Single Exon Research Articles

Self‐Assembled Antibody‐Oligonucleotide Conjugates for Targeted Delivery of Complementary Antisense Oligonucleotides

AbstractAntibody‐oligonucleotide conjugate (AOC) affords preferential cell targeting and enhanced cellular uptake of antisense oligonucleotide (ASO). Here, we have developed a modular AOC (MAOC) approach based on accurate self‐assembly of separately prepared antibody and ASO modules. Homogeneous multimeric AOC with defined ASO‐to‐antibody ratio were generated by L–DNA scaffold mediated precise self‐assembly of antibodies and ASOs. The MAOC approach has been implemented to deliver exon skipping ASOs via transferrin receptor (TfR1) mediated internalization. We discovered an anti‐TfR1 sdAb that can greatly enhance nuclear delivery of ASOs. Cryo‐EM structure of the sdAb‐TfR1 complex showed a new epitope that does not overlap with the binding sites of endogenous TfR1 ligands. In vivo functional analyses of MAOCs with one ASO for single exon skipping and two ASOs for double exon skipping showed that both ASO concentration and exon skipping efficacy of MAOC in cardiac and skeletal muscles are dramatically higher than conventional ASOs in the transgenic human TfR1 mouse model. MAOC treatment was well tolerated in vivo and not associated with any toxicity‐related morbidity or mortality. Collectively, our data suggest that the self‐assembled MAOC is a viable option for broadening the therapeutic application of ASO via multi‐specific targeting and delivery.

Allelic Diversity and Development of Breeder-Friendly Marker Specific to floury2 Gene Regulating the Accumulation of α-Zeins and Essential Amino Acids in Maize Kernel.

Maize zeins lack essential amino acids, such as methionine, lysine, and tryptophan. The floury2 (fl2) mutation reduces zein synthesis and increases methionine and lysine content in kernels. In this study, fl2 gene (1612bp) was sequenced in eight wild-type and two mutant inbreds and detected 218 SNPs and 18 InDels. Transversion of C to T at 343bp position caused the substitution of alanine by valine in the fl2 mutant. A PCR-based marker (FL-SNP-CT) was developed, which distinguished the favorable mutant fl2 allele (T) from the wild-type (C) Fl2 allele. Gene-based diversity analysis using seven gene-based InDel markers grouped 48 inbred lines into three major clusters, with an average genetic dissimilarity coefficient of 0.534. The average major allele frequency, gene diversity, heterozygosity, and polymorphism information content of the InDel markers were 0.701, 0.392, 0.039, and 0.318, respectively. Haplotype analysis revealed 29 haplotypes of fl2 gene among these 48 inbreds. Amino acid substitution (Ala-Val) at the signal peptide cleavage site produced unprocessed 24-kDa mutant protein instead of 22-kDa zein found in normal genotype. Eight paralogues of fl2 detected in the study showed variation in exon lengths (616-1170bp) and translation lengths (135-267 amino acids). Orthologue analysis among 15 accessions of Sorghum bicolor and two accessions of Saccharum spontaneum revealed a single exon in fl2 gene, ranging from 267 to 810bp. The study elucidated the molecular basis of fl2 mutation and reported a breeder-friendly marker for molecular breeding programs. This is the first study to characterize fl2 gene in a set of subtropically adapted inbreds.

Self-Assembled Antibody-Oligonucleotide Conjugates for Targeted Delivery of Complementary Antisense Oligonucleotides.

Antibody-oligonucleotide conjugate (AOC) affords preferential cell targeting and enhanced cellular uptake of antisense oligonucleotide (ASO). Here, we have developed a modular AOC (MAOC) approach based on accurate self-assembly of separately prepared antibody and ASO modules. Homogeneous multimeric AOC with defined ASO-to-antibody ratio were generated by L-DNA scaffold mediated precise self-assembly of antibodies and ASOs. The MAOC approach has been implemented to deliver exon skipping ASOs via transferrin receptor (TfR1) mediated internalization. We discovered an anti-TfR1 sdAb that can greatly enhance nuclear delivery of ASOs. Cryo-EM structure of the sdAb-TfR1 complex showed a new epitope that does not overlap with the binding sites of endogenous TfR1 ligands. In vivo functional analyses of MAOCs with one ASO for single exon skipping and two ASOs for double exon skipping showed that both ASO concentration and exon skipping efficacy of MAOC in cardiac and skeletal muscles are dramatically higher than conventional ASOs in the transgenic Duchenne muscular dystrophy (DMD) mouse model. MAOC treatment was well tolerated in vivo and not associated with any toxicity-related morbidity or mortality. Collectively, our data suggest that the self-assembled MAOC is a viable option for broadening the therapeutic application of ASO via multi-specific targeting and delivery.

