Cis Elements Research Articles

SSA4 Mediates Cd Tolerance via Activation of the Cis Element of VHS1 in Yeast and Enhances Cd Tolerance in Chinese Cabbage.

Identifying key genes involved in Cadmium (Cd) response pathways in plants and developing low-Cd-accumulating cultivars may be the most effective and eco-friendly strategy to tackle the problem of Cd pollution in crops. In our previous study, Stressseventy subfamily A 4 (SSA4) was identified to be associated with Cd tolerance in yeast. Here, we investigated the mechanism of SSA4 in regulating Cd tolerance in yeast. ScSSA4 binds to POre Membrane 34 (POM34), a key component of nuclear pore complex (NPC), and translocates from the cytoplasm to the nucleus, where it regulates the expression of its downstream gene, Viable in a Hal3 Sit4 background 1 (VHS1), resulting in reduced Cd accumulation in yeast cells. Additionally, we identified a Chinese cabbage SSA4 gene, BrSSA4c, which could enhance the Cd tolerance in Chinese cabbage. This study offers new insights into the regulatory mechanisms of Cd tolerance in yeast, a model organism, and paves the way for the genetic enhancement of Cd tolerance in Chinese cabbage.

The role of structure in regulatory RNA elements.

Regulatory RNA elements fulfill functions such as translational regulation, control of transcript levels, and regulation of viral genome replication. Trans-acting factors (i.e. RNA-binding proteins) bind the so-called cis elements and confer functionality to the complex. The specificity during protein-RNA complex (RNP) formation often exploits the structural plasticity of RNA. Functional integrity of cis-trans pairs depends on the availability of properly folded RNA elements, and RNA conformational transitions can cause diseases. Knowledge of RNA structure and the conformational space is needed for understanding complex formation and deducing functional effects. However, structure determination of RNAs under in vivo conditions remains challenging. This review provides an overview of structured eukaryotic and viral RNA cis elements and discusses the effect of RNA structural equilibria on RNP formation. We showcase implications of RNA structural changes for diseases, outline strategies for RNA structure-based drug targeting, and summarize the methodological toolbox for deciphering RNA structures.

Clustering of rRNA operons in E. coli is disrupted by σH.

Chromosomal organization in E. coli as examined by Hi-C methodology indicates that long-range interactions are sparse. Yet, spatial co-localization or 'clustering' of 6/7 ribosomal RNA (rrn) operons distributed over half the 4.6 Mbp genome has been captured by two other methodologies - fluorescence microscopy and Mu transposition. Our current understanding of the mechanism of clustering is limited to mapping essential cis elements. To identify trans elements, we resorted to perturbing the system by chemical and physical means and observed that heat shock disrupts clustering. Levels of σH are known to rise as a cellular response to the shock. We show that elevated expression of σH alone is sufficient to disrupt clustering, independent of heat stress. The anti-clustering activity of σH does not depend on its transcriptional activity but requires core-RNAP interaction and DNA-binding activities. This activity of σH is suppressed by ectopic expression of σD suggesting a competition for core-RNAP. A query of the other five known σ factors of E. coli found that elevated expression of FecI, the ECF σ factor that controls iron citrate transport, also perturbs clustering and is also suppressed by σD. We discuss a possible scenario for how these membrane-associated σ factors participate in clustering of distant rrn loci.

Unraveling barley's PAL gene family: a genome-wide study on defense mechanisms against Puccinia graminis f. sp. tritici

