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1.
Science ; 385(6705): eadm8189, 2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38991068

RESUMO

TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life. IS605-family TnpB homologs function as programmable RNA-guided homing endonucleases in bacteria, driving transposon maintenance through DNA double-strand break-stimulated homologous recombination. In this work, we uncovered molecular mechanisms of the transposition life cycle of IS607-family elements that, notably, also encode group I introns. We identified specific features for a candidate "IStron" from Clostridium botulinum that allow the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts evolved an ability to balance competing and mutually exclusive activities that promote selfish transposon spread while limiting adverse fitness costs on the host. Collectively, this work highlights molecular innovation in the multifunctional utility of transposon-encoded noncoding RNAs.


Assuntos
Elementos de DNA Transponíveis , Íntrons , RNA Guia de Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas/genética , Splicing de RNA , Sistemas CRISPR-Cas , Transposases/metabolismo , Transposases/genética , Recombinação Homóloga
2.
Science ; 385(6704): 105-112, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38963841

RESUMO

Introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. In this work, we studied intron-encoded homing endonuclease gp210 in bacteriophage ΦPA3 and found that it contributes to viral competition by interfering with the replication of a coinfecting phage, ΦKZ. We show that gp210 targets a specific sequence in ΦKZ, which prevents the assembly of progeny viruses. This work demonstrates how a homing endonuclease can be deployed in interference competition among viruses and provide a relative fitness advantage. Given the ubiquity of homing endonucleases, this selective advantage likely has widespread evolutionary implications in diverse plasmid and viral competition as well as virus-host interactions.


Assuntos
Endonucleases , Íntrons , Fagos de Pseudomonas , Pseudomonas aeruginosa , Interferência Viral , Proteínas Virais , Endonucleases/metabolismo , Endonucleases/genética , Interferência Viral/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Montagem de Vírus , Replicação Viral , Fagos de Pseudomonas/enzimologia , Fagos de Pseudomonas/genética , Pseudomonas aeruginosa/virologia
3.
Proc Natl Acad Sci U S A ; 121(28): e2400151121, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38954548

RESUMO

Protein folding and evolution are intimately linked phenomena. Here, we revisit the concept of exons as potential protein folding modules across a set of 38 abundant and conserved protein families. Taking advantage of genomic exon-intron organization and extensive protein sequence data, we explore exon boundary conservation and assess the foldon-like behavior of exons using energy landscape theoretic measurements. We found deviations in the exon size distribution from exponential decay indicating selection in evolution. We show that when taken together there is a pronounced tendency to independent foldability for segments corresponding to the more conserved exons, supporting the idea of exon-foldon correspondence. While 45% of the families follow this general trend when analyzed individually, there are some families for which other stronger functional determinants, such as preserving frustrated active sites, may be acting. We further develop a systematic partitioning of protein domains using exon boundary hotspots, showing that minimal common exons correspond with uninterrupted alpha and/or beta elements for the majority of the families but not for all of them.


Assuntos
Éxons , Dobramento de Proteína , Éxons/genética , Humanos , Proteínas/genética , Proteínas/química , Evolução Molecular , Íntrons/genética
4.
Cell Biochem Funct ; 42(5): e4091, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38973151

RESUMO

The intron retention (IR) is a phenomenon utilized by cells to allow diverse fates at the same mRNA, leading to a different pattern of synthesis of the same protein. In this study, we analyzed the modulation of phosphoinositide-specific phospholipase C (PI-PLC) enzymes by Harpagophytum procumbens extract (HPE) in synoviocytes from joins of osteoarthritis (OA) patients. In some samples, the PI-PLC γ1 isoform mature mRNA showed the IR and, in these synoviocytes, the HPE treatment increased the phenomenon. Moreover, we highlighted that as a consequence of IR, a lower amount of PI-PLC γ1 was produced. The decrease of PI-PLC γ1 was associated with the decrease of metalloprotease-3 (MMP-3), and MMP-13, and ADAMTS-5 after HPE treatment. The altered expression of MMPs is a hallmark of the onset and progression of OA, thus substances able to decrease their expression are very desirable. The interesting outcomes of this study are that 35% of analyzed synovial tissues showed the IR phenomenon in the PI-PLC γ1 mRNA and that the HPE treatment increased this phenomenon. For the first time, we found that the decrease of PI-PLC γ1 protein in synoviocytes interferes with MMP production, thus affecting the pathways involved in the MMP expression. This finding was validated by the silencing of PI-PLC γ1 in synoviocytes where the IR phenomenon was not present. Our results shed new light on the biochemical mechanisms involved in the degrading enzyme production in the joint of OA patients, suggesting a new therapeutic target and highlighting the importance of personalized medicine.