Neonatal sclerosing cholangitis- phenotype genotype observations of novel biallelic variants and single exon deletions in dcdc2 gene in indian children-expanding horizon

Neonatal sclerosing cholangitis- phenotype genotype observations of novel biallelic variants and single exon deletions in dcdc2 gene in indian children-expanding horizon

Evolution of the sex-determination gene Doublesex within the termite lineage

The molecular mechanism of sex determination has long been considered conserved in insects. However, recent studies of hemimetabolous insects have challenged this notion. One notable example is termites. In Reticulitermes speratus, a homolog of sex determination gene, Doublesex (RsDsx), exhibits characteristics that are distinct from those of other insects, including sister-group cockroaches. It comprises a single exon, contains only doublesex/mab-3 DNA-binding domain (DM) but lacks a conserved oligomerization domain (OD), and exhibits transcriptional activity only in males. To investigate whether these characteristics are widespread within the termite lineage, we identified Dsx homologs in three different families. The absence of the conserved OD sequences was observed in all termite species examined, whereas the number of exons and expression patterns between sexes varied among families. Particularly, distinctive differences in Dsx were found in species from the Archotermopsidae and Kalotermitidae, both of which have a linear caste developmental pathway. Our findings indicate that diversification of Dsx structure and expression patterns may have contributed to ecological diversification, such as caste developmental pathways, within the termite lineage.

Characteristics of Rapeseed (Brassica rapa L.) genome DREB family demonstrate their roles in stress

Dehydration-responsive element-binding (DREB) proteins are crucial transcription factors in plant regulatory mechanisms, playing a key role in responding to environmental stresses. Dehydration-responsive element-binding (DREB) proteins are crucial transcription factors in plant regulatory mechanisms, playing a key role in responding to environmental stresses. This study employs bioinformatics research methods to identify and characterize the DREB gene family in rapeseed (Brassica rapa L.) under abiotic stress conditions. The approach facilitates a detailed exploration of the genomic architecture and potential regulatory mechanisms that these genes may exhibit in response to environmental stresses. A whole-genome survey identified 75 DREB genes in rapeseed, which were classified into six subgroups (Ⅰ-Ⅵ) based on phylogenetic comparison with Arabidopsis homologs. The amino acid lengths of these genes ranged from 144 to 530, with predicted molecular weights between 15.63 kDa and 60.36 kDa, and isoelectric points from 4.48 to 9.32. Chromosomal localization revealed that these genes are distributed across 10 chromosomes, predominantly near telomeric regions, with more than 10 DREB genes located on Chr3 and Chr9 respectively. Structural domain analysis indicated that 57 DREB genes possess a single exon. Promoter cis-element analyses suggested that BrDREB genes could respond to diverse stressors, including drought, heat, hormones, and light. Transcriptomic analysis under cold stress revealed significant upregulation of BrDREB17, 24, 49, 50, and 51 in response to low temperature, while five BrDREB genes were not expressed. Specifically, BrDREB17, 24, 49, 50, and 51 showed high expression levels at 3 h of cold stress, whereas BrDREB30, 42, and 67 were highly expressed at 24 h of cold stress. Gene Ontology (GO) analysis demonstrated that the majority of biological processes mediated by BrDREB are associated with plant stress resistance. Protein interaction network analysis revealed that BrDREB9 and BrDREB26 interact with 15 proteins each, underscoring their significant biological roles within the DREB family in rapeseed. These findings provide a theoretical framework for further examination of the physiological and biochemical properties of the DREB gene family components in rapeseed. Our results may help to screen candidate genes for cold resistance in rapeseed and select rapeseed varieties better suited to the Tibetan locality to improve quality and yield in agricultural production.

