Exon Intron Research Articles

Genome-Wide Identification, Characterization of the ORA (Olfactory Receptor Class A) Gene Family, and Potential Roles in Bile Acid and Pheromone Recognition in Mandarin Fish (Siniperca chuatsi)

The ORA (olfactory receptor class A) gene family in teleosts is related to the V1R (vomeronasal 1 receptors) family in mammals and plays a key role in odor detection. Although ORA genes have been identified in several teleosts, their characteristics in mandarin fish (Siniperca chuatsi) have not been explored. In this study, we conducted a comprehensive genomic analysis of the mandarin fish and discovered a complete ORA gene family consisting of five members located on chromosome 2 (ORA1, ORA2, ORA3, ORA4) and chromosome 16 (ORA6). Phylogenetic, synteny, and gene structure analyses revealed typical exon–intron conservation with strong evidence of purifying selection. Tissue expression analysis showed distinct expression profiles for each ORA gene, with some showing sexual dimorphism in specific tissues. The expression of ORA1 and ORA2 in the olfactory epithelium exhibits sexual dimorphism, while ORA3 shows sexual dimorphism in the brain. In situ hybridization confirmed that ORA1, ORA2, ORA3, and ORA6 are expressed in the microvillar sensory neurons of the olfactory epithelium, while ORA4 is expressed in crypt cells. Additionally, molecular docking simulations indicated that the five ORA proteins have a high binding affinity with seven bile acids (LAC, GLAC, CA, TLCA, 3-KLCA, 7-KLCA, and 12-KLCA), with ORAs showing stronger binding affinity with LCA and CA. This study comprehensively characterizes the ORA gene family in mandarin fish, examining its phylogeny, synteny, gene structure, and selection pressure. Furthermore, we found that each ORA displays a distinct expression pattern across multiple tissues, with notable sexual dimorphism, and shows potential binding interactions with specific bile acids and pheromones. Our findings provide valuable insights that enhance the overall understanding of fish ORAs and their potential functions.

Characterization of the SWEET Gene Family in Blueberry (Vaccinium corymbosum L.) and the Role of VcSWEET6 Related to Sugar Accumulation in Fruit Development

Sugars will eventually be exported transporters (SWEETs) are essential transmembrane proteins involved in plant growth, stress responses, and plant–pathogen interactions. Despite their importance, systematic studies on SWEETs in blueberries (Vaccinium corymbosum L.) are limited. Blueberries are recognized for their rapid growth and the significant impact of sugar content on fruit flavor, yet the role of the SWEET gene family in sugar accumulation during fruit development remains unclear. In this study, 23 SWEET genes were identified in blueberry, and their phylogenetic relationships, duplication events, gene structures, cis-regulatory elements, and expression profiles were systematically analyzed. The VcSWEET gene family was classified into four clades. Structural and motif analysis revealed conserved exon–intron organization within each clade. RT-qPCR analysis showed widespread expression of VcSWEETs across various tissues and developmental stages, correlating with promoter cis-elements. VcSWEET6a, in particular, was specifically expressed in fruit and showed reduced expression during fruit maturation. Subcellular localization indicated that VcSWEET6a is located in the endoplasmic reticulum. Functional assays in yeast confirmed its role in glucose and fructose uptake, with transport activity inhibited at higher sugar concentrations. Overexpression of VcSWEET6a in blueberries resulted in reduced sugar accumulation. These findings offer valuable insights into the role of VcSWEETs in blueberry sugar metabolism.

Genome-Wide Identification of the GRAS Transcription Factor Family in Medicago ruthenica and Expression Analysis Under Drought Stress

The GRAS gene family encodes a group of plant-specific transcription factors essential for regulating plant growth, development and stress responses. While the GRAS gene family has been extensively studied in various plant species, a comprehensive characterization of the GRAS gene family in Medicago ruthenica has not yet been conducted. In this study, a total of 62 MrGRAS gene family members were identified through a comprehensive whole-genome analysis of M. ruthenica, and phylogenetic analysis categorized these 62 genes into 13 distinct groups. Gene structure and conserved domain analysis showed that MrGRAS genes from the same evolutionary branch share similar exon–intron architecture and conserved motifs. A large number of hormone-responsive, growth and development and stress-responsive cis-regulatory elements were detected in the upstream sequences of MrGRAS genes. RT-qPCR analysis showed that drought stress significantly induced the expression of nine selected MrGRAS genes. Overall, this study analyzed the phylogenetic relationships, conserved domains, cis-regulatory elements and expression patterns of the GRAS gene family in M. ruthenica, filling the gap in the identification of the MrGRAS gene family and laying the foundation for functional analysis of the MrGRAS gene family.