Phenylalanine ammonia lyase (PAL) is a pivotal enzyme bridging primary and secondary phenylpropanoid metabolism, influencing plant growth, development, and stress responses. Despite extensive studies on PAL genes across various plant species, their investigation in barley, a critical staple food globally, has been relatively scarce. In this study, we have successfully identified 10 HvPAL genes, designated as HvPAL genes, in Hordeum vulgare (barley). These HvPAL genes were categorized based on their conserved sequences, which revealed patterns through MEME analysis and multiple sequence alignment. Interestingly, we found cis elements related to stress in the promoter regions of HvPAL genes, indicating their involvement in the response to pathogens. Furthermore, these gene promoters contained components associated with light, development, and hormone responsiveness. This suggests that they may play a role in hormonal developmental processes. MicroRNAs were also identified as regulators of the HvPAL genes we identified highlighting their significance in barley. To further investigate these gene expression patterns, we analyzed the RNA-seq data revealed the upregulating of HvPAL 2, HvPAL3, and HvPAL8, and downregulating HvPAL 5, HvPAL 6, and HvPAL9 genes in this study. This study focused on the regulation of PAL genes in response to 23 different races of Puccinia graminis f. sp. tritici in barley. These results suggest ways to improve traits and develop barley varieties that are resistant to pathogens by selectively increasing the expression of certain HvPAL genes that were not previously regulated. This thorough investigation aims to expand our knowledge of the versatility of the PAL gene family, providing insights for advancements in host -pathogen genetics.

Deciphering regulation of FOXP3 expression in human conventional T cells.

FOXP3 is a lineage-defining transcription factor that controls differentiation and maintenance of suppressive function of regulatory T cells (Tregs). Foxp3 is exclusively expressed in Tregs in mice. However, in humans, FOXP3 is not only constitutively expressed in Tregs; it is also transiently expressed in stimulated CD4+CD25- conventional T cells (Tconvs)1-3. Mechanisms governing the expression of FOXP3 in human Tconvs are not understood. Here, we performed CRISPR interference (CRISPRi) screens using a 15K-member gRNA library tiling 39 kb downstream of the FOXP3 transcriptional start site (TSS) to 85 kb upstream of the TSS in Treg and Tconvs. The FOXP3 promoter and conserved non-coding sequences (CNS0, CNS1, CNS2 and CNS3), characterized as enhancer elements in murine Tregs, were required for maintenance of FOXP3 in human Tregs. In contrast, FOXP3 in human Tconvs depended on regulation at CNS0 and a novel Tconv-specific noncoding sequence (TcNS+) located upstream of CNS0. Arrayed validations of these sites identified an additional repressive cis-element overlapping with the PPP1R3F promoter (TcNS-). Pooled CRISPR knockouts revealed multiple transcription factors required for proper expression of FOXP3 in Tconvs, including GATA3, STAT5, IRF4, ETS1 and DNA methylation-associated regulators DNMT1 and MBD2. Analysis of ChIP-seq and ATAC-seq paired with knock-out (KO) of GATA3, STAT5, IRF4, and ETS1 revealed regulation of CNS0 and TcNS+ accessibility. Collectively, this work identified Treg-shared and Tconv-specific cis-elements and the trans-factors that interact with them, building a network of regulators controlling FOXP3 expression in human Tconvs.

Chromosome-level genome provides new insight into the overwintering process of Korla pear (Pyrus sinkiangensis Yu)

Korla pear has a unique taste and aroma and is a breeding parent of numerous pear varieties. It is susceptible to Valsa mali var. pyri, which invades bark wounded by freezing injury. Its genetic relationships have not been fully defined and could offer insight into the mechanism for freezing tolerance and disease resistance. We generated a high-quality, chromosome-level genome assembly for Korla pear via the Illumina and PacBio circular consensus sequencing (CCS) platforms and high-throughput chromosome conformation capture (Hi-C). The Korla pear genome is ~ 496.63 Mb, and 99.18% of it is assembled to 17 chromosomes. Collinearity and phylogenetic analyses indicated that Korla might be derived from Pyrus pyrifolia and that it diverged ~ 3.9-4.6 Mya. During domestication, seven late embryogenesis abundant (LEA), two dehydrin (DHN), and 54 disease resistance genes were lost from Korla pear compared with P. betulifolia. Moreover, 21 LEA and 31 disease resistance genes were common to the Korla pear and P. betulifolia genomes but were upregulated under overwintering only in P. betulifolia because key cis elements were missing in Korla pear. Gene deletion and downregulation during domestication reduced freezing tolerance and disease resistance in Korla pear. These results could facilitate the breeding of novel pear varieties with high biotic and abiotic stress resistance.