Assuntos
Fibroblastos , Íntrons , Fosfolipase C gama , RNA Mensageiro , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fosfolipase C gama/metabolismo , Fosfolipase C gama/genética , Células Cultivadas , Osteoartrite/metabolismo , Osteoartrite/patologia , Membrana Sinovial/metabolismo , Membrana Sinovial/citologia , Membrana Sinovial/efeitos dos fármacos , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Proteína ADAMTS5/metabolismo , Proteína ADAMTS5/genética , Sinoviócitos/metabolismo , Sinoviócitos/efeitos dos fármacos , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética
5.
Proc Natl Acad Sci U S A ; 121(29): e2404349121, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-38985764

RESUMO

Intron-containing RNA expressed from the HIV-1 provirus activates type 1 interferon in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with short hairpin RNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte-derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the interferon-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with nontargetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant 2-CARD domain-deletion or phosphomimetic point mutations, indicates that IFIH1 (MDA5) filament formation, dephosphorylation, and association with MAVS are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 (MDA5) and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1 knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes by HIV-1. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 (MDA5), over two orders of magnitude, was revealed by formaldehyde cross-linking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is the innate immune receptor for intron-containing RNA from the HIV-1 provirus and that IFIH1 potentially contributes to chronic inflammation in people living with HIV-1, even in the presence of effective antiretroviral therapy.


Assuntos
Células Dendríticas , HIV-1 , Imunidade Inata , Helicase IFIH1 Induzida por Interferon , Íntrons , Provírus , RNA Viral , Humanos , HIV-1/genética , HIV-1/imunologia , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Provírus/genética , Células Dendríticas/imunologia , Células Dendríticas/virologia , Células Dendríticas/metabolismo , Íntrons/genética , RNA Viral/genética , RNA Viral/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Infecções por HIV/genética , Carioferinas/genética , Carioferinas/metabolismo
6.
RNA Biol ; 21(1): 52-74, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38989833

RESUMO

The aim of this study was to compare the circular transcriptome of divergent tissues in order to understand: i) the presence of circular RNAs (circRNAs) that are not exonic circRNAs, i.e. originated from backsplicing involving known exons and, ii) the origin of artificial circRNA (artif_circRNA), i.e. circRNA not generated in-vivo. CircRNA identification is mostly an in-silico process, and the analysis of data from the BovReg project (https://www.bovreg.eu/) provided an opportunity to explore new ways to identify reliable circRNAs. By considering 117 tissue samples, we characterized 23,926 exonic circRNAs, 337 circRNAs from 273 introns (191 ciRNAs, 146 intron circles), 108 circRNAs from small non-coding genes and nearly 36.6K circRNAs classified as other_circRNAs. Furthermore, for 63 of those samples we analysed in parallel data from total-RNAseq (ribosomal RNAs depleted prior to library preparation) with paired mRNAseq (library prepared with poly(A)-selected RNAs). The high number of circRNAs detected in mRNAseq, and the significant number of novel circRNAs, mainly other_circRNAs, led us to consider all circRNAs detected in mRNAseq as artificial. This study provided evidence of 189 false entries in the list of exonic circRNAs: 103 artif_circRNAs identified by total RNAseq/mRNAseq comparison using two circRNA tools, 26 probable artif_circRNAs, and 65 identified by deep annotation analysis. Extensive benchmarking was performed (including analyses with CIRI2 and CIRCexplorer-2) and confirmed 94% of the 23,737 reliable exonic circRNAs. Moreover, this study demonstrates the effectiveness of a panel of highly expressed exonic circRNAs (5-8%) in analysing the tissue specificity of the bovine circular transcriptome.