Assessing the genetic composition of invasive knotweeds (Reynoutria; Polygonaceae) using data from the first intron and second exon of the nuclear LEAFY gene

Abstract In places where multiple related taxa are invasive and known to hybridize, it is important to have correct identifications to enable an appropriate legal, ecological, and management understanding of each kind of invader. Invasive knotweeds in the genus Reynoutria Houtt. are noxious weeds in Europe, North America, Africa, and Oceania, where they disrupt native plant communities and negatively impact human activities. Two species (R. japonica Houtt., R. sachalinensis (F.Schmidt) Nakai) and their hybrid (known as R. × bohemica Chrtek & Chrtková) have similar invasive tendencies, although there are some noted differences among them in their reproduction potential, ecological tolerance, and effect on native communities. Prior studies demonstrated that there is not only one kind of interspecific hybrid, but in fact at least four kinds that differ in the sequence variants they possess from each parent. Thus, in addition to identifying plants as hybrids, it may become important to distinguish each kind of hybrid when considering control or treatment strategies. In the current study, we expand the available genetic information for invasive knotweeds by providing expanded DNA sequence data for the low-copy nuclear gene LEAFY, which has become important for characterizing hybrids. Our methods recover the same LEAFY genotypes that were identified previously for the commonly sequenced second intron, and we also provide sequence data for the first intron and second exon of the gene.

Advanced perspectives of biocomputational gene discovery

Gene discovery is one of the most significant processes in understanding and analysis of an organism’s genome after its sequencing. The method comprises determining the location of intron structures, exon structures, and open reading frames (ORFs). It computationally specifies all of the genes with near 100% reliability. It can substantially reduce the amount of wet lab experimentation. In eukaryotic genomes, mosaic organization occurs when a gene is divided into exons by intervening noncoding regions (called introns). Several methods are available for gene finding such as laboratory-based approaches, feature-based approaches homology-based approaches, and statistical and HMM-based approaches. In this paper, we aim to discuss in silco approaches for gene prediction with available bioinformatics tools for gene finding.

Genome-Wide Analysis of the Multidrug and Toxic Compound Extrusion Gene Family in the Tea Plant

The multidrug and toxic compound extrusion (MATE) family is the latest class of novel secondary transporters discovered in plants. However, there is currently no comprehensive analysis of the MATE gene family in the tea plant. In this study, 68 CsMATE genes were identified from the tea plant genome using bioinformatic methods. In general, we analyzed the evolutionary relationships, intron–exon structure, distribution in chromosomes, conserved domains, and gene expression patterns in different tissues and stresses of the CsMATE gene family. The 68 CsMATEs were phylogenetically divided into four major clusters (Class I to Class IV). The CsMATE genes within the same class exhibit similar structural features, while displaying certain distinctions across different classes. Evolutionary analysis indicated that the CsMATE gene family expanded mainly through gene duplication events, in addition to proximal duplication. Through the analysis of cis-acting elements, it was found that CsMATE genes may be involved in the growth, development, and stress response. Furthermore, we observed that certain CsMATE genes could be induced to exhibit expression under abiotic stress conditions such as low temperature, high salinity (NaCl), osmotic stress (PEG), and methyl jasmonate treatment (MeJA). The findings presented herein offer a crucial theoretical foundation for elucidating the biological functions of CsMATE genes, particularly in response to abiotic stress, and furnish valuable potential genetic resources for tea plant resistance breeding.