Abstract We142: Identification of the Molecular Determinants for the Circadian Regulation of the Human KCNH2 promoter

Background: KCNH2 encodes a voltage-gated potassium channel (Kv11.1), which conducts the rapid delayed rectifier potassium current (IKr) critical for ventricular repolarization in the heart. Circadian clock factors regulate the expression of the KCNH2 mRNA transcript in the heart. However, the molecular mechanisms for the circadian expression of KCNH2 transcripts have yet to be defined. Hypothesis: A conserved cis-regulatory element in the core KCNH2 promoter drives circadian regulation of human KCNH2 (hKCNH2) promoter activity. Objective: Identify the molecular determinants of the circadian regulation of hKCNH2 promoter activity and determine the impact of single nucleotide polymorphisms (SNPs) in this region. Methods: Real-time bioluminescence (LumiCycle, Actimetrics) and Dual Glo luciferase assays were employed to identify critical cis elements that regulate the circadian expression of the human KCNH2 promoter using promoter-luciferase reporter constructs. We studied several deletion and mutant hKCNH2 promoter reporter vectors transiently transfected in a myogenic cell line (C2C12 cells). Bioluminescence was measured at 10-minute intervals for 7-10 days. Data were analyzed for circadian characteristics (period, phase and amplitude). Mutant constructs included both engineered and constructs containing naturally occurring rare and common SNPs. Results: Lumicycle data demonstrated that the hKCNH2 promoter reporter showed robust circadian and overall activity in C2C12 cells. Deletion and mutational analysis of the hKCNH2 promoter-reporter construct identified a highly conserved tandem E-box element within 1 Kb of the exon 1 start site that was critical for circadian and overall regulation of hKCNH2 promoter activity. Cells expressing hKCNH2 promoter-reporter constructs with different SNPs in this tandem E-box element showed SNP-specific effects. Surprisingly, a relatively common SNP decreased hKCNH2 circadian promoter activity by 66% (n = 6, p=0.0049). Conclusion: We identified a conserved tandem E-box in the proximal promoter of hKCNH2 Exon1 necessary for circadian and overall hKCNH2 promoter activity. Several SNPs in this region suggested a functional impact that might lead to decreased levels of IKr.

The MADS-box transcription factor GmFULc promotes GmZTL4 gene transcription to modulate maturity in soybean.

Flowering time and maturity are crucial agronomic traits that affect the regional adaptability of soybean plants. The development of soybean cultivars with early maturity adapted to longer days and colder climates of high latitudes is very important for ensuring normal ripening before frost begins. FUL belongs to the MADS-box transcription factor family and has several duplicated members in soybeans. In this study, we observed that overexpression of GmFULc in the Dongnong 50 cultivar promoted soybean maturity, while GmFULc knockout mutants exhibited late maturity. Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) revealed that GmFULc could bind to the CArG, bHLH and homeobox motifs. Further investigation revealed that GmFULc could directly bind to the CArG motif in the promoters of the GmZTL3 and GmZTL4 genes. Overexpression of GmZTL4 promoted soybean maturity, whereas the ztl4 mutants exhibited delayed maturity. Moreover, we found that the cis element box 4 motif of the GmZTL4 promoter, a motif of light response elements, played an important role in controlling the growth period. Deletion of this motif shortened the growth period by increasing the expression levels of GmZTL4. Functional investigations revealed that short-day treatment promoted the binding of GmFULc to the promoter of GmZTL4 and inhibited the expression of E1 and E1Lb, ultimately resulting in the promotion of flowering and early maturation. Taken together, these findings suggest a novel photoperiod regulatory pathway in which GmFULc directly activates GmZTL4 to promote earlier maturity in soybean.

Characterization of GPX Gene Family in Pepper (Capsicum annuum L.) under Abiotic Stress and ABA Treatment.

