Alternative Transcripts Research Articles

The Basis of Diversity in Laminopathy Phenotypes Caused by Variants in the Intron 8 Donor Splice Site of the LMNA Gene

Laminopathies are a broad spectrum of hereditary diseases caused by pathogenic variants of the LMNA gene. Such phenotypic diversity is explained by the function of intermediate filaments encoded by the LMNA gene. We examined a family with an overlapping phenotype of cardiac arrhythmia, cardiomyopathy, limb–girdle muscular dystrophy, and partial lipodystrophy. The cause of the disorder was a novel LMNA(NM_170707.4):c.1488+2T>C variant. The analysis of mRNA extracted from the probands’ blood showed a multitude of alternative splicing products, which was the cause of the complex phenotype in affected family members. Aside from that, we used minigene constructs to analyze the c.1488+2T>C variant, as well as other previously described variants affecting the same donor splice site in intron 8 (c.1488+1G>A, c.1488+5G>C, c.1488+5G>A). We demonstrated that these variants result in multiple splicing events, each producing splicing products with varying prevalence. Our experiments suggest that the variety of alternative transcripts contributes to complex phenotypes, while the quantitative ratio of these transcripts influences the varying severity of the disease.

An IS element-driven antisense RNA attenuates the expression of serotype 2 fimbriae and the cytotoxicity of Bordetella pertussis

Insertion sequences (IS) represent mobile genetic elements that have been shown to be associated with bacterial evolution and adaptation due to their effects on genome plasticity. In Bordetella pertussis, the causative agent of whooping cough, the numerous IS elements induce genomic rearrangements and contribute to the diversity of the global B. pertussis population. Previously, we have shown that the majority of IS-specific endogenous promoters induce the synthesis of alternative transcripts and thereby affect the transcriptional landscape of B. pertussis. Here, we describe the regulatory RNA Rfi2, which is transcribed from the Pout promoter of the IS481 gene BP1118 antisense to the adjacent fim2 gene encoding the major serotype 2 fimbrial subunit of B. pertussis. Among the classical bordetellae, Rfi2 is unique to B. pertussis, suggesting its specific role in virulence. We show that Rfi2 RNA attenuates fim2 transcription and, consequently, the production of the Fim2 protein. Interestingly, the mutant that does not produce Rfi2 displayed significantly increased cytotoxicity toward human macrophages compared to the parental strain. This observation suggests that the Rfi2-mediated reduction in cytotoxicity represents an evolutionary adaptation of B. pertussis that fine-tunes its interaction with the human host. Given the immunogenicity of Fim2, we further hypothesize that Rfi2-mediated modulation of Fim2 production contributes to immune evasion. To our knowledge, Rfi2 represents the first functionally characterized IS element-driven antisense RNA that modulates the expression of a virulence gene.

OAS1: A Protective Mechanism for Alzheimer's Disease? An Exploration of Data and Possible Mechanisms.

The immune system and neuroinflammation are now well established in the aetiology of neurodegeneration. Previous studies of transcriptomic and gene association studies have highlighted the potential of the 2'-5' oligoadenylate synthetase 1 (OAS1) to play a role in Alzheimer's disease. OAS1 is a viral response gene, interferon-induced, dsRNA activated enzyme, which binds RNase L to degrade dsRNA, and has been associated with COVID-19 response. This study explores whether a viral defence gene could play a vital role in neurodegeneration pathology. The genotyping of five SNPs across the OAS1 locus was conducted in the Brains for Dementia Research (BDR) Cohort for association with AD. RNA-sequencing data were explored for differences in OAS1 gene expression between phenotypes and genotypes. Finally, levels of dsRNA were measured in control cell lines, prior to and after exposure to amyloid oligomers and in cells harbouring a dementia-relevant mutation. No association of any of the OAS1 SNPs investigated were associated with the AD phenotype in the BDR cohort. However, gene expression data supported the previous observation that the minor allele haplotype was associated with higher levels of the OAS1 gene expression and the presence of an alternative transcript. Further to this, the presence of endogenous dsRNA was found to increase with exposure to amyloid oligomers and in the cell line with a dementia-relevant mutation. The data presented here suggest further exploration of the OAS1 gene in relation to dementia is warranted. Investigations of whether carriers of the protective OAS1 haplotype lower dsRNA presence and in turn lower inflammation and cell death are required to support the role of the gene as a moderator of neurodegeneration.

