliferative signaling via the canonical IGF-2 pathway is, therefore, augmented by IR-A. This study shows that RMS patients express increased IR-A levels compared to control tissues that predominantly express the IR-B isoform. We also found that Hif-1α is significantly increased in RMS tumors, portraying their hypoxic phenotype. Concordantly, the alternative splicing of IR adapts to produce more IR-A in response to hypoxic stress. Upon examining the pre-mRNA structure of the gene, we identified a potential hypoxia-responsive element, which is also the binding site for the RNA-binding protein CUG-BP1 (CELF1). We designed Splice Switching Oligonucleotides (SSO) against this binding site to decrease IR-A levels in RMS cell lines and, consequently, rescue the IR-B expression levels. SSO treatment resulted in a significant reduction in cell proliferation, migration, and angiogenesis. Our data shows promising insight into how impeding the IGF-2 pathway by reducing IR-A expression mitigates tumor growth. It is evident that Rhabdomyosarcomas use IR alternative splicing as yet another survival strategy that can be exploited as a therapeutic intervention in conjunction with already established anti-IGF-1 receptor therapies.

Expression of OsDREB2A in Transgenic Tomato Improves Drought Tolerance

Dehydration responsive element binding (DREB) are important regulatory molecules which have a crucial role in abiotic stress tolerance. The productivity of tomato, as a drought-sensitive crop, is highly restricted by drought stress. The current study aimed at introducing the OsDERB2A gene into two tomato genotypes via Agrobacterium-mediated transformation system. Cotyledonary explants were pre-cultured for two days with Agrobacterium strain LBA4404 harboring pCAMBIA1301 with OsDREB2A driven by the constitutive promoter CaMV35S for transformation. Shoots were directly regenerated on MS medium containing 1 mg l-1 zeatin and 1 mg l-1 BAP, and in presence of 30 mg l-1 hygromycin as selective agent. Only eight weeks were needed to regenerate transgenic tomato using this protocol. An OD600 of 0.4 resulted in 64.3-76.9% transformation efficiency. Stable integration and expression of the OsDREB2A gene were confirmed in transgenic tomato using PCR and RT-PCR analyses, and drought tolerance of T0 transgenic lines was confirmed by leaf disc assay in 300 mM mannitol. The superior biomass, photosynthetic pigments, free soluble sugars and proline accumulation of OsDREB2A transgenic lines over wild type in response to mannitol-stress revealed their enhanced drought tolerance and indicated that the constitutive expression of OsDREB2A might modulate the expression of other drought responsive genes.

Comprehensive Analysis of Jumonji Domain C Family from Citrus grandis and Expression Profilings in the Exocarps of “Huajuhong” (Citrus grandis “Tomentosa”) during Various Development Stages

Citrus grandis “Tomentosa” (“Huajuhong”) is a famous Traditional Chinese Medicine. In this study, a total of 18 jumonji C (JMJC) domain-containing proteins were identified from C. grandis. The 18 CgJMJCs were unevenly located on six chromosomes of C. grandis. Phylogenetic analysis revealed that they could be classified into five groups, namely KDM3, KDM4, KDM5, JMJC, and JMJD6. The domain structures and motif architectures in the five groups were diversified. Cis-acting elements on the promoters of 18 CgJMJC genes were also investigated, and the abscisic acid-responsive element (ABRE) was distributed on 15 CgJMJC genes. Furthermore, the expression profiles of 18 CgJMJCs members in the exocarps of three varieties of “Huajuhong”, for different developmental stages, were examined. The results were validated by quantitative real-time PCR (qRT-PCR). The present study provides a comprehensive characterization of JMJC domain-containing proteins in C. grandis and their expression patterns in the exocarps of C. grandis “Tomentosa” for three varieties with various development stages.

Genome-wide promoter analysis, homology modeling and protein interaction network of Dehydration Responsive Element Binding (DREB) gene family in Solanum tuberosum

Dehydration Responsive Element Binding (DREB) regulates the expression of numerous stress-responsive genes, and hence plays a pivotal role in abiotic stress responses and tolerance in plants. The study aimed to develop a complete overview of the cis-acting regulatory elements (CAREs) present in S. tuberosum DREB gene promoters. A total of one hundred and four (104) cis-regulatory elements (CREs) were identified from 2.5kbp upstream of the start codon (ATG). The in-silico promoter analysis revealed variable sets of cis-elements and functional diversity with the predominance of light-responsive (30%), development-related (20%), abiotic stress-responsive (14%), and hormone-responsive (12%) elements in StDREBs. Among them, two light-responsive elements (Box-4 and G-box) were predicted in 64 and 61 StDREB genes, respectively. Two development-related motifs (AAGAA-motif and as-1) were abundant in StDREB gene promoters. Most of the DREB genes contained one or more Myeloblastosis (MYB) and Myelocytometosis (MYC) elements associated with abiotic stress responses. Hormone-responsive element i.e. ABRE was found in 59 out of 66 StDREB genes, which implied their role in dehydration and salinity stress. Moreover, six proteins were chosen corresponding to A1-A6 StDREB subgroups for secondary structure analysis and three-dimensional protein modeling followed by model validation through PROCHECK server by Ramachandran Plot. The predicted models demonstrated >90% of the residues in the favorable region, which further ensured their reliability. The present study also anticipated pocket binding sites and disordered regions (DRs) to gain insights into the structural flexibility and functional annotation of StDREB proteins. The protein association network determined the interaction of six selected StDREB proteins with potato proteins encoded by other gene families such as MYB and NAC, suggesting their similar functional roles in biological and molecular pathways. Overall, our results provide fundamental information for future functional analysis to understand the precise molecular mechanisms of the DREB gene family in S. tuberosum.