Plant glutathione peroxidases (GPXs) are important enzymes for removing reactive oxygen species in plant cells and are closely related to the stress resistance of plants. This study identified the GPX gene family members of pepper (Capsicum annuum L.), "CM333", at the whole-genome level to clarify their expression patterns and enzyme activity changes under abiotic stress and ABA treatment. The results showed that eight CaGPX genes were unevenly distributed across four chromosomes and one scaffold of the pepper genome, and their protein sequences had Cys residues typical of the plant GPX domains. The analysis of collinearity, phylogenetic tree, gene structure, and conserved motifs indicated that the CaGPX gene sequence is conserved, structurally similar, and more closely related to the sequence structure of Arabidopsis. Meanwhile, many cis elements involved in stress, hormones, development, and light response were found in the promoter region of the CaGPX gene. In addition, CaGPX1/4 and CaGPX6 were basically expressed in all tissues, and their expression levels were significantly upregulated under abiotic stress and ABA treatment. Subcellular localization showed that CaGPX1 and CaGPX4 are localized in chloroplasts. Additionally, the variations in glutathione peroxidase activity (GSH-Px) mostly agreed with the variations in gene expression. In summary, the CaGPXs gene may play an important role in the development of peppers and their response to abiotic stress and hormones.

Single Nucleotide Polymorphisms: A Deep Consideration of Protein Sequence Variation

Introduction: Human genome consists of the three billion base pairs that has about one percent of genetic variation from one person to another، which determines physical، psychological، and susceptibility to diseases. Among the types of genetic diversity, single nucleotide polymorphisms are one of the most important genetic differences between two people. Single nucleotide polymorphism variation is located in the promoter region, exons، introns، untranslated regions and other Deoxyribonucleic acid (DNA) regions. While variation in the exon region can change susceptibility to diseases depending on whether it changes the protein structure or affects translation kinetics. Diversity in the promoter region can affect the interaction of genetic and epigenetic elements. Also، variation in the promoter region can affect the DNA methylation status. Polymorphic variation in the intron region can affect Messenger Ribonucleic acid splicing and the function of cis-regulatory elements. Polymorphic variation in the 5' Untranslated region، region causes a change in translation efficiency,، while a change in the 3' Untranslated region binds micro Ribonucleic acids to their position then affects the effects. In some cases، variations in Transfer Ribonucleic acid (tRNA) and Ribosomal ribonucleic acid (rRNA) affect the function of these regulatory cis elements. Conclusion: From a clinical point of view, a deep knowledge of this type of genetic variation can help the treatment process, manage patients and understand the prognosis based on these SNPs. Private or personalized medicine is also fundamentally based on genetic diversity. In this article, it was reviewed the types of single nucleotide genetic variation and presented examples of types of cancer, neurological and immunological diseases.

Genome-Wide Identification and Expression Analysis of the Casparian Strip Membrane Domain Protein-like Gene Family in Peanut (Arachis hypogea L.) Revealed Its Crucial Role in Growth and Multiple Stress Tolerance.

Casparian strip membrane domain proteins (CASPs), regulating the formation of Casparian strips in plants, serve crucial functions in facilitating plant growth, development, and resilience to abiotic stress. However, little research has focused on the characteristics and functions of AhCASPs in cultivated peanuts. In this study, the genome-wide identification and expression analysis of the AhCASPs gene family was performed using bioinformatics and transcriptome data. Results showed that a total of 80 AhCASPs members on 20 chromosomes were identified and divided into three subclusters, which mainly localized to the cell membrane. Ka/Ks analysis revealed that most of the genes underwent purifying selection. Analysis of cis elements suggested the possible involvement of AhCASPs in hormonal and stress responses, including GA, MeJA, IAA, ABA, drought, and low temperature. Moreover, 20 different miRNAs for 37 different AhCASPs genes were identified by the psRNATarget service. Likewise, transcriptional analysis revealed key AhCASPs responding to various stresses, hormonal processing, and tissue types, including 33 genes in low temperature and drought stress and 41 genes in tissue-specific expression. These results provide an important theoretical basis for the functions of AhCASPs in growth, development, and multiple stress resistance in cultivated peanuts.

Genome-Wide Identification of the PIP5K Gene Family in Camellia sinensis and Their Roles in Metabolic Regulation.