Comprehensive Peptide Mapping Is Crucial for Proteogenomics and Proteomics.

Proteogenomics enables the confirmation and refinement of gene models, the detection of new ones, and the proposition of alternative transcripts using support at the protein level. Such evidence is usually generated using mass spectrometry and subsequent result mapping to various sequence databases. This workflow entails several problems: (1) To speed up the analysis, only a small set of expected proteins is searched; (2) database search tools generally do not provide mapping to the genome; and (3) upon new releases of the sequence databases, expensive rerunning of all results would need to be performed. Therefore, fast and accurate peptide mapping is needed as part of proteogenomic pipelines. Unfortunately, some available tools have technical shortcomings. Thus, a set of test cases was developed to allow tool developers to test their implementations comprehensively. The need for comprehensive testing is exemplified by PGx and PGM, two published tools that could only solve a subset of test cases. Lelantos passed all test cases. A set of comprehensive test cases has been developed to overcome these issues. Many unpublished peptide mapping tools are part of proteogenomic workflows, and such tools would also benefit from comprehensive testing. Finally, peptide mapping may also be crucial for proteomics because sequence databases change over time. In response, peptide remapping should be performed to ensure that peptides identifying a protein are proteotypic in a larger sequence context.

Multi-omics in exploring the pathophysiology of diabetic retinopathy.

Diabetic retinopathy (DR) is a leading global cause of vision impairment, with its prevalence increasing alongside the rising rates of diabetes mellitus (DM). Despite the retina's complex structure, the underlying pathology of DR remains incompletely understood. Single-cell RNA sequencing (scRNA-seq) and recent advancements in multi-omics analyses have revolutionized molecular profiling, enabling high-throughput analysis and comprehensive characterization of complex biological systems. This review highlights the significant contributions of scRNA-seq, in conjunction with other multi-omics technologies, to DR research. Integrated scRNA-seq and transcriptomic analyses have revealed novel insights into DR pathogenesis, including alternative transcription start site events, fluctuations in cell populations, altered gene expression profiles, and critical signaling pathways within retinal cells. Furthermore, by integrating scRNA-seq with genetic association studies and multi-omics analyses, researchers have identified novel biomarkers, susceptibility genes, and potential therapeutic targets for DR, emphasizing the importance of specific retinal cell types in disease progression. The integration of scRNA-seq with metabolomics has also been instrumental in identifying specific metabolites and dysregulated pathways associated with DR. It is highly conceivable that the continued synergy between scRNA-seq and other multi-omics approaches will accelerate the discovery of underlying mechanisms and the development of novel therapeutic interventions for DR.

Piwi regulates the usage of alternative transcription start sites in the Drosophila ovary.

Alternative transcription initiation, which refers to the transcription of a gene from different transcription start sites (TSSs), is prevalent across metazoans and has important biological functions. Although transcriptional regulation has been extensively studied, the mechanism that selects one TSS over others within a gene remains elusive. Using the Cap Analysis of Gene Expression sequencing (CAGE-seq) method, we discovered that Piwi, an RNA-binding protein, regulates TSS usage in at least 87 genes. In piwi-deficient Drosophila ovaries, these genes displayed significantly altered TSS usage (ATU). The regulation of TSS usage occurredin both germline and somatic cells in ovaries, as well as in cultured ovarian somatic cells (OSCs). Correspondingly, RNA Polymerase II (Pol II) initiation and elongation at the TSSs of ATU genes were affected in germline-piwi-knockdown ovaries and piwi-knockdown OSCs. Furthermore, we identified a Facilitates Chromatin Transcription (FACT)complex component, Ssrp, that is essential for mRNA elongation, as a novel interactor of Piwi in the nucleus. Temporally controlled knockdown of ssrp affected TSS usage in ATU genes, whereas overexpression of ssrp partially rescued the TSS usage of ATU genes in piwi mutant ovaries. Thus, Piwi may interact with Ssrp to regulate TSS usage in Drosophila ovaries by affecting Pol II initiation and elongation.