Abstract PR015: Decoding RNA metabolism by RNA-linked CRISPR screening in human cells

Abstract RNAs undergo a complex choreography of metabolic processes in human cells that are regulated by thousands of RNA-associated proteins. While the effects of individual RNA-associated proteins on RNA metabolism have been extensively characterized, the full complement of regulators for most RNA metabolic events remain unknown. Here we present a massively parallel RNA-linked CRISPR (ReLiC) screening approach to measure the responses of diverse RNA metabolic events to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC screens highlight modular interactions between gene networks regulating splicing, translation, and decay of mRNAs. When combined with biochemical fractionation of polysomes, ReLiC reveals striking pathway-specific coupling between growth fitness and mRNA translation. Perturbing different components of the translation and proteostasis machineries have distinct effects on ribosome occupancy, while perturbing mRNA transcription leaves ribosome occupancy largely intact. Isoform-selective ReLiC screens capture differential regulation of intron retention and exon skipping by SF3b complex subunits. Chemogenomic screens using ReLiC decipher translational regulators upstream of mRNA decay and uncover a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a versatile platform for discovering and dissecting regulatory principles of human RNA metabolism. Citation Format: Patrick J Nugent, Heungwon Park, Arvind Rasi Subramaniam. Decoding RNA metabolism by RNA-linked CRISPR screening in human cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR015.

Targeting Poly(U) Binding Splicing Factor 60 (PUF60): A Small-Molecule Inhibitor Shows Anti-Leukemic Activity and Impacts Cell Cycle in Leukemia Models

The emerging role of altered RNA splicing in the development of myeloid neoplasms including MDS and AML has received wide attention. Facilitating genes, including the frequently mutated splicing factors, SF3B1, U2AF1, and SRSF2, have been reported to play a key role in leukemogenesis. Pharmacological intervention targeting cells harboring these mutations has been pursued, but limited agents have been reported. Rare events of more than one hit in splicing factors were found in MDS and AML patients' samples. This suggests that inhibiting another target in the splicing process may overcome the threshold of survival in these vulnerable cells carrying splicing factor mutations and induce toxicity. The concept has been demonstrated in the case of leukemia cells carrying U2AF1 mutations being vulnerable to inhibitors modulating wild-type SF3B1 function. The primary function of U2AF1 is to form a complex with U2AF2 for 3‘ splice site (SS) definition in RNA splicing and lower frequency U2AF2 mutations are also detected in MDS patients, underlying the significance of dysfunctional 3‘ SS definition as a potential facilitator of leukemogenesis. Here, we hypothesize that compounds inhibiting proteins participating in 3' SS selection may disrupt isoform patterns in leukemia cells. These agents may act alone or serve as a second hit in the RNA processing to invoke additional vulnerabilities to leukemia cells carrying the most frequent splicing factor mutations. Definition of 3' SS by U2AF2/U2AF1 is mediated by the recognition of the polypyridine tract (PPT) by U2AF2 and the binding of U2AF1 to the conserved AG dinucleotides at the 3' SS. Like U2AF2, PUF60 contains two zinc-finger domains, recognizing the polyuridine tract preceding 3' SS, and a U2 homology motif (UHM) domain that engages in protein-protein interaction with partner proteins including U2AF2, SF1, and SF3B1. Different from the U2AF1/U2AF2 complex, PUF60 does not have a protein domain to bind to the exon intron boundary junction at the 3' SS. PUF60 has been reported to enhance the splicing activity of U2AF1/U2AF2, but also directly regulates alternative splicing of a subset of genes. A recent finding indicated that knockout of PUF60 leads to alternative splicing switching of CDC25 from isoform A to isoform C to induce cell cycle arrest at G2/M in solid tumors. Leukemia patients carrying PUF60 overexpression have also been found to have less progression free survival (https://ualcan.path.uab.edu/cgi-bin/TCGA-survival1.pl?genenam=PUF60&ctype=LAML) compared to patients with low expression of PUF60. To support our hypothesis, we undertook an inhibitor development campaign to discover small-molecule compounds selectively targeting PUF60. Based on a relatively less selective and weak inhibitor to the UHM domain, SF-153, we conducted chemical modifications and obtained a PUF60 inhibitor, SF2-69, with >15-fold selectivity against U2AF1, RBM39, SPF45, and U2AF2. We found SF2-69 had an IC50 value of ~3 mM in NKM-1 and K562 leukemia cell lines. In the cell cycle analysis, we found SF2-69 increased S and subG1 cell population in NKM-1 at 24h, but induced G1 arrest in K562 at 48h in a dose dependent manner. In comparison, E7820, an RBM39 degrader, increased G2/M phase in both NKM-1 and K562. RNA-seq analysis of NKM-1 treated with SF2-69 after 24h revealed that SF2-69 upregulated a set of genes participating in cholesterol biosynthesis, NME1-NME2 (a tumor suppressor kinase) and downregulated PLK1, a kinase that phosphorylates and activates CDC25C in G2/M transition. Because K562 is p53-null and NKM-1 has wild-type p53, SF2-69 likely induced p53-dependent and p53-independent responses in NKM-1 and K562 cells, respectively. In summary, SF2-69 is a new selective PUF60 inhibitor which disrupts cell cycle progression in leukemia cell lines by modulating p53-mediated responses. Upregulation of cholesterol biosynthesis may be a feedback loop to counteract the inhibition of PUF60 by SF2-69. Further optimization of SF2-69 will allow us to develop more effective and selective chemical probes for studying the role of PUF60 overexpression in leukemia and the potential use of PUF60 inhibitors in MDS and AML cells carrying splicing factor mutations.

