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1.
BMC Med Genomics ; 17(1): 175, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956616

RESUMO

This research analyzes the clinical data, whole-exome sequencing results, and in vitro minigene functional experiments of a child with developmental delay and intellectual disability. The male patient, aged 4, began experiencing epileptic seizures at 3 months post-birth and has shown developmental delay. Rehabilitation training was administered between the ages of one and two. There were no other significant family medical histories. Through comprehensive family exome genetic testing, a hemizygous variant in the 11th exon of the OPHN1 gene was identified in the affected child: c.1025 + 1G > A. Family segregation analysis confirmed the presence of this variant in the patient's mother, which had not been previously reported. According to the ACMG guidelines, this variant was classified as a likely pathogenic variant. In response to this variant, an in vitro minigene functional experiment was designed and conducted, confirming that the mutation affects the normal splicing of the gene's mRNA, resulting in a 56 bp retention on the left side of Intron 11. It was confirmed that OPHN1: c.1025 + 1G > A is the pathogenic cause of X-linked intellectual disabilities in the child, with clinical phenotypes including developmental delay and seizures.


Assuntos
Deficiência Intelectual , Proteínas Nucleares , Splicing de RNA , Humanos , Masculino , Pré-Escolar , Deficiência Intelectual/genética , Proteínas Nucleares/genética , Proteínas do Citoesqueleto/genética , Proteínas Ativadoras de GTPase/genética , Deficiências do Desenvolvimento/genética , Linhagem , Mutação , Sequenciamento do Exoma
2.
Genome Biol ; 25(1): 173, 2024 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956576

RESUMO

BACKGROUND: RNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community's ability to interrogate both forms of RNA variation simultaneously. RESULTS: We employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis. CONCLUSIONS: Ultimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.


Assuntos
Haplótipos , Humanos , Linhagem Celular Tumoral , Polimorfismo de Nucleotídeo Único , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias Pulmonares/genética , Splicing de RNA , Inosina/metabolismo , Inosina/genética , Análise de Sequência de RNA , Adenocarcinoma de Pulmão/genética , Edição de RNA , Software
3.
Wiley Interdiscip Rev RNA ; 15(4): e1866, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38972853

RESUMO

Pre-mRNA splicing, the removal of introns and ligation of flanking exons, is a crucial step in eukaryotic gene expression. The spliceosome, a macromolecular complex made up of five small nuclear RNAs (snRNAs) and dozens of proteins, assembles on introns via a complex pathway before catalyzing the two transesterification reactions necessary for splicing. All of these steps have the potential to be highly regulated to ensure correct mRNA isoform production for proper cellular function. While Saccharomyces cerevisiae (yeast) has a limited set of intron-containing genes, many of these genes are highly expressed, resulting in a large number of transcripts in a cell being spliced. As a result, splicing regulation is of critical importance for yeast. Just as in humans, yeast splicing can be influenced by protein components of the splicing machinery, structures and properties of the pre-mRNA itself, or by the action of trans-acting factors. It is likely that further analysis of the mechanisms and pathways of splicing regulation in yeast can reveal general principles applicable to other eukaryotes. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.


Assuntos
Precursores de RNA , Splicing de RNA , Saccharomyces cerevisiae , Spliceossomos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Spliceossomos/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
4.
PLoS One ; 19(7): e0305012, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38980892

