Splice Isoforms Research Articles

Deletion of Crkl Splice Isoforms in the Mouse Kidney Disrupts Intercompartmental Signaling Required for Progenitor Renewal and Branched Morphogenesis

Deletion of Crkl Splice Isoforms in the Mouse Kidney Disrupts Intercompartmental Signaling Required for Progenitor Renewal and Branched Morphogenesis

SCR106 splicing factor modulates abiotic stress responses by maintaining RNA splicing in rice.

Plants employ sophisticated molecular machinery to fine-tune their responses to growth, developmental, and stress cues. Gene expression influences plant cellular responses through regulatory processes such as transcription and splicing. Pre-mRNA is alternatively spliced to increase the genome coding potential and further regulate expression. Serine/arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize splicing cis-elements and regulate both constitutive and alternative splicing. Several studies have reported SR protein genes in the rice genome, subdivided into six subfamilies based on their domain structures. Here, we identified a new splicing factor in rice with an RNA recognition motif (RRM) and SR-dipeptides, which is related to the SR proteins, subfamily SC. OsSCR106 regulates pre-mRNA splicing under abiotic stress conditions. It localizes to the nuclear speckles, a major site for pre-mRNA splicing in the cell. The loss-of-function scr106 mutant is hypersensitive to salt, abscisic acid, and low-temperature stress, and harbors a developmental abnormality indicated by the shorter length of the shoot and root. The hypersensitivity to stress phenotype was rescued by complementation using OsSCR106 fused behind its endogenous promoter. Global gene expression and genome-wide splicing analysis in wild-type and scr106 seedlings revealed that OsSCR106 regulates its targets, presumably through regulating the alternative 3'-splice site. Under salt stress conditions, we identified multiple splice isoforms regulated by OsSCR106. Collectively, our results suggest that OsSCR106 is an important splicing factor that plays a crucial role in accurate pre-mRNA splicing and regulates abiotic stress responses in plants.

Novel OBSCN variants associated with a risk to exercise-intolerance and rhabdomyolysis

Obscurin, encoded by the OBSCN gene, is a muscle protein consisting of three main splice isoforms, obscurin-A, obscurin-B, and obscurin kinase-only protein (also known as KIAA1639 or Obsc-kin). Obscurin is located at the M-band and Z-disks and interacts with titin and myomesin. It plays an important role in the stability and maintenance of the A- and M-bands and the subsarcolemmal organization of the microtubule network. Furthermore, obscurin is involved in Ca2+ regulation and sarcoplasmic reticulum function and is connected to several other muscle proteins. OBSCN gene variants have been reported to be relatively common in inherited cardiomyopathies. Here we reported two young patients with a history of cramps, myalgia, exercise intolerance, rhabdomyolysis, and myoglobinuria without any evidence of concomitant cardiomyopathy in association with novel OBSCN variants (c.24822C>A and c.2653+1G>C). Obscurin-deficient muscle fibers seem to have increased susceptibility to damage triggered by exercise that may lead to rhabdomyolysis. More studies are needed to clarify the diverse clinical phenotypes and the pathophysiology of OBSCN gene variants.

Modulatory role of RNA helicases in MBNL-dependent alternative splicing regulation

Muscleblind-like splicing regulators (MBNLs) activate or repress the inclusion of alternative splicing (AS) events, enabling the developmental transition of fetal mRNA splicing isoforms to their adult forms. Herein, we sought to elaborate the mechanism by which MBNLs mediate AS related to biological processes. We evaluated the functional role of DEAD-box (DDX) RNA helicases, DDX5 and DDX17 in MBNL-dependent AS regulation. Whole-transcriptome analysis and validation approaches revealed a handful of MBNLs-dependent AS events to be affected by DDX5 and DDX17 in mostly an opposite manner. The opposite expression patterns of these two groups of factors during muscle development and coordination of fetal-to-adult splicing transition indicate the importance of these proteins at early stages of development. The identified pathways of how the helicases modulate MBNL splicing activity include DDX5 and DDX17-dependent changes in the ratio of MBNL splicing isoforms and most likely changes in accessibility of MBNL-binding sites. Another pathway involves the mode of action of the helicases independent of MBNL activity. These findings lead to a deeper understanding of the network of interdependencies between RNA-binding proteins and constitute a valuable element in the discussion on developmental homeostasis and pathological states in which the studied protein factors play a significant role.