Identification and molecular marker development for peel color gene in melon (Cucumis melo L.)

Peel color is an important appearance quality of melons that greatly affects consumer preferences. In this study, a near-isogenic line NIL-G (dark green peel) was generated from B8 (grey-green peel) and B15 (white peel). The F2 population constructed by crossing NIL-G and B15 was used to study the inheritance pattern of peel color, and bulked-segregant analysis sequencing (BSA-seq) was employed to identify the interval in which the target gene was located. Genetic analysis results showed that the dark green peel trait at maturity is controlled by a dominant gene. BSA-seq and molecular markers were used to localize the candidate gene in a 263.7 kb interval of chromosome 4, which contained the CmAPRR2 gene with known functions. Moreover, allelic sequence analysis revealed four SNP variations of the CmAPRR2 gene in B15, of which SNP.G614331A was located at the junction of the 6th exon and 6th intron. The G-to-A mutation caused alternative splicing of the transcript of CmAPRR2 in B15, generating two transcripts (CmAPRR2-A and CmAPRR2-B) with premature termination codons. Furthermore, the Kompetitive Allele Specific PCR (KASP) marker, APRR2-G/A, was developed based on this SNP and shown to co-segregate with the peel color phenotype in the F2 population. Compared to white-peel B15, the expression level of CmAPRR2 in dark green peel NIL-G was higher at each growth stage. Therefore, CmAPRR2 may be the key gene controlling the fruit color of melons. Overall, this study identified a novel allelic variant of CmAPRR2 that leads to white peel formation in mature melons. The study also provides a theoretical basis for further research on the gene regulatory mechanism of melon peel colors and may promote the future use of molecular marker-assisted selection to modify melon peel colors.

In silico analysis of R2R3-MYB transcription factors in the basal eudicot model, Aquilegia coerulea

R2R3-MYBs are an important group of transcription factors that regulate crucial developmental processes across the plant kingdom; yet no comprehensive analysis of the R2R3-MYBs in the early-diverging eudicot clade of Ranunculaceae has been conducted so far. In the present study, Aquilegia coerulea is chosen to understand the extent of conservation and divergence of R2R3-MYBs as a representative of the family by analysing the genomic distribution, organization, gene structure, physiochemical properties, protein architecture, evolution and possible mode of expansion. Genome-wide analysis showed the presence of 82 putative homologues classified into 21 subgroups, based on phylogenetic analysis of full-length protein sequences. The domain has remained largely conserved across all homologues with few differences from the characterized Arabidopsis thaliana R2R3-MYBs. The topology of the phylogenetic tree remains the same when full-length protein sequences are used, indicating that the evolution of R2R3-MYBs is driven by the domain region only. This is supported by the presence of similar structures of exon–intron and conserved motifs within the same subgroup. Furthermore, comparisons of the AqcoeR2R3-MYB members with monocots and core-eudicots revealed the evolutionary expansion of a few functional clades, such as A. thaliana R2R3-MYB subgroup 6 (SG6), the upstream regulatory factors of floral pigment biosynthesis and floral color. The reconstructed evolutionary history of SG6-like genes across angiosperms highlights the occurrence of independent duplication events in the genus Aquilegia. AqcoeR2R3-MYB genes are present in all seven chromosomes of A. coerulea, most of which result from local and segmental duplications. Selection analysis of these duplicated gene pairs indicates purifying selection except one, and the physiochemical analyses of R2R3-MYBs reveal differences among the MYBs signifying their functional diversification. This study paves the way for further investigation of paralogous copies and their probable role in the evolution of different floral traits in A. coerulea. It lays the foundation for functional genomic studies of R2R3-MYBs in the basal eudicots and facilitates comparative studies among angiosperms. The work also provides a framework for deciphering novel genetic regulatory pathways that govern the diversity of floral morphology.