Assuntos
Éxons , RNA Circular , RNA Circular/genética , Animais , Bovinos , Íntrons , Biologia Computacional/métodos , Transcriptoma , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos
7.
Nat Commun ; 15(1): 4980, 2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38898052

RESUMO

The self-splicing group II introns are bacterial and organellar ancestors of the nuclear spliceosome and retro-transposable elements of pharmacological and biotechnological importance. Integrating enzymatic, crystallographic, and simulation studies, we demonstrate how these introns recognize small molecules through their conserved active site. These RNA-binding small molecules selectively inhibit the two steps of splicing by adopting distinctive poses at different stages of catalysis, and by preventing crucial active site conformational changes that are essential for splicing progression. Our data exemplify the enormous power of RNA binders to mechanistically probe vital cellular pathways. Most importantly, by proving that the evolutionarily-conserved RNA core of splicing machines can recognize small molecules specifically, our work provides a solid basis for the rational design of splicing modulators not only against bacterial and organellar introns, but also against the human spliceosome, which is a validated drug target for the treatment of congenital diseases and cancers.


Assuntos
Domínio Catalítico , Íntrons , Splicing de RNA , Spliceossomos , Splicing de RNA/efeitos dos fármacos , Spliceossomos/metabolismo , Spliceossomos/efeitos dos fármacos , Humanos , Íntrons/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química
10.
Nat Commun ; 15(1): 5130, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38879536

RESUMO

Intron retention (IR) is the most common alternative splicing event in Arabidopsis. An increasing number of studies have demonstrated the major role of IR in gene expression regulation. The impacts of IR on plant growth and development and response to environments remain underexplored. Here, we found that IR functions directly in gene expression regulation on a genome-wide scale through the detainment of intron-retained transcripts (IRTs) in the nucleus. Nuclear-retained IRTs can be kept away from translation through this mechanism. COP1-dependent light modulation of the IRTs of light signaling genes, such as PIF4, RVE1, and ABA3, contribute to seedling morphological development in response to changing light conditions. Furthermore, light-induced IR changes are under the control of the spliceosome, and in part through COP1-dependent ubiquitination and degradation of DCS1, a plant-specific spliceosomal component. Our data suggest that light regulates the activity of the spliceosome and the consequent IRT nucleus detainment to modulate photomorphogenesis through COP1.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Núcleo Celular , Regulação da Expressão Gênica de Plantas , Íntrons , Luz , Spliceossomos , Ubiquitina-Proteína Ligases , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Arabidopsis/metabolismo , Íntrons/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Spliceossomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Núcleo Celular/metabolismo , Plântula/crescimento & desenvolvimento , Plântula/genética , Plântula/efeitos da radiação , Plântula/metabolismo , Processamento Alternativo , Ubiquitinação
11.
BMC Genomics ; 25(1): 649, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38943073

RESUMO

Despite the fact that introns mean an energy and time burden for eukaryotic cells, they play an irreplaceable role in the diversification and regulation of protein production. As a common feature of eukaryotic genomes, it has been reported that in protein-coding genes, the longest intron is usually one of the first introns. The goal of our work was to find a possible difference in the biological function of genes that fulfill this common feature compared to genes that do not. Data on the lengths of all introns in genes were extracted from the genomes of six vertebrates (human, mouse, koala, chicken, zebrafish and fugu) and two other model organisms (nematode worm and arabidopsis). We showed that more than 40% of protein-coding genes have the relative position of the longest intron located in the second or third tertile of all introns. Genes divided according to the relative position of the longest intron were found to be significantly increased in different KEGG pathways. Genes with the longest intron in the first tertile predominate in a range of pathways for amino acid and lipid metabolism, various signaling, cell junctions or ABC transporters. Genes with the longest intron in the second or third tertile show increased representation in pathways associated with the formation and function of the spliceosome and ribosomes. In the two groups of genes defined in this way, we further demonstrated the difference in the length of the longest introns and the distribution of their absolute positions. We also pointed out other characteristics, namely the positive correlation between the length of the longest intron and the sum of the lengths of all other introns in the gene and the preservation of the exact same absolute and relative position of the longest intron between orthologous genes.