Geneapp: A web application for visualizing alternative splicing for biomedicine

Alternative Splicing (AS) is an essential mechanism for eukaryotes. However, the consequences of deleting a single exon can be dramatic for the organism and can lead to cancer in humans. Additionally, alternative 5′ and 3′ splice sites, which define the boundaries of exons, also play key roles to human disorders. Therefore, Investigating AS events is crucial for understanding the molecular basis of human diseases and developing therapeutic strategies. Workflow for AS event analysis can be sampling followed by data analysis with bioinformatics to identify the different AS events in the control and case samples, data visualization for curation, and selection of relevant targets for experimental validation. The raw output of the analysis software does not favor the inspection of events by bioinformaticians requiring custom scripts for data visualization. In this work, we propose the Geneapp application with three modules: GeneappScript, GeneappServer, and GeneappExplorer. GeneappScript is a wrapper that assists in identifying AS in samples compared in two different approaches, while GeneappServer integrates data from AS analysis already performed by the user. In GeneappExplorer, the user visualizes the previous dataset by exploring AS events in genes with functional annotation. This targeted screens that Geneapp allows to perform helps in the identification of targets for experimental validation to confirm the hypotheses under study. The Geneapp is freely available for non-commercial use at https://geneapp.net to advance research on AS for bioinformatics.

Loss of TDP-43 induces synaptic dysfunction that is rescued by UNC13A splice-switching ASOs.

TDP-43 loss of function induces multiple splicing changes, including a cryptic exon in the amyotrophic lateral sclerosis and fronto-temporal lobar degeneration risk gene UNC13A, leading to nonsense-mediated decay of UNC13A transcripts and loss of protein. UNC13A is an active zone protein with an integral role in coordinating pre-synaptic function. Here, we show TDP-43 depletion induces a severe reduction in synaptic transmission, leading to an asynchronous pattern of network activity. We demonstrate that these deficits are largely driven by a single cryptic exon in UNC13A. Antisense oligonucleotides targeting the UNC13A cryptic exon robustly rescue UNC13A protein levels and restore normal synaptic function, providing a potential new therapeutic approach for ALS and other TDP-43-related disorders.

Wheat bread making (WBM)-like seed proteins (WSPN): A new family of small prolamins in barley

Expression level of the gene Wheat Breadmaking (TaWBM) has been shown to be correlated with bread quality, and influences the rising of the bread loaf. We found that a family of the wbm homologous genes are present not only in wheat, but also in oat, rye and barley. However, a WBM protein has never been demonstrated experimentally. We have identified the existence of a family of WBM-like Seed Proteins (WSPN) from the prolamin fraction of barley flour, encoded by three closely linked genes on the long arm of chromosome 7H, analysing the gene expression by qPCR and relative protein levels by mass spectroscopy. All three genes are expressed specifically in seeds with no detectable expression in the leaves. The barley WSPNs share a common gene structure with a single exon containing an open reading frame in the size 253–313 bp, with the first 80 bp predicted by TargetP 2.0 to encode a sorting peptide. Furthermore, they all contain the conserved motif C–P-X-G-X4-C-X(4–8)-C-X-C. Structure prediction with Alphafold 2.0 suggest that this motif is a structurally conserved microdomain consisting of two antiparallel strands where the cysteines align.