Characterization of Superoxide Dismutase (SOD) Gene Family and Their Responses to Salinity Stress and Fruit Development in Octoploid Strawberry

Superoxide dismutases (SODs), as the first line of defense against reactive oxygen species (ROS), play an essential role in protecting plants from adverse elicitors during plant growth and development. However, little is known about the SOD gene family and their response to salinity stress and fruit development in cultivated strawberries (Fragaria × ananassa). In this study, 32 SOD genes consisting of 16 Cu/ZnSODs, 11 FeSODs, and 5 MnSOD were identified, which presented three well-resolved clades in the phylogenetic tree. Each clade had similar motifs, and exon–intron structures, which in turn supported the evolutionary classification. Cis-acting element analysis suggested that FaSOD genes might be involved in the plant response to abiotic and biotic stresses, hormones, and light. The analysis of previously published transcriptome data revealed that FaSOD genes are expressed variably under salt stress. Among these SODs, FaMSD5 was expressed at relatively high levels in strawberry root and leaf, and its transcript abundance significantly increased after salt treatment. Some transcription factors related to photomorphogenesis, hormone signaling pathways, and hyperosmotic salinity response were predicted to bind to the FaMSD5 promoter. These outcomes implied that FaMSD5 might play an important role in protection against salt stress. In addition, the comprehensive transcriptome analysis of FaSOD genes in strawberry fruit showed that almost all FaCSDs and FaMSDs were more highly expressed than FaFSDs at different developmental stages, and the expression patterns of FaCSD1, FaCSD2, FaCSD7, FaCSD8, and FaCSD10 suggested that they were likely to be involved in fruit development and ripening. This study provides a basis for further exploration of the function of the FaSOD gene family in strawberry and provides candidate FaSOD genes for enhancing salinity tolerance and regulating fruit development and ripening.

SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing.

O-GlcNAcylation is the reversible post-translational addition of β-N-acetylglucosamine to serine and threonine residues of nuclear and cytoplasmic proteins. It plays an important role in several cellular processes through the modification of thousands of protein substrates. O-GlcNAcylation in humans is mediated by a single essential enzyme, O-GlcNAc transferase (OGT). OGT, together with the sole O-GlcNAcase OGA, form an intricate feedback loop to maintain O-GlcNAc homeostasis in response to changes in cellular O-GlcNAc using a dynamic mechanism involving nuclear retention of its fourth intron. However, the molecular mechanism of this dynamic regulation remains unclear. Using an O-GlcNAc responsive GFP reporter cell line, we identify SFSWAP, a poorly characterized splicing factor, as a trans-acting factor regulating OGT intron detention. We show that SFSWAP is a global regulator of retained intron splicing and exon skipping that primarily acts as a negative regulator of splicing. In contrast, knockdown of SFSWAP leads to reduced inclusion of a 'decoy exon' present in the OGT retained intron which may mediate its role in OGT intron detention. Global analysis of decoy exon inclusion in SFSWAP and UPF1 double knockdown cells indicate altered patterns of decoy exon usage. Together, these data indicate a role for SFSWAP as a global negative regulator of pre-mRNA splicing and positive regulator of intron retention.