Spider mite infestation has a severe impact on tea growth and quality. In this study, we conducted a deep exploration of the functions and regulations of the CsPIP5K gene family using chromosomal localization and collinearity analysis. Additionally, we carefully examined the cis elements within these genes. To fully understand the metabolic response of CsPIP5K under spider mite infection, we integrated previously published metabolomic and transcriptomic data. Our analysis revealed that multiple CsPIP5K genes are associated with phospholipid metabolism, with CsPIP5K06 showing the strongest correlation. Therefore, we employed qPCR and subcellular localization techniques to determine the expression pattern of this gene and its functional location within the cell. Overall, this study not only comprehensively elucidated the characteristics, structure, and evolution of the CsPIP5K gene family but also identified several candidate CsPIP5K genes related to phospholipid biosynthesis and associated with spider mites based on previously published data. This research makes a significant contribution to enhancing the resistance of tea to spider mite and maintaining optimal tea quality.

Genome-wide analysis of the calmodulin-binding transcription activator (CAMTA) gene family in Sesamum indicum L., and its role in abiotic stress tolerance traits

Calmodulin-binding transcription activator (CAMTA) is one of the key transcription factor families possessing calcium receptors (calmodulins, CaM). It modulates the expression levels of genes associated with ontogeny and various biotic and abiotic stress factors. The CAMTA family genes were known to be involved in different abiotic stress in several crop species. However, their functional relevance in sesame remains unexplored. To understand the role of CAMTA in stress tolerance in sesame, we performed a genome-wide analysis to identify the members of the SiCAMTA gene family. We have identified and reported here the five SiCAMTA genes localized on four chromosomes within the sesame genome. In silico analysis of the putative 2-kilobase (kb) promoter regions for these five SiCAMTA genes showed that phytohormone and stress response-related cis-elements were predominated in SiCAMTA2 and SiCAMTA5. Also, we studied its modulated expression levels, with special reference to drought and waterlogging stress. It revealed that the SiCAMTA5 and SiCAMTA2 genes were the most responsive to the studied stress factors. The target prediction and network analysis suggested that SiCAMTAs could bind the CGCG cis element in the target gene promoters and predicted 1202 SiCAMTA target genes in the sesame genome, including abiotic stress-responsive genes viz. LEA, PIP1–2, PPO1, SAP, ARF17, and GA3OX1. These findings were validated using qPCR analysis for five CAMTA and 10 CAMTA target genes and establish a foundation for future functional research of SiCAMTA genes towards sesame stress tolerance.

Regulation of thiamine and pyruvate decarboxylase genes by Pdc2 in Nakaseomyces glabratus (Candida glabrata) is complex.

Thiamine (vitamin B1) is essential for glucose catabolism. In the yeast species, Nakaseomyces glabratus (formerly Candida glabrata) and Saccharomyces cerevisiae, the transcription factor Pdc2 (with Thi3 and Thi2) upregulates pyruvate decarboxylase (PDC) genes and thiamine biosynthetic and acquisition (THI) genes during starvation. There have not been genome-wide analyses of Pdc2 binding. Previously, we identified small regions of Pdc2-regulated genes sufficient to confer thiamine regulation. Here, we performed deletion analyses on these regions. We observed that when the S. cerevisiae PDC5 promoter is introduced into N. glabratus, it is thiamine starvation inducible but does not require the Thi3 coregulator. The ScPDC5 promoter contains a 22-bp duplication with an AT-rich spacer between the 2 repeats, which are important for regulation. Loss of the first 22-bp element does not eliminate regulation, but the promoter becomes Thi3 dependent, suggesting cis architecture can generate a Thi3-independent, thiamine starvation inducible response. Whereas many THI promoters only have 1 copy of this element, addition of the first 22-bp element to a Thi3-dependent promoter confers Thi3 independence. Finally, we performed fluorescence anisotropy and chromatin immunoprecipitation sequencing. Pdc2 and Thi3 bind to regions that share similarity to the 22-bp element in the ScPDC5 promoter and previously identified cis elements in N. glabratus promoters. Also, while Pdc2 binds to THI and PDC promoters, neither Pdc2 nor Thi3 appears to bind the evolutionarily new NgPMU3 promoter that is regulated by Pdc2. Further study is warranted because PMU3 is required for cells to acquire thiamine from environments where thiamine is phosphorylated, such as in the human bloodstream.