Splice site and de novo variants can cause PLCG2-associated immune dysregulation with cold urticaria (PLAID-CU).

Phospholipase Cγ2 (PLCγ2) is an important signaling molecule that receives and transmits signals from various cell surface receptors in most hematopoietic lineages. Variants of PLCG2 cause PLCγ2-associated immune dysregulation (PLAID), a family of conditions that are classified by mutational effect. PLAID with cold urticaria (PLAID-CU) is caused by in-frame deletions of PLCG2 that are dominant negative at physiologic temperatures but become spontaneously active at sub-physiologic temperatures. To identify genetic lesions that cause PLAID by combining RNA sequencing of full-length PLCG2 with whole genome sequencing. We studied nine probands with antibody deficiency and a positive evaporative cooling test, together with two known PLAID-CU patients and three healthy subjects. Illumina sequencing was performed on full-length PLCG2 cDNA synthesized from peripheral blood mononuclear cell RNA and whole genome sequencing was used to identify genetic lesions. Novel alternate transcripts were overexpressed in the Plcg2-deficient DT40 cell overexpression system. ERK phosphorylation was quantified by flow cytometry with and without BCR crosslinking. Two probands expressed novel alternative transcripts of PLCG2 with in-frame deletions. Proband 1, expressing PLCG2 without exons 18-19, carried a splice site mutation in intron 19. Proband 2, expressing PLCG2 without exons 19-22, carried a 14kb de novo deletion of PLCG2. DT40 cells overexpressing the exon 18-19 or exon 19-22 deletions failed to phosphorylate ERK in response to BCR crosslinking. In addition to autosomal dominant genomic deletions, de novo deletions and splice site mutations of PLCG2 can also cause PLAID-CU. All of these can be identified by cDNA-based sequencing.

Targeted Variant Assessments of Human Endogenous Retroviral Regions in Whole Genome Sequencing Data Reveal Retroviral Variants Associated with Papillary Thyroid Cancer.

Papillary thyroid cancer (PTC) is one of the fastest-growing cancers worldwide, lacking established causal factors or validated early diagnostics. Human endogenous retroviruses (HERVs), comprising 8% of human genomes, have potential as PTC biomarkers due to their comparably high baseline expression in healthy thyroid tissues, indicating homeostatic roles. However, HERV regions are often overlooked in genome-wide association studies because of their highly repetitive nature, low sequence coverage, and decreased sequencing quality. Using targeted whole-genome sequence analysis in conjunction with high sequencing depth to overcome methodological limitations, we identified associations of specific HERV variants with PTC. Analyzing WGS data from 138 patients with PTC generated through The Cancer Genome Atlas project and 2015 control samples from the 1000 Genomes Project, we examined the mutational variation in HERVs within a 20 kb radius of known cancer predisposition genes (CPGs) differentially expressed in PTC. We discovered 15 common and 13 rare germline HERV variants near or within 20 CPGs that distinguish patients with PTC from healthy controls. We identified intragenic-intronic HERV variants within RYR2, LRP1B, FN1, MET, TCRVB, UNC5D, TRPM3, CNTN5, CD70, RYR1, RUNX1, CRLF2, and PCDH1X, and three variants downstream of SERPINA1 and RUNX1T1. Sanger sequencing analyses of 20 thyroid and 5 non-thyroid cancer cell lines confirmed associations with PTC, particularly for MSTA HERV-L variant rs200077102 within the FN1 gene and HERV-L MLT1A LTR variant rs78588384 within the CNTN5 gene. Variant rs78588384, in particular, was shown in our analyses to be located within a POL2 binding site regulating an alternative transcript of CNTN5. In addition, we identified 16 variants that modified the poly(A) region in Alu elements, potentially altering the potential to retrotranspose. In conclusion, this study serves as a proof-of-concept for targeted variant analysis of HERV regions and establishes a basis for further exploration of HERVs in thyroid cancer development.