Assuntos
Íntrons , Íntrons/genética , Animais , Humanos , Arabidopsis/genética , Spliceossomos/genética , Spliceossomos/metabolismo
12.
Iran J Immunol ; 21(2): 166-175, 2024 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-38912647

RESUMO

Background: Ankylosing spondylitis (AS) is a chronic autoimmune disorder characterized by the fusion of vertebral joints and axial arthritis. The programmed death-1 (PD-1) inhibitory receptor has a pivotal role in controlling T cell function and may have a significant impact on the pathogenesis of autoimmune diseases such as AS pathogenesis. Objective: To investigate PD-1 gene expression and its epigenetic regulation by detecting methylated CpG islands in the regulatory sites of the gene. This will provide insight into the mechanisms involved in the disease. Methods: 30 AS patients and 30 healthy individuals were examined to detect the 16 CpG islands in intron 1 using bisulfite conversion and methylation-specific PCR technique. In addition, RNA samples were isolated from fresh peripheral blood mononuclear cells (PBMCs), and after complementary DNA (cDNA) synthesis, the expression level of the PD-1 gene was evaluated using Real-Time PCR. Results: The CpG islands located in the intronic zone of the PD-1 gene were hyper-methylated in both the patients with AS and the healthy controls. The gene expression of PD-1 was significantly downregulated in AS patients compared with the controls (p=0.017). A negative correlation between the Bath Ankylosing Spondylitis Disease Activity Index and PD-1 gene expression was also revealed. Conclusion: The low level of PD-1 gene expression is implicated in the pathogenesis of AS. However, in both groups, the methylation level of the intron 1 CpG islands of the PD-1 gene suggests that other regulatory mechanisms are more relevant to PD-1 gene expression than methylation in the intron.


Assuntos
Ilhas de CpG , Metilação de DNA , Epigênese Genética , Receptor de Morte Celular Programada 1 , Espondilite Anquilosante , Humanos , Espondilite Anquilosante/genética , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Masculino , Feminino , Adulto , Ilhas de CpG/genética , Transcriptoma , Regulação da Expressão Gênica , Pessoa de Meia-Idade , Perfilação da Expressão Gênica , Sequências Reguladoras de Ácido Nucleico/genética , Adulto Jovem , Íntrons/genética
13.
PLoS Genet ; 20(6): e1011316, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38833506

RESUMO

Splicing is an important step of gene expression regulation in eukaryotes, as there are many mRNA precursors that can be alternatively spliced in different tissues, at different cell cycle phases or under different external stimuli. We have developed several integrated fluorescence-based in vivo splicing reporter constructs that allow the quantification of fission yeast splicing in vivo on intact cells, and we have compared their splicing efficiency in a wild type strain and in a prp2-1 (U2AF65) genetic background, showing a clear dependency between Prp2 and a consensus signal at 5' splicing site (5'SS). To isolate novel genes involved in regulated splicing, we have crossed the reporter showing more intron retention with the Schizosaccharomyces pombe knock out collection. Among the candidate genes involved in the regulation of splicing, we have detected strong splicing defects in two of the mutants -Δcwf12, a member of the NineTeen Complex (NTC) and Δsaf5, a methylosome subunit that acts together with the survival motor neuron (SMN) complex in small nuclear ribonucleoproteins (snRNP) biogenesis. We have identified that strains with mutations in cwf12 have inefficient splicing, mainly when the 5'SS differs from the consensus. However, although Δsaf5 cells also have some dependency on 5'SS sequence, we noticed that when one intron of a given pre-mRNA was affected, the rest of the introns of the same pre-mRNA had high probabilities of being also affected. This observation points Saf5 as a link between transcription rate and splicing.