Proteopathy Linked to Exon-Skipping Isoform of RGR-Opsin Contributes to the Pathogenesis of Age-Related Macular Degeneration.

Proteopathy is believed to contribute to age-related macular degeneration (AMD). Much research indicates that AMD begins in the retinal pigment epithelium (RPE), which is associated with formation of extracellular drusen, a clinical hallmark of AMD. Human RPE produces a drusen-associated abnormal protein, the exon Ⅵ-skipping splice isoform of retinal G protein-coupled receptor (RGR-d). In this study, we investigate the detrimental effects of RGR-d on cultured cells and mouse retina. ARPE-19 cells were stably infected by lentivirus overexpressing RGR or RGR-d and were treated with MG132, sometimes combined with or without endoplasmic reticulum (ER) stress inducer, tunicamycin. RGR and RGR-d protein expression, degeneration pathway, and potential cytotoxicity were explored. Homozygous RGR-d mice aged 8 or 14months were fed with a high-fat diet for 3months and then subjected to ocular examination and histopathology experiments. We confirm that RGR-d is proteotoxic under various conditions. In ARPE-19 cells, RGR-d is misfolded and almost completely degraded via the ubiquitin-proteasome system. Unlike normal RGR, RGR-d increases ER stress, triggers the unfolded protein response, and exerts potent cytotoxicity. Aged RGR-d mice manifest disrupted RPE cell integrity, apoptotic photoreceptors, choroidal deposition of complement C3, and CD86+CD32+ proinflammatory cell infiltration into retina and RPE-choroid. Furthermore, the AMD-like phenotype of RGR-d mice can be aggravated by a high-fat diet. Our study confirmed the pathogenicity of the RGR splice isoform and corroborated a significant role of proteopathy in AMD. These findings may contribute to greater comprehension of the multifactorial causes of AMD.

The Roles of transformer-2 (tra-2) in the Sex Determination and Fertility of Riptortus pedestris, a Hemimetabolous Agricultural Pest.

In most holometabolous insects, transformer-2 (tra-2) is an auxiliary gene required for sex determination, exerting a crucial role in regulating sexual differentiation; however, the study of tra-2 in hemimetabolous insects remains very sparse and limited to just a few species. In this study, we investigated the sequence and expression profile of the tra-2 gene in the bean bug, Riptortus pedestris, an agricultural pest belonging to the Heteroptera order. Three non-sex-specific splicing isoforms of Rptra-2 were found, Rptra-2293, Rptra-2284, and Rptra-2299, which shared most exons and exhibited similar expression throughout all stages of development, with particularly elevated levels in the embryo, ovary, and testis. RNAi knockdown experiments revealed that the suppression of Rptra-2 in nymphs led to abnormal females, characterized the formation of male-specific external genital, and also caused longer nymph duration. Knockdown of the expression of the Rptra-2 gene in newly emergent virgin females would cause ovarian arrest, and injecting the 8th-day virgin females with dsRptra-2 also caused a noticeable decline in the offspring numbers. Conversely, in dsRptra-2-treated males, the testes maintained normal morphology but experienced impaired reproductive capacity, attributed to diminished sperm viability. These findings highlight the crucial role of Rptra-2 in the sex determination and fertility of R. pedestris, providing valuable insights into the sex determination mechanisms of hemimetabolous insects.