PVACview: an interactive visualization tool for efficient neoantigen prioritization and selection

BackgroundNeoantigen-targeting therapies including personalized vaccines have shown promise in the treatment of cancers, particularly when used in combination with checkpoint blockade therapy. At least 100 clinical trials involving these therapies have been initiated globally. Accurate identification and prioritization of neoantigens is crucial for designing these trials, predicting treatment response, and understanding mechanisms of resistance. With the advent of massively parallel DNA and RNA sequencing technologies, it is now possible to computationally predict neoantigens based on patient-specific variant information. However, numerous factors must be considered when prioritizing neoantigens for use in personalized therapies. Complexities such as alternative transcript annotations, various binding, presentation and immunogenicity prediction algorithms, and variable peptide lengths/registers all potentially impact the neoantigen selection process. There has been a rapid development of computational tools that attempt to account for these complexities. While these tools generate numerous algorithmic predictions for neoantigen characterization, results from these pipelines are difficult to navigate and require extensive knowledge of the underlying tools for accurate interpretation. This often leads to over-simplification of pipeline outputs to make them tractable, for example, limiting prediction to a single RNA isoform or only summarizing the top ranked of many possible peptide candidates. In addition to variant detection, gene expression, and predicted peptide binding affinities, recent studies have also demonstrated the importance of mutation location, allele-specific anchor locations, and variation of T-cell response to long versus short peptides. Due to the intricate nature and number of salient neoantigen features, presenting all relevant information to facilitate candidate selection for downstream applications is a difficult challenge that current tools fail to address.ResultsWe have created pVACview, the first interactive tool designed to aid in the prioritization and selection of neoantigen candidates for personalized neoantigen therapies including cancer vaccines. pVACview has a user-friendly and intuitive interface where users can upload, explore, select, and export their neoantigen candidates. The tool allows users to visualize candidates at multiple levels of detail including variant, transcript, peptide, and algorithm prediction information.ConclusionspVACview will allow researchers to analyze and prioritize neoantigen candidates with greater efficiency and accuracy in basic and translational settings. The application is available as part of the pVACtools software at pvactools.org and as an online server at pvacview.org.

Noncanonical transcription initiation is primarily tissue specific and epigenetically tuned in paleopolyploid plants.

Alternative transcription initiation (ATI) appears to be a ubiquitous regulatory mechanism of gene expression in eukaryotes. However, the extent to which it affects the products of gene expression and how it evolves and is regulated remain unknown. Here, we report genome-wide identification and analysis of transcription start sites (TSSs) in various soybean (Glycine max) tissues using a survey of transcription initiation at promoter elements with high-throughput sequencing (STRIPE-seq). We defined 193,579 TSS clusters/regions (TSRs) in 37,911 annotated genes, with 56.5% located in canonical regulatory regions and 43.5% from start codons to 3' untranslated regions, which were responsible for changes in open reading frames of 24,131 genes. Strikingly, 6,845 genes underwent ATI within coding sequences (CDSs). These CDS-TSRs were tissue-specific, did not have TATA-boxes typical of canonical promoters, and were embedded in nucleosome-free regions flanked by nucleosomes with enhanced levels of histone marks potentially associated with intragenic transcriptional initiation, suggesting that ATI within CDSs was epigenetically tuned and associated with tissue-specific functions. Overall, duplicated genes possessed more TSRs, exhibited lower degrees of tissue specificity, and underwent stronger purifying selection than singletons. This study highlights the significance of ATI and the genomic and epigenomic factors shaping the distribution of ATI in CDSs in a paleopolyploid eukaryote.

Cell-type-specific splicing of transcription regulators and Ptbp1 by Rbfox1/2/3 in the developing neocortex.