Glutamine Synthetase and Glutamate Synthase Family Perform Diverse Physiological Functions in Exogenous Hormones and Abiotic Stress Responses in Pyrus betulifolia Bunge (P.be)

The unscientific application of nitrogen (N) fertilizer not only increases the economic input of pear growers but also leads to environmental pollution. Improving plant N use efficiency (NUE) is the most effective economical method to solve the above problems. The absorption and utilization of N by plants is a complicated process. Glutamine synthetase (GS) and glutamate synthase (GOGAT) are crucial for synthesizing glutamate from ammonium in plants. However, their gene family in pears has not been documented. This study identified 29 genes belonging to the GS and GOGAT family in the genomes of Pyrus betulaefolia (P.be, 10 genes), Pyrus pyrifolia (P.py, 9 genes), and Pyrus bretschneideri (P.br, 10 genes). These genes were classified into two GS subgroups (GS1 and GS2) and two GOGAT subgroups (Fd–GOGAT and NADH–GOGAT). The similar exon–intron structures and conserved motifs within each cluster suggest the evolutionary conservation of these genes. Meanwhile, segmental duplication has driven the expansion and evolution of the GS and GOGAT gene families in pear. The tissue–specific expression dynamics of PbeGS and PbeGOGAT genes suggest significant roles in pear growth and development. Cis–acting elements of the GS and GOGAT gene promoters are crucial for plant development, hormonal responses, and stress reactions. Furthermore, qRT–PCR analysis indicated that PbeGSs and PbeGOGATs showed differential expression under exogenous hormones (GA3, IAA, SA, ABA) and abiotic stress (NO3− and salt stress). In which, the expression of PbeGS2.2 was up–regulated under hormone treatment and down–regulated under salt stress. Furthermore, physiological experiments demonstrated that GA3 and IAA promoted GS, Fd–GOGAT, and NADH–GOGAT enzyme activities, as well as the N content. Correlation analysis revealed a significant positive relationship between PbeGS1.1, PbeGS2.2, PbeNADH–GOGATs, and the N content. Therefore, PbeGS1.1, PbeGS2.2, and PbeNADH–GOGATs could be key candidate genes for improving NUE under plant hormone and abiotic stress response. To the best of our knowledge, our study provides valuable biological information about the GS and GOGAT family in the pear for the first time and establishes a foundation for molecular breeding aimed at developing high NUE pear rootstocks.

Identification of Ascorbate Oxidase Genes and Their Response to Cold Stress in Citrus sinensis

Ascorbate oxidase (AAO) plays an important role in maintaining cellular redox homeostasis, thereby influencing plant growth, development, and responses to both biotic and abiotic stresses. However, there has been no systematic characterization of AAO genes in Citrus, especially their roles in response to cold stress. In the present study, nine AAO genes were identified in C. sinensis through bioinformatics analyses, exhibiting uneven distribution across four chromosomes. All CsAAOs possessed three conserved domains and were predicted to localize in the apoplast. The CsAAO gene family displayed varied intron–exon patterns. Phylogenetic analysis categorized the CsAAO family into three main clades (Clade A–C), suggesting distinct biological functions. Collinearity and Ka/Ks analysis revealed three duplicate gene pairs within the CsAAO gene family, with all duplicated CsAAOs primarily evolving under purifying selection. Analysis of cis-acting elements showed the presence of multiple hormone response elements and stress response elements within the CsAAO promoters. The computational analysis of microRNA target transcripts suggested that CsAAO9 may be a target of csi-miR156. RNA-Seq data demonstrated high expression levels of CsAAOs in roots and young fruits, while qRT-PCR analysis showed significant upregulation of six CsAAOs in response to cold treatment. Furthermore, the activities of CsAAOs exhibited a pattern of initial decrease followed by an increase after exposure to low temperatures. These findings offer important insights into the role of CsAAOs in response to cold stress. Furthermore, AAOs could be target genes for breeding crops with better cold resistance.

Functional characterization vs in silico prediction for TBX5 missense and splice variants in Holt-Oram syndrome