Comparative analyses of dynamic transcriptome profiles highlight key response genes and dominant isoforms for muscle development and growth in chicken

BackgroundModern breeding strategies have resulted in significant differences in muscle mass between indigenous chicken and specialized broiler. However, the molecular regulatory mechanisms that underlie these differences remain elusive. The aim of this study was to identify key genes and regulatory mechanisms underlying differences in breast muscle development between indigenous chicken and specialized broiler.ResultsTwo time-series RNA-sequencing profiles of breast muscles were generated from commercial Arbor Acres (AA) broiler (fast-growing) and Chinese indigenous Lushi blue-shelled-egg (LS) chicken (slow-growing) at embryonic days 10, 14, and 18, and post-hatching day 1 and weeks 1, 3, and 5. Principal component analysis of the transcriptome profiles showed that the top four principal components accounted for more than 80% of the total variance in each breed. The developmental axes between the AA and LS chicken overlapped at the embryonic stages but gradually separated at the adult stages. Integrative investigation of differentially-expressed transcripts contained in the top four principal components identified 44 genes that formed a molecular network associated with differences in breast muscle mass between the two breeds. In addition, alternative splicing analysis revealed that genes with multiple isoforms always had one dominant transcript that exhibited a significantly higher expression level than the others. Among the 44 genes, the TNFRSF6B gene, a mediator of signal transduction pathways and cell proliferation, harbored two alternative splicing isoforms, TNFRSF6B-X1 and TNFRSF6B-X2. TNFRSF6B-X1 was the dominant isoform in both breeds before the age of one week. A switching event of the dominant isoform occurred at one week of age, resulting in TNFRSF6B-X2 being the dominant isoform in AA broiler, whereas TNFRSF6B-X1 remained the dominant isoform in LS chicken. Gain-of-function assays demonstrated that both isoforms promoted the proliferation of chicken primary myoblasts, but only TNFRSF6B-X2 augmented the differentiation and intracellular protein content of chicken primary myoblasts.ConclusionsFor the first time, we identified several key genes and dominant isoforms that may be responsible for differences in muscle mass between slow-growing indigenous chicken and fast-growing commercial broiler. These findings provide new insights into the regulatory mechanisms underlying breast muscle development in chicken.

Abl2 repairs microtubules and phase separates with tubulin to promote microtubule nucleation

Abl family kinases are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation, both invitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics are unclear. We report here that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. We find that Abl2 promotes MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo liquid-liquid phase separation (LLPS) and form co-condensates with tubulin. Abl2 binds to regions adjacent to MT damage, facilitates MT repair via fresh tubulin recruitment, and increases MT rescue frequency and lifetime. Cryo-EM analyses strongly support a model in which Abl2 engages tubulin C-terminal tails along an extended MT lattice conformation at damage sites to facilitate repair via fresh tubulin recruitment. Abl2Δ688-790, which closely mimics a naturally occurring splice isoform, retains binding to the MT lattice but does not bind tubulin, promote MT nucleation, or increase rescue frequency. In COS-7 cells, MT reassembly after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared with wild-type cells, and these defects are rescued by re-expression of Abl2, but not Abl2Δ688-790. We propose that Abl2 locally concentrates tubulin to promote MT nucleation and recruits it to defects in the MT lattice to enable repair and rescue.

Comprehensive Profiling of Alternative Splicing and Alternative Polyadenylation during Fruit Ripening in Watermelon (Citrullus lanatus).

Fruit ripening is a highly complicated process that is accompanied by the formation of fruit quality. In recent years, a series of studies have demonstrated post-transcriptional control play important roles in fruit ripening and fruit quality formation. Till now, the post-transcriptional mechanisms for watermelon fruit ripening have not been comprehensively studied. In this study, we conducted PacBio single-molecule long-read sequencing to identify genome-wide alternative splicing (AS), alternative polyadenylation (APA) and long non-coding RNAs (lncRNAs) in watermelon fruit. In total, 6,921,295 error-corrected and mapped full-length non-chimeric (FLNC) reads were obtained. Notably, more than 42,285 distinct splicing isoforms were derived from 5,891,183 intron-containing full-length FLNC reads, including a large number of AS events associated with fruit ripening. In addition, we characterized 21,506 polyadenylation sites from 11,611 genes, 8703 of which have APA sites. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that fructose and mannose metabolism, starch and sucrose metabolism and carotenoid biosynthesis were both enriched in genes undergoing AS and APA. These results suggest that post-transcriptional regulation might potentially have a key role in regulation of fruit ripening in watermelon. Taken together, our comprehensive PacBio long-read sequencing results offer a valuable resource for watermelon research, and provide new insights into the molecular mechanisms underlying the complex regulatory networks of watermelon fruit ripening.