Assuntos
Splicing de RNA , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Transcrição Gênica , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Regulação Fúngica da Expressão Gênica , Íntrons/genética , Mutação , Processamento Alternativo/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo
14.
BMC Genomics ; 25(1): 595, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38872102

RESUMO

BACKGROUND: Nuclear introns in Euglenida have been understudied. This study aimed to investigate nuclear introns in Euglenida by identifying a large number of introns in Euglena gracilis (E. gracilis), including cis-spliced conventional and nonconventional introns, as well as trans-spliced outrons. We also examined the sequence characteristics of these introns. RESULTS: A total of 28,337 introns and 11,921 outrons were identified. Conventional and nonconventional introns have distinct splice site features; the former harbour canonical GT/C-AG splice sites, whereas the latter are capable of forming structured motifs with their terminal sequences. We observed that short introns had a preference for canonical GT-AG introns. Notably, conventional introns and outrons in E. gracilis exhibited a distinct cytidine-rich polypyrimidine tract, in contrast to the thymidine-rich tracts observed in other organisms. Furthermore, the SL-RNAs in E. gracilis, as well as in other trans-splicing species, can form a recently discovered motif called the extended U6/5' ss duplex with the respective U6s. We also describe a novel type of alternative splicing pattern in E. gracilis. The tandem repeat sequences of introns in this protist were determined, and their contents were comparable to those in humans. CONCLUSIONS: Our findings highlight the unique features of E. gracilis introns and provide insights into the splicing mechanism of these introns, as well as the genomics and evolution of Euglenida.


Assuntos
Euglena gracilis , Íntrons , Euglena gracilis/genética , Sítios de Splice de RNA , Processamento Alternativo , Splicing de RNA
15.
BMC Genomics ; 25(1): 599, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38877397

RESUMO

BACKGROUND: Tubulins play crucial roles in numerous fundamental processes of plant development. In flowering plants, tubulins are grouped into α-, ß- and γ-subfamilies, while α- and ß-tubulins possess a large isotype diversity and gene number variations among different species. This circumstance leads to insufficient recognition of orthologous isotypes and significantly complicates extrapolation of obtained experimental results, and brings difficulties for the identification of particular tubulin isotype function. The aim of this research is to identify and characterize tubulins of an emerging biofuel crop Camelina sativa. RESULTS: We report comprehensive identification and characterization of tubulin gene family in C. sativa, including analyses of exon-intron organization, duplicated genes comparison, proper isotype designation, phylogenetic analysis, and expression patterns in different tissues. 17 α-, 34 ß- and 6 γ-tubulin genes were identified and assigned to a particular isotype. Recognition of orthologous tubulin isotypes was cross-referred, involving data of phylogeny, synteny analyses and genes allocation on reconstructed genomic blocks of Ancestral Crucifer Karyotype. An investigation of expression patterns of tubulin homeologs revealed the predominant role of N6 (A) and N7 (B) subgenomes in tubulin expression at various developmental stages, contrarily to general the dominance of transcripts of H7 (C) subgenome. CONCLUSIONS: For the first time a complete set of tubulin gene family members was identified and characterized for allohexaploid C. sativa species. The study demonstrates the comprehensive approach of precise inferring gene orthology. The applied technique allowed not only identifying C. sativa tubulin orthologs in model Arabidopsis species and tracking tubulin gene evolution, but also uncovered that A. thaliana is missing orthologs for several particular isotypes of α- and ß-tubulins.


Assuntos
Evolução Molecular , Genoma de Planta , Família Multigênica , Filogenia , Tubulina (Proteína) , Tubulina (Proteína)/genética , Brassicaceae/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sintenia , Regulação da Expressão Gênica de Plantas , Duplicação Gênica , Íntrons/genética , Éxons/genética
16.
Mol Genet Metab ; 142(3): 108511, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38878498