PurposePredicting effects of genomic variants has become a real challenge in the diagnosis of rare human diseases. Holt-Oram syndrome is an autosomal condition characterized by the association of radial and heart defects, due to variants in TBX5. Most variants are predicted to be truncating and result in haploinsufficiency. The pathogenicity of missense or splice variants is harder to demonstrate. MethodsFourteen TBX5 variants of uncertain significance (5 missense, 9 splice) and 6 likely pathogenic missense variants were selected for functional testing, depending on the variant-type (immunolocalization, western blot, reporter assays, minigene splice assays, and reverse transcription-polymerase chain reaction). Results were compared with in silico predictions. ResultsFunctional tests allowed to reclassify 9/14 variants of uncertain significance in TBX5 as likely pathogenic, confirming their role in Holt-Oram syndrome. We demonstrated loss of function (n = 8) or gain of function (n = 1) for 9 of the 11 missense variants, whereas no functional impact was shown for the 2 variants: p.(Gly195Ala) and p.(Ser261Cys), as suggested by contradictory predictions of in silico approaches. Of 9 splice variants predicted to affect splicing by SpliceAI, we observed partial or complete exon skipping (n = 6), intron retention (n = 2) or exon shortening (n = 1), inducing frame shifting with premature stop codons. ConclusionBioinformatic and biological approaches are complementary, together with a good knowledge of clinical conditions, for accurate American College of Medical Genetics and Genomics classification in human rare diseases.

Genome-wide identification and expression analysis of CmoADHs in Cucurbita moschata—Critical role of CmoADH9 in drought tolerance

In plants, alcohol dehydrogenases (ADHs) are involved in stress response, organ development, fruit ripening, and metabolite synthesis. However, little is known regarding ADH-encoding genes (ADHs) in Cucurbita moschata which is usually used as a rootstock for cucumber, melon, watermelon, and other cucurbit crops to resist soil-borne diseases and abiotic stresses. We identified 11 CmoADHs in the C. moschata genome that were unevenly distributed across seven chromosomes. These genes were predicted to encode stable cytoplasmic acidic proteins, sharing a low degree of identity with each other. The genes exhibited different intron–exon structures. Analysis of cis-acting regulatory elements showed that CmoADHs contain environmental stress-, hormone response-, light response-, and development/tissue specificity-related elements in their promoters. Expression pattern analysis revealed that CmoADH2, CmoADH3, CmoADH4, CmoADH9, CmoADH10, and CmoADH11 had the highest expression levels in the roots, which were significantly higher than those in the other tested tissues. These six genes may play important roles in the growth and development of roots, and in related abiotic stress responses. CmoADH1, CmoADH5, CmoADH6, CmoADH7, CmoADH8 had the highest expression in the apical region and could be involved in the differentiation of newly formed tissues. To study the role of CmoADHs in abiotic stress, salt, drought, low temperature, and ethephon treatments were performed. Under drought conditions, CmoADHs showed different expression trends. The expression levels of CmoADH1, CmoADH2, CmoADH3, and CmoADH9 increased significantly and peaked after 1 h of drought treatment, indicating that these four genes are more sensitive to drought stress. Under salt treatment, all CmoADHs showed a significant increase or decrease in expression within 6 h, except for CmoADH5 and CmoADH10, which were insensitive to salt treatment. The expression of most of the CmoADHs was significantly downregulated by low-temperature treatment. Ethephon treatment significantly induced the expression of all the CmoADHs, except CmoADH2, to different degrees within 12 h. CmoADH9 was found to be involved in root growth and drought stress resistance. Identification of these ADH genes can provide useful resources for conferring stress resistance in other economically important crops.

Disruption of nuclear speckle integrity dysregulates RNA splicing in C9ORF72-FTD/ALS

Expansion of an intronic (GGGGCC)n repeat within the C9ORF72 gene is the most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (C9-FTD/ALS), characterized with aberrant repeat RNA foci and noncanonical translation-produced dipeptide repeat (DPR) protein inclusions. Here, we elucidate that the (GGGGCC)n repeat RNA co-localizes with nuclear speckles and alters their phase separation properties and granule dynamics. Moreover, the essential nuclear speckle scaffold protein SRRM2 is sequestered into the poly-GR cytoplasmic inclusions in the C9-FTD/ALS mouse model and patient postmortem tissues, exacerbating the nuclear speckle dysfunction. Impaired nuclear speckle integrity induces global exon skipping and intron retention in human iPSC-derived neurons and causes neuronal toxicity. Similar alternative splicing changes can be found in C9-FTD/ALS patient postmortem tissues. This work identified novel molecular mechanisms of global RNA splicing defects caused by impaired nuclear speckle function in C9-FTD/ALS and revealed novel potential biomarkers or therapeutic targets.

Synaptic gene expression changes in frontotemporal dementia due to the MAPT 10+ 16 mutation.