MacroH2A1.1 as a crossroad between epigenetics, inflammation and metabolism of mesenchymal stromal cells in myelodysplastic syndromes

Ineffective hematopoiesis is a hallmark of myelodysplastic syndromes (MDS). Hematopoietic alterations in MDS patients strictly correlate with microenvironment dysfunctions, eventually affecting also the mesenchymal stromal cell (MSC) compartment. Stromal cells are indeed epigenetically reprogrammed to cooperate with leukemic cells and propagate the disease as “tumor unit”; therefore, changes in MSC epigenetic profile might contribute to the hematopoietic perturbations typical of MDS. Here, we unveil that the histone variant macroH2A1 (mH2A1) regulates the crosstalk between epigenetics and inflammation in MDS-MSCs, potentially affecting their hematopoietic support ability. We show that the mH2A1 splicing isoform mH2A1.1 accumulates in MDS-MSCs, correlating with the expression of the Toll-like receptor 4 (TLR4), an important pro-tumor activator of MSC phenotype associated to a pro-inflammatory behavior. MH2A1.1-TLR4 axis was further investigated in HS-5 stromal cells after ectopic mH2A1.1 overexpression (mH2A1.1-OE). Proteomic data confirmed the activation of a pro-inflammatory signature associated to TLR4 and nuclear factor kappa B (NFkB) activation. Moreover, mH2A1.1-OE proteomic profile identified several upregulated proteins associated to DNA and histones hypermethylation, including S-adenosylhomocysteine hydrolase, a strong inhibitor of DNA methyltransferase and of the methyl donor S-adenosyl-methionine (SAM). HPLC analysis confirmed higher SAM/SAH ratio along with a metabolic reprogramming. Interestingly, an increased LDHA nuclear localization was detected both in mH2A1.1-OE cells and MDS-MSCs, probably depending on MSC inflammatory phenotype. Finally, coculturing healthy mH2A1.1-OE MSCs with CD34+ cells, we found a significant reduction in the number of CD34+ cells, which was reflected in a decreased number of colony forming units (CFU-Cs). These results suggest a key role of mH2A1.1 in driving the crosstalk between epigenetic signaling, inflammation, and cell metabolism networks in MDS-MSCs.

Metabolic regulation of forkhead box P3 alternative splicing isoforms and their impact on health and disease.

Forkhead Box P3 (FOXP3) is crucial for the development and suppressive function of human regulatory T cells (Tregs). There are two predominant FOXP3 splicing isoforms in healthy humans, the full-length isoform and the isoform lacking exon 2, with different functions and regulation mechanisms. FOXP3 splicing isoforms show distinct abilities in the cofactor interaction and the nuclear translocation, resulting in different effects on the differentiation, cytokine secretion, suppressive function, linage stability, and environmental adaptation of Tregs. The balance of FOXP3 splicing isoforms is related to autoimmune diseases, inflammatory diseases, and cancers. In response to environmental challenges, FOXP3 transcription and splicing can be finely regulated by T cell antigen receptor stimulation, glycolysis, fatty acid oxidation, and reactive oxygen species, with various signaling pathways involved. Strategies targeting energy metabolism and FOXP3 splicing isoforms in Tregs may provide potential new approaches for the treatment of autoimmune diseases, inflammatory diseases, and cancers. In this review, we summarize recent discoveries about the FOXP3 splicing isoforms and address the metabolic regulation and specific functions of FOXP3 splicing isoforms in Tregs.