Ras signaling and its effector RREB1 are required for the dissociation of MEE cells in palatogenesis

Cleft palate is one of the major congenital craniofacial birth defects. The etiology underlying the pathogenesis of cleft palate has largely remained unelucidated. Dissociation of the medial edge epithelium (MEE) at the contacting region of palatal shelves and subsequent migration or apoptosis of MEE cells is required for the proper MEE removal. Ras Responsive Element Binding Protein 1 (RREB1), a RAS transcriptional effector, has recently been shown to play a crucial role in developmental EMT, in which loss of epithelial characteristics is an initial step, during mid-gastrulation of embryonic development. Interestingly, the involvement of RREB1 in cleft palate has been indicated in humans. Here, we demonstrated that pan-Ras inhibitor prevents the dissociation of MEE during palatal fusion. Rreb1 is expressed in the palatal epithelium during palatal fusion, and knockdown of Rreb1 in palatal organ culture resulted in palatal fusion defects by inhibiting the dissociation of MEE cells. Our present findings provide evidence that RREB1-mediated Ras signaling is required during palatal fusion. Aberrant RREB1-mediated Ras signaling might be involved in the pathogenesis of cleft palate.

Genetically Modified Sugarcane Intercropping Soybean Impact on Rhizosphere Bacterial Communities and Co-occurrence Patterns

Strategies involving genes in the dehydration-responsive element binding (DREB) family, which participates in drought stress regulation, and intercropping with legumes are becoming prominent options in promoting sustainable sugarcane cultivation. An increasing number of studies focusing on root interactions in intercropping systems, particularly involving transgenic crops, are being conducted to better understand and thus, harness beneficial soil microbes to enhance plant growth. We designed experiments to investigate the characteristics of two intercropping patterns, soybean with wild-type (WT) sugarcane and soybean with genetically modified (GM) Ea-DREB2B-overexpressing sugarcane, to assess the response of the rhizosphere microbiota to the different cropping patterns. Bacterial diversity in the rhizosphere microbial community differed between the two intercropping pattens. In addition, the biomass of GM sugarcane that intercropped with soybean was significantly improved compared with WT sugarcane, and the aboveground biomass and root biomass of GM soybean intercropping sugarcane increased by 49.15 and 46.03% compared with monoculture. Furthermore, a beneficial rhizosphere environment for the growth of Actinobacteria was established in the systems intercropped with GM sugarcane. Improving the production mode of crops by genetic modification is a key strategy to improving crop yields and provides new opportunities to further investigate the effects of intercropping on plant roots and soil microbiota. Thus, this study provides a basis for selecting suitable sugarcane–soybean intercropping patterns and a theoretical foundation for a sustainable sugarcane production.

Gpr19 is a circadian clock-controlled orphan GPCR with a role in modulating free-running period and light resetting capacity of the circadian clock

Gpr19 encodes an evolutionarily conserved orphan G-protein-coupled receptor (GPCR) with currently no established physiological role in vivo. We characterized Gpr19 expression in the suprachiasmatic nucleus (SCN), the locus of the master circadian clock in the brain, and determined its role in the context of the circadian rhythm regulation. We found that Gpr19 is mainly expressed in the dorsal part of the SCN, with its expression fluctuating in a circadian fashion. A conserved cAMP-responsive element in the Gpr19 promoter was able to produce circadian transcription in the SCN. Gpr19−/− mice exhibited a prolonged circadian period and a delayed initiation of daily locomotor activity. Gpr19 deficiency caused the downregulation of several genes that normally peak during the night, including Bmal1 and Gpr176. In response to light exposure at night, Gpr19−/− mice had a reduced capacity for light-induced phase-delays, but not for phase-advances. This defect was accompanied by reduced response of c-Fos expression in the dorsal region of the SCN, while apparently normal in the ventral area of the SCN, in Gpr19−/− mice. Thus, our data demonstrate that Gpr19 is an SCN-enriched orphan GPCR with a distinct role in circadian regulation and may provide a potential target option for modulating the circadian clock.

Whole-Genome Sequencing on 220 Alfalfa (Medicago sativa L.) Accessions Identified Loci Associated with Fall Dormancy

Fall dormancy (FD) is one of the most important traits of alfalfa (Medicago sativa) for cultivar selection to overcome winter damage. Regrowth plant height following autumn clipping is an indirect way to evaluate FD. Although transcriptomics, proteomics analysis, and QTL mapping have revealed some important genes correlated with FD, the genetic architecture of this trait is still unclear. There are no applicable genes or markers for selection, which hinders progress in the genetic research and molecular breeding for the trait. We conducted whole-genome sequencing (WGS) on 220 alfalfa accessions at 10x depth. Among the 875,023 SNPs, seven of them were associated with FD using genome-wide association study (GWAS). One SNP located on chromosome 6 is in linkage disequilibrium with dehydration-responsive element-binding protein 1C (DREB1C). Furthermore, seven DREB genes are clustered in this region, one of which has previously been shown to enhance freezing tolerance in the model plant Medicago truncatula. The candidate genes uncovered by our research will benefit the transgenic and CRISPR-Cas9 research of FD in alfalfa. This gene will also be useful for marker development and assisted selection of FD for alfalfa.