Investigation of genotype-phenotype and familial features of Turkish dystrophinopathy patients.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive allelic muscle diseases caused by dystrophin gene mutations. Eight hundred thirty-seven patients admitted between 1997 and 2022 were included in the study. Two hundred twenty patients were analyzed by multiplex PCR (mPCR) alone. Five hundred ninety-five patients were investigated by multiplex ligation-dependent probe amplification (MLPA), and 54 patients were examined by sequencing. Deletion was detected in 60% (132/220) of the cases in the mPCR group only and in 58.3% (347/595) of the cases with MLPA analysis. The rates of deletion and duplication were 87.7% and 12.3%, respectively, in the MLPA analysis. Single exon deletions were the most common mutation type. The introns 43-55 (81.8%) and exons 2-21 (13.1%) regions were detected as hot spots in deletions. It was determined that 89% of the mutations were suitable for exon skipping therapy. The reading frame rule did not hold in 7.6% of D/BMD cases (17/224). We detected twenty-five pathogenic/likely pathogenic variants in sequencing, five of which were novel variants. Nonsense mutation was the most common small mutation (44%). 21% of DMD patients were familial. We detected germline mosaicism in four families (4.3%) in the large rearrangement group and one gonosomal mosaicism in a family with a nonsense mutation. This is the largest study examining genotype and phenotype data in Turkish D/BMD families investigated by MLPA analysis. The reading frame hypothesis is not valid in all cases. Sharing the genotype and phenotype characteristics of these cases in the literature will shed light on the molecular structure of DMD and guide gene therapy research. In genetic counseling, carrier screening in the family and possible gonadal mosaicism should be emphasized.

Biallelic Deletion of PEX26 Exon 4 in a Boy with Phenotypic Features of both Zellweger Syndrome and Infantile Refsum Disease

Introduction: Peroxisome biogenesis disorders (PBDs) encompass a group of diseases marked by clinical and genetic heterogeneity. Phenotypes linked to PBDs include Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease (IRD), rhizomelic chondrodysplasia punctata type 1, and Heimler syndrome. PBD phenotypes manifest through hypotonia, developmental delay, facial dysmorphism, seizures, liver dysfunction, sensorineural hearing loss, and retinal dystrophy. Methods: The proband underwent comprehensive clinical evaluation, followed by whole-exome sequencing (WES) coupled with copy number analysis (CNV), aimed at identifying potential disease-causing variants aligning with the observed phenotype. Results: Our findings detail an individual exhibiting developmental delay, hearing loss, visual impairment, hepatomegaly, and splenomegaly, attributed to a biallelic deletion of exon 4 in the PEX26 gene. The WES analysis of the index case did not uncover any pathogenic/likely pathogenic single-nucleotide variations that could account for the observed clinical findings. However, the CNV data derived from WES revealed a homozygous deletion in exon 4 of the PEX26 gene (NM_001127649.3), providing a plausible explanation for the patient’s clinical features. The exon 4 region of PEX26 encodes the transmembrane domain of the protein. The transmembrane domain plays a crucial role in anchoring the protein within lipid bilayers, and its absence can disrupt proper localization and functioning. As a result, this structural alteration may impact the protein’s ability to facilitate essential cellular processes related to peroxisome biogenesis and function. Conclusion: The index patient, which presented with hearing loss, retinal involvement and hepatic dysfunction in adolescence age, has atypical clinical course that can be considered unusual for Zellweger syndrome (ZS) and IRD phenotypes, and its rare genotypic data (in-frame single exon deletion) expands the PBD disease spectrum. This study revealed for the first time that PEX26 protein transmembrane domain loss exhibits an unusual course with clinical findings of IRD and ZS phenotypes. WES studies, incorporating CNV analyses, empower the identification of novel genetic alterations in genes seldom associated with gross deletion/duplication variations, such as those in the PEX26 gene. This not only enhances diagnostic rates in rare diseases but also contributes to broadening the spectrum of causal mutations.

Genome-wide identification and characterization of DIRIGENT gene family (CcDIR) in pigeonpea (Cajanus cajan L.) provide insights on their spatial expression pattern and relevance to stress response