Identification and functional regulation of three alternative splicing isoforms of the fthl27 gene in miiuy croaker, Miichthys miiuy

Alternative splicing is an important basic mechanism for eukaryotes to control gene expression. Different forms of alternative splicing may lead to the production of protein subtypes with different functions, leading to the expansion of protein diversity in organisms, affecting cell production and metabolism, and is even related to the occurrence of many diseases. Many studies have shown that ferritin is usually associated with inflammation, vascular proliferation, and tumors, which is the focus of immunological research. It not only plays a role in iron metabolism and storage in the body, but also plays an important regulatory role in pathways related to immune and inflammatory regulation. However, there are few studies on alternative splicing events of the ferritin gene nowadays. Therefore, this study identified three different splicing isoforms in its ferritin gene fthl27 of Miichthys miiuy through Sanger sequencing, qRT-PCR, and other experimental techniques, and we found that three different splicing isoforms of the ferritin gene fthl27 in M. Miiuy cells showed an upregulation trend after being stimulated by Lipopolysaccharide (LPS) and poly (I: C). The experiment also found that the three isoforms may have different regulatory effects on the expression of inflammatory factors and antiviral immune factors, playing an important role in the innate immune response of fish.

Crosstalk between m6A modification and alternative splicing during cancer progression.

Background N6-methyladenosine (m6A), the most prevalent internal mRNA modification in eukaryotes, isadded bym6Amethyltransferases, removed by m6A demethylases and recognised by m6A-binding proteins. This modification significantly influences carious facets of RNA metabolism and plays a pivotal role in cellular and physiological processes. Main body Pre-mRNA alternative splicing, a process that generates multiple splice isoforms from multi-exon genes, contributes significantly to the protein diversity in mammals. Moreover, the presence of crosstalk between m6A modification and alternative splicing, with m6A modifications on pre-mRNAs exerting regulatory control, has been established. The m6A modification modulates alternative splicing patterns by recruiting specific RNA-binding proteins (RBPs) that regulate alternative splicing or by directly influencing the interaction between RBPs and their target RNAs. Conversely, alternative splicing can impact the deposition or recognition of m6A modification on mRNAs. The integration of m6A modifications has expanded the scope of therapeutic strategies for cancer treatment, while alternative splicing offers novel insights into the mechanistic role of m6A methylation in cancer initiation and progression. Conclusion This review aims to highlight the biological functions of alternative splicing of m6A modification machinery and its implications in tumourigenesis. Furthermore, we discuss the clinical relevance of understanding m6A-dependent alternative splicing in tumour therapies.

A robust pipeline for high-content, high-throughput immunophenotyping reveals age- and genetics-dependent changes in blood leukocytes

High-dimensional flow cytometry is the gold standard to study the human immune system in large cohorts. However, large sample sizes increase inter-experimental variation because of technical and experimental inaccuracies introduced by batch variability. Our high-throughput sample processing pipeline in combination with 28-color flow cytometry focuses on increased throughput (192 samples/experiment) and high reproducibility. We implemented quality control checkpoints to reduce technical and experimental variation. Finally, we integrated FlowSOM clustering to facilitate automated data analysis and demonstrate the reproducibility of our pipeline in a study with 3,357 samples. We reveal age-associated immune dynamics in 2,300 individuals, signified by decreasing T and B cell subsets with age. In addition, by combining genetic analyses, our approach revealed unique immune signatures associated with a single nucleotide polymorphism (SNP) that abrogates CD45 isoform splicing. In summary, we provide a versatile and reliable high-throughput, flow cytometry-based pipeline for immune discovery and exploration in large cohorts.