RESUMO

The diagnosis of Mendelian disorders has notably advanced with integration of whole exome and genome sequencing (WES and WGS) in clinical practice. However, challenges in variant interpretation and uncovered variants by WES still leave a substantial percentage of patients undiagnosed. In this context, integrating RNA sequencing (RNA-seq) improves diagnostic workflows, particularly for WES inconclusive cases. Additionally, functional studies are often necessary to elucidate the impact of prioritized variants on gene expression and protein function. Our study focused on three unrelated male patients (P1-P3) with ATP6AP1-CDG (congenital disorder of glycosylation), presenting with intellectual disability and varying degrees of hepatopathy, glycosylation defects, and an initially inconclusive diagnosis through WES. Subsequent RNA-seq was pivotal in identifying the underlying genetic causes in P1 and P2, detecting ATP6AP1 underexpression and aberrant splicing. Molecular studies in fibroblasts confirmed these findings and identified the rare intronic variants c.289-233C > T and c.289-289G > A in P1 and P2, respectively. Trio-WGS also revealed the variant c.289-289G > A in P3, which was a de novo change in both patients. Functional assays expressing the mutant alleles in HAP1 cells demonstrated the pathogenic impact of these variants by reproducing the splicing alterations observed in patients. Our study underscores the role of RNA-seq and WGS in enhancing diagnostic rates for genetic diseases such as CDG, providing new insights into ATP6AP1-CDG molecular bases by identifying the first two deep intronic variants in this X-linked gene. Additionally, our study highlights the need to integrate RNA-seq and WGS, followed by functional validation, in routine diagnostics for a comprehensive evaluation of patients with an unidentified molecular etiology.


Assuntos
Íntrons , RNA Mensageiro , Humanos , Masculino , Íntrons/genética , RNA Mensageiro/genética , ATPases Vacuolares Próton-Translocadoras/genética , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/patologia , Mutação , Sequenciamento Completo do Genoma , Sequenciamento do Exoma , Análise de Sequência de RNA , Deficiência Intelectual/genética , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/patologia , Criança , Splicing de RNA/genética , Pré-Escolar
17.
Nat Commun ; 15(1): 4963, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862535

RESUMO

Image-based lineage tracing enables tissue turnover kinetics and lineage potentials of different adult cell populations to be investigated. Previously, we reported a genetic mouse model system, Red2Onco, which ectopically expressed mutated oncogenes together with red fluorescent proteins (RFP). This system enabled the expansion kinetics and neighboring effects of oncogenic clones to be dissected. We now report Red2Flpe-SCON: a mosaic knockout system that uses multicolor reporters to label both mutant and wild-type cells. We develop the Red2Flpe mouse line for red clone-specific Flpe expression, as well as the FRT-based SCON (Short Conditional IntrON) method to facilitate tunable conditional mosaic knockouts in mice. We use the Red2Flpe-SCON method to study Sox2 mutant clonal analysis in the esophageal epithelium of adult mice which reveal that the stem cell gene, Sox2, is less essential for adult stem cell maintenance itself, but rather for stem cell proliferation and differentiation.


Assuntos
Proteínas Luminescentes , Camundongos Knockout , Proteína Vermelha Fluorescente , Fatores de Transcrição SOXB1 , Animais , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Camundongos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mosaicismo , Diferenciação Celular , Proliferação de Células/genética , Esôfago/metabolismo , Esôfago/patologia , Linhagem da Célula/genética , Íntrons/genética , Feminino , Masculino
18.
J Peripher Nerv Syst ; 29(2): 262-274, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38860315