How master splicing regulators crosstalk with each other and to what extent transcription regulators are differentially spliced remain unclear in the developing brain. Here, cell-type-specific RNA-Seq analyses of the developing neocortex uncover variable expression of the Rbfox1/2/3 genes and enriched splicing events in transcription regulators, altering protein isoforms or inducing nonsense-mediated mRNA decay. Transient expression of Rbfox proteins in radial glial progenitors induces neuronal splicing events preferentially in transcription regulators such as Meis2 and Tead1 Surprisingly, Rbfox proteins promote the inclusion of a mammal-specific alternative exon and a previously undescribed poison exon in Ptbp1 Simultaneous ablation of Rbfox1/2/3 in the neocortex downregulates neuronal isoforms and disrupts radial neuronal migration. Furthermore, the progenitor isoform of Meis2 promotes Tgfb3 transcription, while the Meis2 neuron isoform promotes neuronal differentiation. These observations indicate that transcription regulators are differentially spliced between cell types in the developing neocortex. [The sex has not been reported to affect cortical neurogenesis in mice, and embryos of both sexes were studied without distinguishing one or the other.]Significance Statement How alternative splicing regulates cell-type-specific gene expression in the developing neocortex remains understudied. Here, analyses of sorted cell types and single-cell long-reads uncover cell-type-specific splicing that is enriched in transcription regulators. Rbfox proteins, including the pan-neuronal marker NeuN/Rbfox3, preferentially switch splice forms of transcription regulators and are required for radial neuronal migration. We further show that the progenitor and neuron isoforms of a transcription regulator Meis2 function differently. Overall, this study suggests a cross-talk between alternative splicing and transcription for neuronal gene regulation.

A WFS1 variant disrupting acceptor splice site uncovers the impact of alternative splicing on beta cell apoptosis in a patient with Wolfram syndrome

Aims/hypothesisWolfram syndrome 1 (WS1) is an inherited condition mainly manifesting in childhood-onset diabetes mellitus and progressive optic nerve atrophy. The causative gene, WFS1, encodes wolframin, a master regulator of several cellular responses, and the gene’s mutations associate with clinical variability. Indeed, nonsense/frameshift variants correlate with more severe symptoms than missense/in-frame variants. As achieving a genotype–phenotype correlation is crucial for dealing with disease outcome, works investigating the impact of transcriptional and translational landscapes stemming from such mutations are needed. Therefore, we sought to elucidate the molecular determinants behind the pathophysiological alterations in a WS1 patient carrying compound heterozygous mutations in WFS1: c.316-1G>A, affecting the acceptor splice site (ASS) upstream of exon 4; and c.757A>T, introducing a premature termination codon (PTC) in exon 7.MethodsBioinformatic analysis was carried out to infer the alternative splicing events occurring after disruption of ASS, followed by RNA-seq and PCR to validate the transcriptional landscape. Patient-derived induced pluripotent stem cells (iPSCs) were used as an in vitro model of WS1 and to investigate the WFS1 alternative splicing isoforms in pancreatic beta cells. CRISPR/Cas9 technology was employed to correct ASS mutation and generate a syngeneic control for the endoplasmic reticulum stress induction and immunotoxicity assays.ResultsWe showed that patient-derived iPSCs retained the ability to differentiate into pancreatic beta cells. We demonstrated that the allele carrying the ASS mutation c.316-1G>A originates two PTC-containing alternative splicing transcripts (c.316del and c.316–460del), and two open reading frame-conserving mRNAs (c.271–513del and c.316–456del) leading to N-terminally truncated polypeptides. By retaining the C-terminal domain, these isoforms sustained the endoplasmic reticulum stress response in beta cells. Otherwise, PTC-carrying transcripts were regulated by the nonsense-mediated decay (NMD) in basal conditions. Exposure to cell stress inducers and proinflammatory cytokines affected expression levels of the NMD-related gene SMG7 (>twofold decrease; p<0.001) without eliciting a robust unfolded protein response in WFS1 beta cells. This resulted in a dramatic accumulation of the PTC-containing isoforms c.316del (>100-fold increase over basal; p<0.001) and c.316–460del (>20-fold increase over basal; p<0.001), predisposing affected beta cells to undergo apoptosis. Cas9-mediated recovery of ASS retrieved the canonical transcriptional landscape, rescuing the normal phenotype in patient-derived beta cells.Conclusions/interpretationThis study represents a new model to study wolframin, highlighting how each single mutation of the WFS1 gene can determine dramatically different functional outcomes. Our data point to increased vulnerability of WFS1 beta cells to stress and inflammation and we postulate that this is triggered by escaping NMD and accumulation of mutated transcripts and truncated proteins. These findings pave the way for further studies on the molecular basis of genotype–phenotype relationship in WS1, to uncover the key determinants that might be targeted to ameliorate the clinical outcome of patients affected by this rare disease.Data availabilityThe in silico predicted N-terminal domain structure file of WT wolframin was deposited in the ModelArchive, together with procedures, ramachandran plots, inter-residue distance deviation and IDDT scores, and Gromacs configuration files (doi/10.5452/ma-cg3qd). The deep-sequencing data as fastq files used to generate consensus sequences of AS isoforms of WFS1 are available in the SRA database (BioProject PRJNA1109747).Graphical