Multiple Sgip1 splice variants inhibit cannabinoid receptor 1 internalization

Alternative splicing can often result in the expression of distinct protein isoforms from a single gene, with specific composition and properties. SH3-containing GRB2-like protein 3-interacting protein 1 (Sgip1) is a brain-enriched protein that regulates clathrin-mediated endocytosis and interferes with the internalization of cannabinoid receptor 1. Several research groups have studied the physiological importance of Sgip1, and four Sgip1 protein isoforms have been described to date, while the NCBI Gene database predicts the expression of 20 splice variants from the Sgip1 gene in mice. In this work, we cloned 15 Sgip1 splice variants from the mouse brain, including 11 novel splice variants. The cloned splice variants differed in exon composition within two Sgip1 regions: the membrane phospholipid-binding domain and the proline-rich region. All the Sgip1 splice isoforms had similar stability and comparable ability to inhibit the internalization of cannabinoid receptor 1. None of the isoforms influenced the internalization of the µ-opioid receptor. We confirm the expression of Sgip1 splice variants described in previous studies or predicted in silico. Our data provide a basis for further studies exploring the significance of Sgip1 splicing, and we suggest a new classification of Sgip1 splice variants to unify their nomenclature.

Identification of herpesvirus transcripts from genomic regions around the replication origins

Long-read sequencing (LRS) techniques enable the identification of full-length RNA molecules in a single run eliminating the need for additional assembly steps. LRS research has exposed unanticipated transcriptomic complexity in various organisms, including viruses. Herpesviruses are known to produce a range of transcripts, either close to or overlapping replication origins (Oris) and neighboring genes related to transcription or replication, which possess confirmed or potential regulatory roles. In our research, we employed both new and previously published LRS and short-read sequencing datasets to uncover additional Ori-proximal transcripts in nine herpesviruses from all three subfamilies (alpha, beta and gamma). We discovered novel long non-coding RNAs, as well as splice and length isoforms of mRNAs. Moreover, our analysis uncovered an intricate network of transcriptional overlaps within the examined genomic regions. We demonstrated that herpesviruses display distinct patterns of transcriptional overlaps in the vicinity of or at the Oris. Our findings suggest the existence of a ‘super regulatory center’ in the genome of alphaherpesviruses that governs the initiation of both DNA replication and global transcription through multilayered interactions among the molecular machineries.

Long-read sequencing reveals the landscape of aberrant alternative splicing and novel therapeutic target in colorectal cancer

BackgroundAlternative splicing complexity plays a vital role in carcinogenesis and cancer progression. Improved understanding of novel splicing events and the underlying regulatory mechanisms may contribute new insights into developing new therapeutic strategies for colorectal cancer (CRC).MethodsHere, we combined long-read sequencing technology with short-read RNA-seq methods to investigate the transcriptome complexity in CRC. By using experiment assays, we explored the function of newly identified splicing isoform TIMP1 Δ4-5. Moreover, a CRISPR/dCasRx-based strategy to induce the TIMP1 exon 4–5 exclusion was introduced to inhibit neoplasm growth.ResultsA total of 90,703 transcripts were identified, of which > 62% were novel compared with current transcriptome annotations. These novel transcripts were more likely to be sample specific, expressed at relatively lower levels with more exons, and oncogenes displayed a characteristic to generate more transcripts in CRC. Clinical outcome data analysis showed that 1472 differentially expressed alternative splicing events (DEAS) were tightly associated with CRC patients’ prognosis, and many novel isoforms were likely to be important determinants for patient survival. Among these, newly identified splicing isoform TIMP1 Δ4-5 was significantly downregulated in CRC. Further in vitro and in vivo assays demonstrated that ectopic expression of TIMP1 Δ4-5 significantly suppresses tumor cell growth and metastasis. Serine/arginine-rich splicing factor 1 (SRSF1) acts as a onco-splicing regulator through sustaining the inclusion of TIMP1 exon 4–5. Furthermore, CRISPR/dCasRx-based strategies designed to induce TIMP1 exon 4–5 exclusion have the potential to restrain the CRC growth.ConclusionsThis data provides a rich resource for deeper studies of gastrointestinal malignancies. Newly identified splicing isoform TIMP1 Δ4-5 plays an important role in mediating CRC progression and may be a potential therapy target in CRC.

High-quality single-cell transcriptomics from ovarian histological sections during folliculogenesis.