RESUMO

Pre-messenger RNA (pre-mRNA) splicing modulation is an attractive approach for investigating the mechanisms of genetic disorders caused by mis-splicing. Previous reports have indicated that a modified U7 small nuclear RNA (U7 snRNA) is a prospective tool for modulating splicing both in vitro and in vivo. To date, very few studies have investigated the role of antisense sequence length in modified U7 snRNA. In this study, we designed a series of antisense sequences with various lengths and evaluated their efficiency in inducing splicing modulation. To express modified U7 snRNAs, we constructed a series of plasmid DNA sequences which codes cytomegalovirus (CMV) enhancer, human U1 promoter, and modified mouse U7 snRNAs with antisense sequences of different lengths. We evaluated in vitro splicing modulation efficiency using a luciferase reporter system for simple and precise evaluation as well as reverse transcription-polymerase chain reaction to monitor splicing patterns. Our in vitro assay findings suggest that antisense sequences of modified mouse U7 snRNAs have an optimal length for efficient splicing modulation, which depends on the target exon. In addition, antisense sequences that were either too long or too short decreased splicing modulation efficiency. To confirm reproducibility, we performed an in vitro assay using two target genes, mouse Fas and mouse Dmd. Together, our data suggests that the antisense sequence length should be optimized for modified mouse U7 snRNAs to induce efficient splicing modulation.


Assuntos
Precursores de RNA , Splicing de RNA , RNA Nuclear Pequeno , RNA Nuclear Pequeno/genética , Animais , Camundongos , Humanos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sequência de Bases , Éxons/genética , RNA Antissenso/genética
5.
BMC Biol ; 22(1): 153, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38982460

RESUMO

Pre-mRNA splicing is a significant step for post-transcriptional modifications and functions in a wide range of physiological processes in plants. Human NHP2L binds to U4 snRNA during spliceosome assembly; it is involved in RNA splicing and mediates the development of human tumors. However, no ortholog has yet been identified in plants. Therefore, we report At4g12600 encoding the ortholog NHP2L protein, and AtSNU13 associates with the component of the spliceosome complex; the atsnu13 mutant showed compromised resistance in disease resistance, indicating that AtSNU13 is a positive regulator of plant immunity. Compared to wild-type plants, the atsnu13 mutation resulted in altered splicing patterns for defense-related genes and decreased expression of defense-related genes, such as RBOHD and ALD1. Further investigation shows that AtSNU13 promotes the interaction between U4/U6.U5 tri-snRNP-specific 27 K and the motif in target mRNAs to regulate the RNA splicing. Our study highlights the role of AtSNU13 in regulating plant immunity by affecting the pre-mRNA splicing of defense-related genes.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Imunidade Vegetal , Precursores de RNA , Splicing de RNA , Imunidade Vegetal/genética , Arabidopsis/genética , Arabidopsis/imunologia , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Spliceossomos/metabolismo , Spliceossomos/genética , Doenças das Plantas/genética , Doenças das Plantas/imunologia
7.
Cell ; 187(13): 3284-3302.e23, 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38843832

RESUMO

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.


Assuntos
Diferenciação Celular , Spliceossomos , Animais , Humanos , Camundongos , Blastocisto/metabolismo , Blastocisto/citologia , Blastômeros/metabolismo , Blastômeros/citologia , Reprogramação Celular , Desenvolvimento Embrionário/genética , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Splicing de RNA , Spliceossomos/metabolismo , Células-Tronco Totipotentes/metabolismo , Células-Tronco Totipotentes/citologia , Zigoto/metabolismo , Células Cultivadas , Modelos Moleculares , Estrutura Terciária de Proteína , Genoma Humano , Análise de Célula Única , Fator 15 de Diferenciação de Crescimento/química , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Epigenômica , Linhagem da Célula
8.
Mol Neurodegener ; 19(1): 45, 2024 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-38853250

RESUMO

BACKGROUND: Cytoplasmic inclusions and loss of nuclear TDP-43 are key pathological features found in several neurodegenerative disorders, suggesting both gain- and loss-of-function mechanisms of disease. To study gain-of-function, TDP-43 overexpression has been used to generate in vitro and in vivo model systems. METHODS: We analyzed RNA-seq datasets from mouse and human neurons overexpressing TDP-43 to explore species specific splicing patterns. We explored the dynamics between TDP-43 levels and exon repression in vitro. Furthermore we analyzed human brain samples and publicly available RNA datasets to explore the relationship between exon repression and disease. RESULTS: Our study shows that excessive levels of nuclear TDP-43 protein lead to constitutive exon skipping that is largely species-specific. Furthermore, while aberrant exon skipping is detected in some human brains, it is not correlated with disease, unlike the incorporation of cryptic exons that occurs after loss of TDP-43. CONCLUSIONS: Our findings emphasize the need for caution in interpreting TDP-43 overexpression data and stress the importance of controlling for exon skipping when generating models of TDP-43 proteinopathy.