RNA/DNA Binding Protein TDP43 Regulates DNA Mismatch Repair Genes with Implications for Genome Stability.

TAR DNA-binding protein 43 (TDP43) is increasingly recognized for its involvement in neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP43 proteinopathy, characterized by dysregulated nuclear export and cytoplasmic aggregation, is present in most ALS/FTD cases and is associated with a loss of nuclear function and genomic instability in neurons. Building on prior evidence linking TDP43 pathology to DNA double-strand breaks (DSBs), this study identifies a novel regulatory role for TDP43 in the DNA mismatch repair (MMR) pathway. We demonstrate that depletion or overexpression of TDP43 affects the expression of key MMR genes, including MLH1, MSH6, MSH2, MSH3, and PMS2. Specifically, TDP43 modulates the expression of MLH1 and MSH6 proteins through alternative splicing and transcript stability. These findings are validated in ALS mice models, patient-derived neural progenitor cells and autopsied brain tissues from ALS patients. Furthermore, MMR depletion showed a partial rescue of TDP43-induced DNA damage in neuronal cells. Bioinformatics analysis of TCGA cancer database reveals significant correlations between TDP43 and MMR gene expressions and mutational burden across various cancer subtypes. These results collectively establish TDP43 as a critical regulator of the MMR pathway, with broad implications for understanding the genomic instability underlying neurodegenerative and neoplastic diseases.

Molecular evolution and genetic diversity of defective chorion 1 in Anastrepha fraterculus and Anastrepha obliqua (Diptera, Tephritidae).

The family Tephritidae comprises numerous fruit fly species, some of which are economically significant, such as several in the genus Anastrepha. Most pest species in this genus belong to the fraterculus group, characterized by closely related species that are difficult to differentiate due to recent divergence and gene flow. Identifying genetic markers for their study is paramount for understanding the group's evolution and eventual phytosanitary control. Because there is variation in eggshell morphology among species in the genus, the study of the rapidly evolving defective chorion 1 (dec-1) gene, which is crucial for chorion formation and reproduction, could provide relevant information for Anastrepha differentiation. We compared transcriptome sequences of dec-1 from two of the most important pest species in the genus, Anastrepha fraterculus and Anastrepha obliqua to dec-1 sequences from Anastrepha ludens, which was used for structure prediction. Furthermore, we amplified a conserved exon across populations of these species. These data revealed three alternative transcripts in A. fraterculus and A. obliqua, consistent with patterns found in other Tephritidae; we obtained orthologous sequences for these other tephritids from NCBI to investigate patterns of selection affecting this gene at different hierarchical levels using different methods. These analyses show a general pattern of purifying selection across the whole gene and throughout its history at different hierarchical levels, from populations to more distantly related species. That notwithstanding, we still found evidence of positive and episodic diversifying selection at different levels. Different parts of the gene have shown distinct evolutionary rates, which were associated with the diverse proproteins produced by posttranslational changes of DEC-1, with proproteins that are incorporated in the chorion earlier in egg formation being in general more conserved than others that are incorporated later. This correlation appears more evident in certain lineages, including the branch that separates Anastrepha, as well as other internal branches that differentiate species within the genus. Our data showed that this gene shows remarkable variation across its different exons, which has proven to be informative at different evolutionary levels. These changes hold promise not only for studying differentiation in Anastrepha but also for the eventual management of selected pest species.

Characterisation of LGP2 complex multitranscript system in humans: role in the innate immune response and evolution from non-human primates.

Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I, MDA5 and LGP2, recognize viral RNA to mount an antiviral interferon (IFN) response RLRs share three different protein domains: C-terminal domain, DExD/H box RNA helicase domain, and an N-terminal domain with two tandem repeats (CARDs). LGP2 lacks tandem CARD and is not able to induce an IFN response. However, LGP2 positively enhances MDA5 and negatively regulates RIG-I signaling. In this study, we determined the LGP2 alternative transcripts in humans to further comprehend the mechanism of its regulation, their evolutionary origin, and the isoforms functionallity. The results showed new eight alternative transcripts in the samples tested. The presence of these transcripts demonstrated that the main mechanisms for the regulation of LGP2 expression are both by insertion of introns and by the loss of exons. The phylogenetic analysis of the comparison between sequences from exon 1 to exon 3 of humans and those previously described in non-human primates showed three well-differentiated groups (lineages) originating from gorillas, suggesting that the transspecies evolution has been maintained for 10 million years. The corresponding protein models (isoforms) were also established, obtaining four isoforms: one complete and three others lacking the C-terminal domain or this domain and the partial or total He2 Helicase domain, which would compromise the functionality of LGP2. In conclusion, this is the first study that elucidate the large genomic organization and complex transcriptional regulation of human LGP2, its pattern of sequence generation, and a mode of evolutionary inheritance across species.

Alternative transcripts recode human genes to express overlapping, frameshifted microproteins.

Overlapping genes were thought to be essentially absent from the human genome until the discovery of abundant, frameshifted internal open reading frames (iORFs) nested within annotated protein coding sequences. However, it is currently unclear how many functional human iORFs exist and how they are expressed. We demonstrate that, in hundreds of cases, alternative transcript variants that bypass the start codon of annotated coding sequences (CDSs) can recode a human gene to express the iORF-encoded microprotein. While many human genes generate such non- coding alternative transcripts, they are poorly annotated. Here we develope a new analysis pipeline enabling the assignment of translated human iORFs to alternative transcripts, and provide long- read sequencing and molecular validation of their expression in dozens of cases. Finally, we demonstrate that a conserved DEDD2 iORF switches the function of this gene from pro- to anti- apoptotic. This work thus demonstrates that alternative transcript variants can broadly reprogram human genes to express frameshifted iORFs, revealing new levels of complexity in the human transcriptome and proteome.

Re-appraising the evidence for the source, regulation and function of p53-family isoforms.

The p53 family of proteins evolved from a common ancestor into three separate genes encoding proteins that act as transcription factors with distinct cellular roles. Isoforms of each member that lack specific regions or domains are suggested to result from alternative transcription start sites, alternative splicing or alternative translation initiation, and have the potential to exponentially increase the functional repertoire of each gene. However, evidence supporting the presence of individual protein variants at functional levels is often limited and is inferred by mRNA detection using highly sensitive amplification techniques. We provide a critical appraisal of the current evidence for the origins, expression, functions and regulation of p53-family isoforms. We conclude that despite the wealth of publications, several putative isoforms remain poorly established. Future research with improved technical approaches and the generation of isoform-specific protein detection reagents is required to establish the physiological relevance of p53-family isoforms in health and disease. In addition, our analyses suggest that p53-family variants evolved partly through convergent rather than divergent evolution from the ancestral gene.

Alternative Splicing of the Last TKFC Intron Yields Transcripts Differentially Expressed in Human Tissues That Code In Vitro for a Protein Devoid of Triokinase and FMN Cyclase Activity.