This study is a thorough characterization of pigeonpea dirigent gene (CcDIR) family, an important component of the lignin biosynthesis pathway. Genome-wide analysis identified 25 CcDIR genes followed by a range of analytical approaches employed to unravel their structural and functional characteristics. Structural examination revealed a classic single exon and no intron arrangement in CcDIRs contributing to our understanding on evolutionary dynamics. Phylogenetic analysis elucidated evolutionary relationships among CcDIR genes with six DIR sub-families, while motif distribution analysis displayed and highlighted ten conserved protein motifs in CcDIRs. Promoter analyses of all the dirigent genes detected 18 stress responsive cis-acting elements offering insights into transcriptional regulation. While spatial expression analyses across six plant tissues showed preferential expression of CcDIR genes, exposure to salt (CcDIR2 and CcDIR9) and herbivory (CcDIR1, CcDIR2, CcDIR3 and CcDIR11), demonstrated potential roles of specific DIRs in plant defense. Interestingly, increased gene expression during herbivory, also correlated with increased lignin content authenticating the specific response. Furthermore, exogenous application of stress hormones, SA and MeJA on leaves significantly induced the expression of CcDIRs that responded to herbivory. Taken together, these findings contribute to a comprehensive understanding of CcDIR genes impacting development and stress response in the important legume pigeonpea.

Optimized MLPA workflow for spinal muscular atrophy diagnosis: identification of a novel variant, NC_000005.10:g.(70919941_70927324)del in isolated exon 1 of SMN1 gene through long-range PCR

BackgroundSpinal muscular atrophy (SMA) is a rare autosomal recessive hereditary neuromuscular disease caused by survival motor neuron 1 (SMN1) gene deletion or mutation. Homozygous deletions of exon 7 in SMN1 result in 95% of SMA cases, while the remaining 5% are caused by other pathogenic variants of SMN1.MethodsWe analyzed two SMA-suspected cases that were collected, with no SMN1 gene deletion and point mutation in whole-exome sequencing. Exon 1 deletion of the SMN gene was detected using Multiplex ligation-dependent probe amplification (MLPA) P021. We used long-range polymerase chain reaction (PCR) to isolate the SMN1 template, optimized-MLPA P021 for copy number variation (CNV) analysis within SMN1 only, and validated the findings via third-generation sequencing.ResultsTwo unrelated families shared a genotype with one copy of exon 7 and a novel variant, g.70919941_70927324del, in isolated exon 1 of the SMN1 gene. Case F1-II.1 demonstrated no exon 1 but retained other exons, whereas F2-II.1 had an exon 1 deletion in a single SMN1 gene. The read coverage in the third-generation sequencing results of both F1-II.1 and F2-II.1 revealed a deletion of approximately 7.3 kb in the 5’ region of SMN1. The first nucleotide in the sequence data aligned to the 7385 bp of NG_008691.1.ConclusionRemarkably, two proband families demonstrated identical SMN1 exon 1 breakpoint sites, hinting at a potential novel mutation hotspot in Chinese SMA, expanding the variation spectrum of the SMN1 gene and corroborating the specificity of isolated exon 1 deletion in SMA pathogenesis. The optimized-MLPA P021 determined a novel variant (g.70919941_70927324del) in isolated exon 1 of the SMN1 gene based on long-range PCR, enabling efficient and affordable detection of SMN gene variations in patients with SMA, providing new insight into SMA diagnosis to SMN1 deficiency and an optimized workflow for single exon CNV testing of the SMN gene.

Characterization of rice aspartic protease genes and induced expression by phytohormones and Xanthomonas oryzae pv. oryzae

Aspartic proteases (APs) are present in both monocotyledonous and dicotyledonous plants. APs consist of nine different families of proteolytic enzymes. APs play important functions in several physiological processes including defense. However, there are limited reports on APs in disease resistance in rice (Oryza sativa). Here, we report that 104 APs were discovered in rice genome. They can be classified into three clades (I, II and III) with APs from Arabidopsis thaliana, and Nymphaea colorata according to phylogenetic relationships. Tandem and segmental duplications were observed to lead to the expansion of OsAP genes in rice. Analysis of gene structure revealed that numerous OsAPs contain multiple exons and introns, however, several OsAPs harbor a single exon and no introns. Synteny analyses demonstrated that four AP genes among O. sativa, A. thaliana, and N. colorata may evolve from a common ancestor of land plants. Ten conserved motifs of amino acids were uncovered from the OsAPs, and two of the motifs are the catalytic sequences, with the conserved Asp-Thr and Ser-Gly amino acids. Within the OsAP promoter sequences, cis-regulatory elements responsive to light, abscisic acid, auxin, gibberellins, methyl jasmonate, and salicylic acid were identified. Stress-responsive elements and tissue-specific expressed elements were also found. Majority of OsAP genes were found to be expressed in diverse tissues and organs. Some of the OsAP genes were observed to be induced by brassinosteroids, jasmonic acid, and salicylic acid. Further, some OsAP genes can be induced by Xanthomonas oryzae pv. oryzae (Xoo), the cause of rice bacterial leaf blight. Among them, OsAP21 was strongly activated by the pathogen, suggesting its potential function in response to the bacterial agent. The results shed light in the potential functions of OsAP genes in rice disease resistance to Xoo.