High-quality, straightforward single-cell RNA sequencing (RNA-seq) with spatial resolution remains challenging. Here, we developed DRaqL (direct RNA recovery and quenching for laser capture microdissection), an experimental approach for efficient cell lysis of tissue sections, directly applicable to cDNA amplification. Single-cell RNA-seq combined with DRaqL allowed transcriptomic profiling from alcohol-fixed sections with efficiency comparable with that of profiling from freshly dissociated cells, together with effective exon-exon junction profiling. The combination of DRaqL with protease treatment enabled robust and efficient single-cell transcriptome analysis from formalin-fixed tissue sections. Applying this method to mouse ovarian sections, we were able to predict the transcriptome of oocytes by their size and identified an anomaly in the size-transcriptome relationship relevant to growth retardation of oocytes, in addition to detecting oocyte-specific splice isoforms. Furthermore, we identified differentially expressed genes in granulosa cells in association with their proximity to the oocytes, suggesting distinct epigenetic regulations and cell-cycle activities governing the germ-soma relationship. Thus, DRaqL is a versatile, efficient approach for high-quality single-cell RNA-seq from tissue sections, thereby revealing histological heterogeneity in folliculogenic transcriptome.

Alternative splicing variants of stimulator of interferon genes (STING) from Asian seabass (Lates calcarifer) and their immune response against red spotted grouper nervous necrosis virus (RGNNV)

The Stimulator of Interferon Genes (STING, also known as MITA/ERYS/MPYS) is an adaptor molecule that plays a crucial role in the RLR pathway and responds to DNA and RNA viruses. In the present study, we have identified two novel isoforms of STING (the canonical form named as LcSTINGa and its alternative splicing isoform named as LcSTINGb) from teleost Lates calcarifer. LcSTINGa has an ORF of 1230 bp, encoding a 409 amino acid protein, while its alternative splicing variant, LcSTINGb, features an ORF of 987 bp, encoding 328 amino acids. LcSTINGa is predicted to contain four transmembrane helices, whereas LcSTINGb has only two. The Lates STING protein showed about 86.85% identity with Perca flavescens, 86.45% with Seriola and 39.51% with Homo sapiens. The tissue distribution studies revealed that the STING variants were constitutively expressed in all the tissues examined, with the highest expression in blood. In-vivo upregulation of LcSTINGa and LcSTINGb mRNA following immune challenge with poly (I:C), Red-spotted grouper nervous necrosis virus (RGNNV) and zymosan A suggests its significance in the immune response.

A geraniol synthase regulates plant defense via alternative splicing in tea plants.

Geraniol is an important contributor to the pleasant floral scent of tea products and one of the most abundant aroma compounds in tea plants; however, its biosynthesis and physiological function in response to stress in tea plants remain unclear. The proteins encoded by the full-length terpene synthase (CsTPS1) and its alternative splicing isoform (CsTPS1-AS) could catalyze the formation of geraniol when GPP was used as a substrate in vitro, whereas the expression of CsTPS1-AS was only significantly induced by Colletotrichum gloeosporioides and Neopestalotiopsis sp. infection. Silencing of CsTPS1 and CsTPS1-AS resulted in a significant decrease of geraniol content in tea plants. The geraniol content and disease resistance of tea plants were compared when CsTPS1 and CsTPS1-AS were silenced. Down-regulation of the expression of CsTPS1-AS reduced the accumulation of geraniol, and the silenced tea plants exhibited greater susceptibility to pathogen infection than control plants. However, there was no significant difference observed in the geraniol content and pathogen resistance between CsTPS1-silenced plants and control plants in the tea plants infected with two pathogens. Further analysis showed that silencing of CsTPS1-AS led to a decrease in the expression of the defense-related genes PR1 and PR2 and SA pathway-related genes in tea plants, which increased the susceptibility of tea plants to pathogens infections. Both in vitro and in vivo results indicated that CsTPS1 is involved in the regulation of geraniol formation and plant defense via alternative splicing in tea plants. The results of this study provide new insights into geraniol biosynthesis and highlight the role of monoterpene synthases in modulating plant disease resistance via alternative splicing.