Genomic Evolution Strategy in SARS-CoV-2 Lineage B: Coevolution of Cis Elements.

In the SARS-CoV-2 lineage, RNA elements essential for its viral life cycle, including genome replication and gene expression, have been identified. Still, the precise structures and functions of these RNA regions in coronaviruses remain poorly understood. This lack of knowledge points out the need for further research to better understand these crucial aspects of viral biology and, in time, prepare for future outbreaks. In this research, the in silico analysis of the cis RNA structures that act in the alpha-, beta-, gamma-, and deltacoronavirus genera has provided a detailed view of the presence and adaptation of the structures of these elements in coronaviruses. The results emphasize the importance of these cis elements in viral biology and their variability between different viral variants. Some coronavirus variants in some groups, depending on the cis element (stem-loop1 and -2; pseudoknot stem-loop1 and -2, and s2m), exhibited functional adaptation. Additionally, the conformation flexibility of the s2m element in the SARS variants was determined, suggesting a coevolution of this element in this viral group. The variability in secondary structures suggests genomic adaptations that may be related to replication processes, genetic regulation, as well as the specific pathogenicity of each variant. The results suggest that RNA structures in coronaviruses can adapt and evolve toward different viral variants, which has important implications for viral adaptation, pathogenicity, and future therapeutic strategies.

Endothelial-specific Enhancer as a Cis Element of PLAUR Regulation by TNF-alpha, IL-1beta, and VEGF.

The expression of human PLAUR gene, which encodes the urokinase plasminogen activator receptor (uPAR), is cell- and process-specific and elevated in inflammation, cancer and senescence. Its tight regulation is achieved by regulatory elements in the gene locus, such as the promoter and several enhancers. The promoter activity is not specific to a particular cell type and has been described earlier. The proximal enhancer is endothelial-specific and responsible for the PLAUR expression pattern in endothelial cells. In this study we described the enhancer activity and its cis-regulatory elements based on the published data. We showed a possible connection of the enhancer activity with known cellular phenotypes.

The Many Roads from Alternative Splicing to Cancer: Molecular Mechanisms Involving Driver Genes.

Cancer driver genes are either oncogenes or tumour suppressor genes that are classically activated or inactivated, respectively, by driver mutations. Alternative splicing-which produces various mature mRNAs and, eventually, protein variants from a single gene-may also result in driving neoplastic transformation because of the different and often opposed functions of the variants of driver genes. The present review analyses the different alternative splicing events that result in driving neoplastic transformation, with an emphasis on their molecular mechanisms. To do this, we collected a list of 568 gene drivers of cancer and revised the literature to select those involved in the alternative splicing of other genes as well as those in which its pre-mRNA is subject to alternative splicing, with the result, in both cases, of producing an oncogenic isoform. Thirty-one genes fall into the first category, which includes splicing factors and components of the spliceosome and splicing regulators. In the second category, namely that comprising driver genes in which alternative splicing produces the oncogenic isoform, 168 genes were found. Then, we grouped them according to the molecular mechanisms responsible for alternative splicing yielding oncogenic isoforms, namely, mutations in cis splicing-determining elements, other causes involving non-mutated cis elements, changes in splicing factors, and epigenetic and chromatin-related changes. The data given in the present review substantiate the idea that aberrant splicing may regulate the activation of proto-oncogenes or inactivation of tumour suppressor genes and details on the mechanisms involved are given for more than 40 driver genes.

Sequence reliance of the Drosophila context-dependent transcription factor CLAMP.

Despite binding similar cis elements in multiple locations, a single transcription factor (TF) often performs context-dependent functions at different loci. How factors integrate cis sequence and genomic context is still poorly understood and has implications for off-target effects in genetic engineering. The Drosophila context-dependent TF chromatin-linked adaptor for male-specific lethal proteins (CLAMP) targets similar GA-rich cis elements on the X-chromosome and at the histone gene locus but recruits very different, locus-specific factors. We discover that CLAMP leverages information from both cis element and local sequence to perform context-specific functions. Our observations imply the importance of other cues, including protein-protein interactions and the presence of additional cofactors.