High-Throughput RNA-HCR-FISH Detection of Endogenous Pre-mRNA Splice Variants.

RNA-fluorescence in situ hybridization (RNA-FISH) is an essential and widely used tool for visualizing RNA molecules in intact cells. Recent advances have increased RNA-FISH sensitivity, signal detection efficiency, and throughput. However, detection of endogenous mRNA splice variants has been challenging due to the limits of visualization of RNA-FISH fluorescence signals and due to the limited number of RNA-FISH probes per target. HiFENS (high-throughput FISH detection of endogenous pre-mRNA splicing isoforms) is a method that enables visualization and relative quantification of mRNA splice variants at single-cell resolution in an automated high-throughput manner. HiFENS incorporates HCR (hybridization chain reaction) signal amplification strategies to enhance the fluorescence signal generated by low abundance transcripts or a small number of FISH probes targeting short stretches of RNA, such as single exons. The technique offers a significant advance in high-throughput FISH-based RNA detection and provides a powerful tool that can be used as a readout in functional genomics screens to discover and dissect cellular pathways regulating gene expression and alternative pre-mRNA splicing events.

Assessing the impact of storage conditions on RNA from human saliva and its application to the identification of mRNA biomarkers for asthma.

Introduction: Human saliva was used to develop non-invasive liquid biopsy biomarkers to establish saliva as an alternate to blood and plasma in translational research. The present study focused on understanding the impact of sample storage conditions on the extraction of RNA from saliva and the RNA yield, to be applied in clinical diagnosis. In this study, genes related to asthma were used to test the method developed. Methods: Salivary RNA was extracted from three subjects using the Qiazol® based method and quantified by both spectrophotometric (NanoDrop) and fluorometric (Qubit®) methods. RNA integrity was measured using a bioanalyzer. Quantitative PCR was used to monitor the impact of storage conditions on the expression of housekeeping genes: GAPDH and β-actin, and the asthma related genes: POSTN and FBN2. In addition, an independent cohort of 38 asthmatics and 10 healthy controls were used to validate the expression of POSTN and FBN2 as mRNA salivary biomarkers. Results: Approximately 2µg of total RNA was obtained from the saliva stored at 40°C without any preservative for 2weeks showing consistent gene expression with RNA stored at room temperature (RT) for 48h with RNAlater. Although saliva stored with RNAlater showed a substantial increase in the yield (110 to 234ng/μL), a similar Cq (15.6 ± 1.4) for the 18s rRNA gene from saliva without preservative showed that the RNA was stable enough. Gene expression analysis from the degraded RNA can be performed by designing the assay using a smaller fragment size spanning a single exon as described below in the case of the POSTN and FBN2 genes in the asthma cohort. Conclusion: This study showed that samples stored at room temperature up to a temperature of 40°C without any preservative for 2weeks yielded relatively stable RNA. The methodology developed can be employed to transport samples from the point of collection to the laboratory, under non-stringent storage conditions enabling the execution of gene expression studies in a cost effective and efficient manner.