The 18-exon human TKFC gene codes for dual-activity triokinase and FMN cyclase (TKFC) in an ORF, spanning from exon 2 to exon 18. In addition to TKFC-coding transcripts (classified as tkfc type by their intron-17 splice), databases contain evidence for alternative TKFC transcripts, but none of them has been expressed, studied, and reported in the literature. A novel full-ORF transcript was cloned from brain cDNA and sequenced (accession no. DQ344550). It results from an alternative 3' splice-site in intron 17. The cloned cDNA contains an ORF also spanning from exon 2 to exon 18 of the TKFC gene but with a 56-nt insertion between exons 17 and 18 (classified as tkfc_ins56 type). This insertion introduces an in-frame stop, and the resulting ORF codes for a shorter TKFC variant, which, after expression, is enzymatically inactive. TKFC intron-17 splicing was found to be differentially expressed in human tissues. In a multiple-tissue northern blot using oligonucleotide probes, the liver showed a strong expression of the tkfc-like splice of intron 17, and the heart preferentially expressed the tkfc_ins56-like splice. Through a comparison to global expression data from massive-expression studies of human tissues, it was inferred that the intestine preferentially expresses TKFC transcripts that contain neither of those splices. An analysis of transcript levels quantified by RNA-Seq in the GTEX database revealed an exception to this picture due to the occurrence of a non-coding short transcript with a tkfc-like splice. Altogether, the results support the occurrence of potentially relevant transcript variants of the TKFC gene, differentially expressed in human tissues. (This work is dedicated in memoriam to Professor Antonio Sillero, 1938-2024, for his lifelong mentoring and his pioneering work on triokinase).

Identification of candidate causal variants and target genes at 41 breast cancer risk loci through differential allelic expression analysis

Understanding breast cancer genetic risk relies on identifying causal variants and candidate target genes in risk loci identified by genome-wide association studies (GWAS), which remains challenging. Since most loci fall in active gene regulatory regions, we developed a novel approach facilitated by pinpointing the variants with greater regulatory potential in the disease’s tissue of origin. Through genome-wide differential allelic expression (DAE) analysis, using microarray data from 64 normal breast tissue samples, we mapped the variants associated with DAE (daeQTLs). Then, we intersected these with GWAS data to reveal candidate risk regulatory variants and analysed their cis-acting regulatory potential. Finally, we validated our approach by extensive functional analysis of the 5q14.1 breast cancer risk locus. We observed widespread gene expression regulation by cis-acting variants in breast tissue, with 65% of coding and noncoding expressed genes displaying DAE (daeGenes). We identified over 54 K daeQTLs for 6761 (26%) daeGenes, including 385 daeGenes harbouring variants previously associated with BC risk. We found 1431 daeQTLs mapped to 93 different loci in strong linkage disequilibrium with risk-associated variants (risk-daeQTLs), suggesting a link between risk-causing variants and cis-regulation. There were 122 risk-daeQTL with stronger cis-acting potential in active regulatory regions with protein binding evidence. These variants mapped to 41 risk loci, of which 29 had no previous report of target genes and were candidates for regulating the expression levels of 65 genes. As validation, we identified and functionally characterised five candidate causal variants at the 5q14.1 risk locus targeting the ATG10 and ATP6AP1L genes, likely acting via modulation of alternative transcription and transcription factor binding. Our study demonstrates the power of DAE analysis and daeQTL mapping to identify causal regulatory variants and target genes at breast cancer risk loci, including those with complex regulatory landscapes. It additionally provides a genome-wide resource of variants associated with DAE for future functional studies.

Identification of a novel splicing variant of thyroid hormone receptor interaction protein 13 (TRIP13) in female infertility characterized by oocyte maturation arrest.

As a cause of primary female infertility, oocyte maturation arrest (OMA) is characterized by failure to obtain mature oocytes due to abnormal meiosis. We aimed to identify pathogenic variants in two sisters with OMA phenotype from a non-consanguineous family. Whole-exome sequencing and Sanger sequencing were conducted to identify and validate the disease-causing gene variant. Additionally, we performed a minigene assay, quantitative reverse transcription PCR, and Western blotting to assess the effects of the variant. We identified a novel homozygous splicing variant (c.1021-11T>C) in TRIP13, which followed a recessive inheritance pattern. Minigene assay showed that the variant could disrupt the integrity of TRIP13 mRNA, as evidenced by the production of an alternative transcript with intron10 intermediate retention of 79 bp. Compared to normal controls, the expression of TRIP13 mRNA and abundance of TRIP13 protein were also significantly decreased in Epstein-Barr virus-immortalized lymphoblastoid cells derived from affected individuals. Our findings confirm the contribution of genetic factors to OMA and expand the mutation spectrum of TRIP13 in female infertility.