Comparative genomic profiling of transport inhibitor Response1/Auxin signaling F-box (TIR1/AFB) genes in eight Pyrus genomes revealed the intraspecies diversity and stress responsiveness patterns.

In the genomics of plants and the phytoecosystem, Pyrus (pear) is among the most nutritious fruits and contains fiber that has great health benefits to humans. It is mostly cultivated in temperate regions and is one of the most cultivated pome fruits globally. Pears are highly subjected to biotic and abiotic stresses that affect their yield. TIR1/AFB proteins act as auxin co-receptors during the signaling of nuclear auxins and play a primary role in development-related regulatory processes and responses to biotic and abiotic stresses. However, this gene family and its members have not been explored in Pyrus genomes, and understanding these genes will help obtain useful insights into stress tolerance and ultimately help maintain a high yield of pears. This study reports a pangenome-wide investigation of TIR1/AFB genes from eight Pyrus genomes: Cuiguan (Pyrus pyrifolia), Shanxi Duli (P. betulifolia), Zhongai 1 [(P. ussuriensis × communis) × spp.], Nijisseiki (P. pyrifolia), Yunhong No.1 (P. pyrifolia), d'Anjou (P. communis), Bartlett v2.0 (P. communis), and Dangshansuli v.1.1 (P. bretschneideri). These genes were randomly distributed on 17 chromosomes in each genome. Based on phylogenetics, the identified TIR1/AFB genes were divided into six groups. Their gene structure and motif pattern showed the intraspecific structural conservation as well as evolutionary patterns of Pyrus TIR1/AFBs. The expansion of this gene family in Pyrus is mainly caused by segmental duplication; however, a few genes showed tandem duplication. Moreover, positive and negative selection pressure equally directed the gene's duplication process. The GO and PPI analysis showed that Pyrus TIR1/AFB genes are associated with abiotic stress- and development-related signaling pathways. The promoter regions of Pyrus TIR1/AFB genes were enriched in hormone-, light-, development-, and stress-related cis elements. Furthermore, publicly available RNA-seq data analysis showed that DaTIR1/AFBs have varied levels of expression in various tissues and developmental stages, fruit hardening disease conditions, and drought stress conditions. This indicated that DaTIR1/AFB genes might play critical roles in response to biotic and abiotic stresses. The DaTIR1/AFBs have similar protein structures, which show that they are involved in the same function. Hence, this study will broaden our knowledge of the TIR1/AFB gene family in Pyrus, elucidating their contribution to conferring resistance against various environmental stresses, and will also provide valuable insights for future researchers.

Deciphering the role of alternative splicing in neoplastic diseases for immune-oncological therapies.

Alternative splicing (AS) is an important molecular biological mechanism regulated by complex mechanisms involving a plethora of cis and trans-acting elements. Furthermore, AS is tissue specific and altered in various pathologies, including infectious, inflammatory, and neoplastic diseases. Recently developed immuno-oncological therapies include monoclonal antibodies (mAbs) and chimeric antigen receptor (CAR) T cells targeting, among others, immune checkpoint (ICP) molecules. Despite therapeutic successes have been demonstrated, only a limited number of patients showed long-term benefit from these therapies with tumor entity-related differential response rates were observed. Interestingly, splice variants of common immunotherapeutic targets generated by AS are able to completely escape and/or reduce the efficacy of mAb- and/or CAR-based tumor immunotherapies. Therefore, the analyses of splicing patterns of targeted molecules in tumor specimens prior to therapy might help correct stratification, thereby increasing therapy success by antibody panel selection and antibody dosages. In addition, the expression of certain splicing factors has been linked with the patients' outcome, thereby highlighting their putative prognostic potential. Outstanding questions are addressed to translate the findings into clinical application. This review article provides an overview of the role of AS in (tumor) diseases, its molecular mechanisms, clinical relevance, and therapy response.