Analyzing the evolution of insect TMED gene and the expression pattern of silkworm TMED gene

Transmembrane emp24 domain (TMED) gene is closely related to immune response, signal transduction, growth and disease development in mammals. However, only the Drosophila TMED gene has been reported on insects. We identified the TMED family genes of silkworm, Tribolium castaneum, tobacco moth and Italian bee from their genomes, and found that the TMED family gene composition patterns of one α-class, one β-class, one δ-class and several γ-classes arose in the common ancestor of pre-divergent Hymenoptera insects, while the composition of Drosophila TMED family members has evolved in a unique pattern. Insect TMED family γ-class genes have evolved rapidly, diverging into three separate subclasses, TMED6-like, TMED5-like and TMED3-like. The TMED5-like gene was lost in Hymenoptera, duplicated in the ancestors of Lepidoptera and duplicated in Drosophila. Insect TMED protein not only has typical structural characteristics of TMED, but also has obvious signal peptide. There are seven TMED genes in silkworm, distributed in six chromosomes. One of seven is single exon and others are multi-exons. The complete open reading frame (ORF) sequences of seven TMED genes of silkworm were cloned from larval tissues and registered in GenBank database. BmTMED1, BmTMED2 and BmTMED6 were expressed in all stages and tissues of the silkworm, and all genes were expressed in the 4th and 5th instar and silk gland of the silkworm. The present study revealed the composition pattern of TMED family members, their γ class differentiation and their evolutionary history, providing a basis for further studies on TMED genes in silkworm and other insects.

Identifying PTPRJ As a Novel Mediator of CEBPA-Mutated AML

10-20% of acute myeloid leukaemia (AML) cases in children and adults carry mutations in the transcription factor CCAAT/enhancer binding protein alpha (CEBPA). CEBPA is transcribed from a single exon and produces two isoforms, p30 and p42, with characteristic mutations, either frameshift mutations in the N-terminal portion ( CEBPA Nterm) leading to production of only the p30 isoform, or in frame mutations in the C-terminal portion ( CEBPA Cterm), disrupting DNA binding. The most frequent secondary driver mutation in CEBPA-mutated AML, is the second allele of CEBPA. Additionally, in rare germline CEBPA cases, the founding mutation is usually CEBPA Nterm, with allindividuals showing acquisition of a second hit mutation in the CEBPA Cterm on the alternate CEBPA allele at the time of AML diagnosis, which has near 100% penetrance in these families. This highlights a strong selective pressure for this very specific biallelic mutation pattern. We have developed zebrafish models with germline cebpa Nterm and cebpa Cterm mutations that faithfully recapitulates the disease, with all homozygous and double heterozygous mutants showing loss of mature myeloid cells, and impaired survival. All double or compound mutants die by 10 weeks of age, with haematopoietic stem and progenitor cell (HSPC) expansion, and leukaemia in around 30% of these animals. Our model also demonstrates distinct phenotypes for cebpa Nterm and cebpa Cterm mutations during developmental haematopoiesis prior to leukaemia onset. We observed that cebpa Nterm and cebpa Cterm mutations have opposite effects on the expression of c-myb, a transcription factor expressed by proliferating HSPC, from 3 days post fertilization. cebpa Nterm/Nterm mutants show an increase in c-myb while cebpa Cterm/Cterm show reduced c-myb expression during development. To uncover the mechanisms by which cebpa Nterm and cebpa Cterm effect HSPCs we undertook RNASeq of sorted Tg(cd41:eGFP) lo HSPCs from fish of all possible genotype combinations. We identified both shared and unique expression profiles between genotypes. We also identified 4 genes that that were differentially regulated in both cebpa Nterm/Nterm and cebpa Cterm/Cterm HSPC, but in opposing vectors (upregulated in cebpa Cterm/Cterm and downregulated in cebpa Nterm/Nterm) (Figure 1). We hypothesised that these 4 genes are candidate drivers of selective pressure for the common CEBPA Cterm/Ntermmutation combination in human AML, and thus the malignant phenotype it confers. One of these candidate genes, PTPRJ, has also recently been identified as a frequently mutated gene in CEBPA-mutated AML in children thereby validating the relevance of our dataset and our approach. To define the effects of these 4 candidate genes we then conducted an F0 CRISPR knock-out screen in all cebpa mutant combinations in our zebrafish model, using c-myb expression as a phenotypic readout. We identified ptprja as our primary candidate gene and showed that loss of ptprja alone and in combination with cebpa Cterm/+ reduced expression of c-myb but had no effect on expression of c-myb in cebpa Cterm/Cterm, indicating that ptprja loss reduces c- myb-expressing HSPC and that this is dependent on functional cebpa DNA binding. We then examined the impact of ptprja knockout in juvenile zebrafish. Homozygous and double heterozygous mutants develop a pre-leukaemia expansion of HSPC before they succumb to death. We showed that that loss of ptprja in cebpa Cterm/Cterm fish accelerates this pre-leukaemic expansion of Tg(cd41:eGFP) lo HSPCs in 4 week fish (Figure 2). We also showed an opposing effect in the cebpa Nterm/Nterm mutants at 2 weeks, where loss of ptprja resulted in decreased pre-leukemic expansion of HSPC. These data indicate a role for PTPRJ in mediating HSPC expansion in CEBPA-mutated AML and suggest that PTPRJ may mediate the mechanism of clonal selection in those with a CEBPA mutation that drives the acquisition of a second CEBPA mutation on the opposing allele, leading to the common CEBPA Cterm/Nterm in patients with CEBPA-mutated AML.