Assuntos
Proteínas de Ligação a DNA , Éxons , Humanos , Éxons/genética , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Camundongos , Neurônios/metabolismo , Encéfalo/metabolismo , Splicing de RNA/genética , Núcleo Celular/metabolismo , Proteinopatias TDP-43/genética , Proteinopatias TDP-43/metabolismo , Proteinopatias TDP-43/patologia
9.
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
10.
PLoS Genet ; 20(6): e1011241, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38870220

RESUMO

Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of co-transcriptional splicing.


Assuntos
Drosophila melanogaster , Íntrons , Splicing de RNA , Transcrição Gênica , Splicing de RNA/genética , Animais , Íntrons/genética , Drosophila melanogaster/genética , Cromossomo Y/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Masculino , Humanos
11.
BMC Genomics ; 25(1): 600, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38877417

RESUMO

BACKGROUND: Splicing variants are a major class of pathogenic mutations, with their severity equivalent to nonsense mutations. However, redundant and degenerate splicing signals hinder functional assessments of sequence variations within introns, particularly at branch sites. We have established a massively parallel splicing assay to assess the impact on splicing of 11,191 disease-relevant variants. Based on the experimental results, we then applied regression-based methods to identify factors determining splicing decisions and their respective weights. RESULTS: Our statistical modeling is highly sensitive, accurately annotating the splicing defects of near-exon intronic variants, outperforming state-of-the-art predictive tools. We have incorporated the algorithm and branchpoint information into a web-based tool, SpliceAPP, to provide an interactive application. This user-friendly website allows users to upload any genetic variants with genome coordinates (e.g., chr15 74,687,208 A G), and the tool will output predictions for splicing error scores and evaluate the impact on nearby splice sites. Additionally, users can query branch site information within the region of interest. CONCLUSIONS: In summary, SpliceAPP represents a pioneering approach to screening pathogenic intronic variants, contributing to the development of precision medicine. It also facilitates the annotation of splicing motifs. SpliceAPP is freely accessible using the link https://bc.imb.sinica.edu.tw/SpliceAPP . Source code can be downloaded at https://github.com/hsinnan75/SpliceAPP .


Assuntos
Internet , Mutação , Splicing de RNA , Software , Humanos , Algoritmos , Íntrons/genética , Sítios de Splice de RNA/genética , Biologia Computacional/métodos
12.
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
13.
Theranostics ; 14(8): 3317-3338, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38855188