Mutations in the MAPT gene encoding tau protein can cause autosomal dominant neurodegenerative tauopathies including frontotemporal dementia (often with Parkinsonism). In Alzheimer's disease, the most common tauopathy, synapse loss is the strongest pathological correlate of cognitive decline. Recently, Positron Emission Tomography (PET) imaging with synaptic tracers revealed clinically relevant loss of synapses in primary tauopathies; however, the molecular mechanisms leading to synapse degeneration in primary tauopathies remain largely unknown. In this study, we examined post-mortem brain tissue from people who died with frontotemporal dementia with tau pathology (FTDtau) caused by the MAPT intronic exon 10+ 16 mutation, which increases splice variants containing exon 10 resulting in higher levels of tau with four microtubule-binding domains. We used RNA sequencing and histopathology to examine temporal cortex and visual cortex, to look for molecular phenotypes compared to age, sex and RNA integrity matched participants who died without neurological disease (n= 12 FTDtau10+ 16 and 13 controls). Bulk tissue RNA sequencing reveals substantial downregulation of gene expression associated with synaptic function. Upregulated biological pathways in human MAPT 10+ 16 brain included those involved in transcriptional regulation, DNA damage response and neuroinflammation. Histopathology confirmed increased pathological tau accumulation in FTDtau10 + 16 cortex as well as a loss of presynaptic protein staining and region-specific increased colocalization of phospho-tau with synapses in temporal cortex. Our data indicate that synaptic pathology likely contributes to pathogenesis in FTDtau10 + 16 caused by the MAPT 10+ 16 mutation.

Decoding RNA Metabolism by RNA-linked CRISPR Screening in Human Cells.

RNAs undergo a complex choreography of metabolic processes in human cells that are regulated by thousands of RNA-associated proteins. While the effects of individual RNA-associated proteins on RNA metabolism have been extensively characterized, the full complement of regulators for most RNA metabolic events remain unknown. Here we present a massively parallel RNA-linked CRISPR (ReLiC) screening approach to measure the responses of diverse RNA metabolic events to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC screens highlight modular interactions between gene networks regulating splicing, translation, and decay of mRNAs. When combined with biochemical fractionation of polysomes, ReLiC reveals striking pathway-specific coupling between growth fitness and mRNA translation. Perturbing different components of the translation and proteostasis machineries have distinct effects on ribosome occupancy, while perturbing mRNA transcription leaves ribosome occupancy largely intact. Isoform-selective ReLiC screens capture differential regulation of intron retention and exon skipping by SF3b complex subunits. Chemogenomic screens using ReLiC decipher translational regulators upstream of mRNA decay and uncover a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a versatile platform for discovering and dissecting regulatory principles of human RNA metabolism.

Local and systemic transcriptome and spliceome reprogramming induced by the root-knot nematode Meloidogyne incognita in tomato.

Root-knot nematodes (Meloidogyne spp.) are widely spread root parasites that infect thousands of vascular plant species. These highly polyphagous nematodes engage in sophisticated interactions with host plants that results in the formation of knot-like structures known as galls whose ontogeny remains largely unknown. Here, we determined transcriptome changes and alternative splicing variants induced by Megalaima incognita in galls and neighboring root cells at two distinct infective stages. M. incognita induced substantial transcriptome changes in tomato roots both locally in galls and systemically in neighboring cells. A considerable parallel regulation of gene expression in galls and neighboring cells were detected, indicative of effective intercellular communications exemplified by suppression of basal defense responses particularly during the early stage of infection. The transcriptome analysis also revealed that M. incognita exerts a tight control over the cell cycle process as a whole that results in an increase of ploidy levels in the feeding sites and accelerated mitotic activity of the gall cells. Alternative splicing analysis indicated that M. incognita significantly modulates pre-mRNA splicing as a total of 9064 differentially spliced events from 2898 genes were identified where intron retention and exon skipping events were largely suppressed. Furthermore, a number of differentially spliced events were functionally validated using transgenic hairy root system and found to impact gall formation and nematode egg mass production. Together, our data provide unprecedented insights into the transcriptome and spliceome reprogramming induced by M. incognita in tomato with respect to gall ontogeny and nematode parasitism.