Identification of Novel Transcripts and Potential Therapeutic Targets for Acute Leukemia

Acute leukemia is a heterogenous disease with many genomic scars. Recent advances in targeted therapy and immunotherapy have improved the prognosis of this disease. However, prognosis still remains poor, and the identification of novel leukemia-specific transcripts may provide new strategies for leukemia therapy. We developed a novel long-read transcriptome method for sequencing full-length RNAs by directly capturing the 5′-end cap structures and the 3′-end poly(A)-tails of individual RNA molecules. We applied this method to bone marrow samples with various acute leukemias, including acute myeloid leukemia, acute lymphoid leukemia, and other rare types of acute leukemia. We covered full-length poly(A)+ RNAs with an average length distribution of over 3,000 bp, a much longer size distribution than previously reported. This not only led to the discovery of a wide array of uncharacterized transcript isoforms of known genes, but also to the discovery of 1,903 novel genes that are not annotated in the current human gene database. We also found that more than 60% of these new human genes were single exon genes and that many of them emerged from primates. This implicates that these new genes may contribute to human-specific leukemia biology. In addition, among these new human genes, 485 genes were predicted to have the potential to encode putative proteins using a GeneMarkS-T program. A fraction of new genes identified in this study were highly leukemia specific as shown by bulk CAGE-seq analysis of over 100 blood tumor samples and by single-cell RNA sequencing analysis of more than 300,000 bone marrow cells from leukemia patients, highlighting their potential as useful biomarkers and novel therapeutic targets. In sum, we constructed a comprehensive atlas of full-length RNA molecules in acute leukemia and identified a large number of uncharacterized ones. Our study provides a versatile framework for exploring novel transcripts and future therapeutic strategies in human diseases.

Genomic assembly, characterization, and quantification of DICER-like gene family in Okra plants under dehydration conditions.

Okra is a plant farmed for its pods, leaves, and stems all of which are edible. It is famous for its ability to tolerate long desiccation periods. It belongs to the Malvaceae family and is a sister species to hibiscus, cotton, and cacao plants. In the current study, okra plants were used as a model to sequence, assemble, and analyze the evolutionary and functional characteristics of the Dicer-like protein gene family (DCL) based on DNAseq and qPCR techniques. Four Dicer-like (DCL) single-copy genes of the okra plant Abelmoschus esculentus (L.) Moench (AeDCL) were successfully assembled. The lengths of the AeDCL copies were 8,494, 5,214, 4,731, and 9,329 bp. The detected exons in these samples ranged from a single exon in AeDCL3 to 24 exons in AeDCL4. AeDCLs had five functional domains of two DEAD-like helicase superfamilies, N and C; one Dicer domain; one ribonuclease III domain (a and b); and one double-stranded RNA-binding domain. The PAZ domain was completely annotated only for AeDCL1 and AeDCL3. All AeDCLs were up-regulated under drought conditions, with leaves showing more extensive fold changes than roots. The study focused on a comprehensive genome-wide identification and analysis of the DCL gene family in naturally drought-tolerant okra plants, an orphan crop that can be used as a model for further genomic and transcriptomic studies on drought-tolerance mechanisms in plants.