RESUMO

Metastasis is one of the key factors of treatment failure in late-stage colorectal cancer (CRC). Metastatic CRC frequently develops resistance to chemotherapeutic agents. This study aimed to identify the novel regulators from "hidden" proteins encoded by long noncoding RNAs (lncRNAs) involved in tumor metastasis and chemoresistance. Methods: CRISPR/Cas9 library functional screening was employed to identify the critical suppressor of cancer metastasis in highly invasive CRC models. Western blotting, immunofluorescence staining, invasion, migration, wound healing, WST-1, colony formation, gain- and loss-of-function experiments, in vivo experimental metastasis models, multiplex immunohistochemical staining, immunohistochemistry, qRT-PCR, and RT-PCR were used to assess the functional and clinical significance of FOXP3, PRDM16-DT, HNRNPA2B1, and L-CHEK2. RNA-sequencing, co-immunoprecipitation, qRT-PCR, RT-PCR, RNA affinity purification, RNA immunoprecipitation, MeRIP-quantitative PCR, fluorescence in situ hybridization, chromatin immunoprecipitation and luciferase reporter assay were performed to gain mechanistic insights into the role of PRDM16-DT in cancer metastasis and chemoresistance. An oxaliplatin-resistant CRC cell line was established by in vivo selection. WST-1, colony formation, invasion, migration, Biacore technology, gain- and loss-of-function experiments and an in vivo experimental metastasis model were used to determine the function and mechanism of cimicifugoside H-1 in CRC. Results: The novel protein PRDM16-DT, encoded by LINC00982, was identified as a cancer metastasis and chemoresistance suppressor. The down-regulated level of PRDM16-DT was positively associated with malignant phenotypes and poor prognosis of CRC patients. Transcriptionally regulated by FOXP3, PRDM16-DT directly interacted with HNRNPA2B1 and competitively decreased HNRNPA2B1 binding to exon 9 of CHEK2, resulting in the formation of long CHEK2 (L-CHEK2), subsequently promoting E-cadherin secretion. PRDM16-DT-induced E-cadherin secretion inhibited fibroblast activation, which in turn suppressed CRC metastasis by decreasing MMP9 secretion. Cimicifugoside H-1, a natural compound, can bind to LEU89, HIS91, and LEU92 of FOXP3 and significantly upregulated PRDM16-DT expression to repress CRC metastasis and reverse oxaliplatin resistance. Conclusions: lncRNA LINC00982 can express a new protein PRDM16-DT to function as a novel regulator in cancer metastasis and drug resistance of CRC. Cimicifugoside H-1 can act on the upstream of the PRDM16-DT signaling pathway to alleviate cancer chemoresistance.


Assuntos
Neoplasias Colorretais , Proteínas de Ligação a DNA , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Metástase Neoplásica , RNA Longo não Codificante , Fatores de Transcrição , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Oxaliplatina/farmacologia , Oxaliplatina/uso terapêutico , Splicing de RNA/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética
14.
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
15.
Genome Biol ; 25(1): 166, 2024 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-38918865

RESUMO

Nucleotide conversion RNA sequencing techniques interrogate chemical RNA modifications in cellular transcripts, resulting in mismatch-containing reads. Biases in mapping the resulting reads to reference genomes remain poorly understood. We present splice_sim, a splice-aware RNA-seq simulation and evaluation pipeline that introduces user-defined nucleotide conversions at set frequencies, creates mixture models of converted and unconverted reads, and calculates mapping accuracies per genomic annotation. By simulating nucleotide conversion RNA-seq datasets under realistic experimental conditions, including metabolic RNA labeling and RNA bisulfite sequencing, we measure mapping accuracies of state-of-the-art spliced-read mappers for mouse and human transcripts and derive strategies to prevent biases in the data interpretation.


Assuntos
RNA-Seq , Camundongos , Animais , Humanos , RNA-Seq/métodos , Splicing de RNA , Análise de Sequência de RNA/métodos , Software , Nucleotídeos/genética , Simulação por Computador
16.
Genes (Basel) ; 15(6)2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38927615

RESUMO

X-linked hypophosphatemia (XLH) is a rare inherited disorder of renal phosphate wasting with a highly variable phenotype caused by loss-of-function variants in the PHEX gene. The diagnosis of individuals with mild phenotypes can be challenging and often delayed. Here, we describe a three-generation family with a very mild clinical presentation of XLH. The diagnosis was unexpectedly found in a 39-year-old woman who was referred for genetic testing due to an unclear childhood diagnosis of a tubulopathy. Genetic testing performed by next-generation sequencing using a kidney disease gene panel identified a novel non-canonical splice site variant in the PHEX gene. Segregation analysis detected that the consultand's father, who presented with hypophosphatemia and decreased tubular phosphate reabsorption, and the consultand's son also carried this variant. RNA studies demonstrated that the non-canonical splice site variant partially altered the splicing of the PHEX gene, as both wild-type and aberrant splicing transcripts were detected in the two male members with only one copy of the PHEX gene. In conclusion, this case contributes to the understanding of the relationship between splicing variants and the variable expressivity of XLH disease. The mild phenotype of this family can be explained by the coexistence of PHEX transcripts with aberrant and wild-type splicing.