RESUMO

BACKGROUND: Loss-of-function variants in MME (membrane metalloendopeptidase) are a known cause of recessive Charcot-Marie-Tooth Neuropathy (CMT). A deep intronic variant, MME c.1188+428A>G (NM_000902.5), was identified through whole genome sequencing (WGS) of two Australian families with recessive inheritance of axonal CMT using the seqr platform. MME c.1188+428A>G was detected in a homozygous state in Family 1, and in a compound heterozygous state with a known pathogenic MME variant (c.467del; p.Pro156Leufs*14) in Family 2. AIMS: We aimed to determine the pathogenicity of the MME c.1188+428A>G variant through segregation and splicing analysis. METHODS: The splicing impact of the deep intronic MME variant c.1188+428A>G was assessed using an in vitro exon-trapping assay. RESULTS: The exon-trapping assay demonstrated that the MME c.1188+428A>G variant created a novel splice donor site resulting in the inclusion of an 83 bp pseudoexon between MME exons 12 and 13. The incorporation of the pseudoexon into MME transcript is predicted to lead to a coding frameshift and premature termination codon (PTC) in MME exon 14 (p.Ala397ProfsTer47). This PTC is likely to result in nonsense mediated decay (NMD) of MME transcript leading to a pathogenic loss-of-function. INTERPRETATION: To our knowledge, this is the first report of a pathogenic deep intronic MME variant causing CMT. This is of significance as deep intronic variants are missed using whole exome sequencing screening methods. Individuals with CMT should be reassessed for deep intronic variants, with splicing impacts being considered in relation to the potential pathogenicity of variants.


Assuntos
Doença de Charcot-Marie-Tooth , Íntrons , Linhagem , Splicing de RNA , Humanos , Doença de Charcot-Marie-Tooth/genética , Masculino , Feminino , Splicing de RNA/genética , Íntrons/genética , Metaloendopeptidases/genética , Adulto , Mutação
19.
Proc Natl Acad Sci U S A ; 121(27): e2406710121, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38917004

RESUMO

The essential role of U4 snRNP in pre-messenger RNA (mRNA) splicing has been well established. In this study, we utilized an antisense morpholino oligonucleotide (AMO) specifically targeting U4 snRNA to achieve functional knockdown of U4 snRNP in HeLa cells. Our results showed that this knockdown resulted in global intronic premature cleavage and polyadenylation (PCPA) events, comparable to the effects observed with U1 AMO treatment, as demonstrated by mRNA 3'-seq analysis. Furthermore, our study suggested that this may be a common phenomenon in both human and mouse cell lines. Additionally, we showed that U4 AMO treatment disrupted transcription elongation, as evidenced by chromatin immunoprecipitation sequencing (ChIP-seq) analysis for RNAPII. Collectively, our results identified a unique role for U4 snRNP in the inhibition of PCPA and indicated a model wherein splicing intrinsically inhibits intronic cleavage and polyadenylation in the context of cotranscriptional mRNA processing.


Assuntos
Poliadenilação , Precursores de RNA , Splicing de RNA , Humanos , Precursores de RNA/metabolismo , Precursores de RNA/genética , Células HeLa , Camundongos , Animais , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Íntrons/genética
20.
Int J Mol Sci ; 25(11)2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38892127

RESUMO

ABCA4 is the most frequently mutated gene leading to inherited retinal disease (IRD) with over 2200 pathogenic variants reported to date. Of these, ~1% are copy number variants (CNVs) involving the deletion or duplication of genomic regions, typically >50 nucleotides in length. An in-depth assessment of the current literature based on the public database LOVD, regarding the presence of known CNVs and structural variants in ABCA4, and additional sequencing analysis of ABCA4 using single-molecule Molecular Inversion Probes (smMIPs) for 148 probands highlighted recurrent and novel CNVs associated with ABCA4-associated retinopathies. An analysis of the coverage depth in the sequencing data led to the identification of eleven deletions (six novel and five recurrent), three duplications (one novel and two recurrent) and one complex CNV. Of particular interest was the identification of a complex defect, i.e., a 15.3 kb duplicated segment encompassing exon 31 through intron 41 that was inserted at the junction of a downstream 2.7 kb deletion encompassing intron 44 through intron 47. In addition, we identified a 7.0 kb tandem duplication of intron 1 in three cases. The identification of CNVs in ABCA4 can provide patients and their families with a genetic diagnosis whilst expanding our understanding of the complexity of diseases caused by ABCA4 variants.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Variações do Número de Cópias de DNA , Doenças Retinianas , Humanos , Transportadores de Cassetes de Ligação de ATP/genética , Doenças Retinianas/genética , Feminino , Masculino , Linhagem , Íntrons/genética , Éxons/genética , Duplicação Gênica
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