Assuntos
Raquitismo Hipofosfatêmico Familiar , Endopeptidase Neutra Reguladora de Fosfato PHEX , Linhagem , Sítios de Splice de RNA , Humanos , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Adulto , Feminino , Raquitismo Hipofosfatêmico Familiar/genética , Masculino , Sítios de Splice de RNA/genética , Splicing de RNA/genética , Fenótipo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação
17.
PLoS One ; 19(6): e0298965, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38829854

RESUMO

Familial Dysautonomia (FD) is a rare disease caused by ELP1 exon 20 skipping. Here we clarify the role of RNA Polymerase II (RNAPII) and chromatin on this splicing event. A slow RNAPII mutant and chromatin-modifying chemicals that reduce the rate of RNAPII elongation induce exon skipping whereas chemicals that create a more relaxed chromatin exon inclusion. In the brain of a mouse transgenic for the human FD-ELP1 we observed on this gene an age-dependent decrease in the RNAPII density profile that was most pronounced on the alternative exon, a robust increase in the repressive marks H3K27me3 and H3K9me3 and a decrease of H3K27Ac, together with a progressive reduction in ELP1 exon 20 inclusion level. In HEK 293T cells, selective drug-induced demethylation of H3K27 increased RNAPII elongation on ELP1 and SMN2, promoted the inclusion of the corresponding alternative exons, and, by RNA-sequencing analysis, induced changes in several alternative splicing events. These data suggest a co-transcriptional model of splicing regulation in which age-dependent changes in H3K27me3/Ac modify the rate of RNAPII elongation and affect processing of ELP1 alternative exon 20.


Assuntos
Processamento Alternativo , Cromatina , Disautonomia Familiar , Éxons , RNA Polimerase II , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Disautonomia Familiar/genética , Disautonomia Familiar/metabolismo , Humanos , Éxons/genética , Animais , Cromatina/metabolismo , Cromatina/genética , Camundongos , Células HEK293 , Histonas/metabolismo , Camundongos Transgênicos , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Cinética , Splicing de RNA , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo
18.
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
19.
Nat Commun ; 15(1): 4697, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38824128

RESUMO

Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.


Assuntos
Axonema , Íntrons , Proteínas de Protozoários , Splicing de RNA , Proteínas de Ligação a RNA , Íntrons/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Masculino , Axonema/metabolismo , Feminino , Gametogênese/genética , Spliceossomos/metabolismo , Spliceossomos/genética , Plasmodium berghei/genética , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/metabolismo , Malária/parasitologia , Plasmodium/genética , Plasmodium/metabolismo
20.
Int J Biol Sci ; 20(8): 3173-3184, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38904016

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges in terms of prognosis and treatment. Recent research has identified splicing deregulation as a new cancer hallmark. Herein, we investigated the largely uncharacterized alternative splicing profile and the key splicing factor SF3B1 in PDAC pancreatic cells and tissues as a potential discovery source of plausible drug targets and new predictive biomarkers of clinical outcome. The research involved a transcriptome-wide analysis, comparing profiles of splicing profiles in PDAC primary cells with normal ductal cells. This revealed more than 400 significant differential splicing events in genes involved in regulation of gene expression, primarily related to mRNA splicing, and metabolism of nucleic acids. PDAC cultures were highly sensitive to the SF3B1 modulators, E7107 and Pladienolide-B, showing IC50s in the low nanomolar range. These compounds induced apoptosis, associated to induction of the MCL-1/S splice variant. and reduced cell migration, associated to RON mis-splicing. In an orthotopic mouse model, E7107 showed promising results. Furthermore, we evaluated SF3B1 expression in specimens from 87 patients and found a significant association of SF3B1 expression with progression-free and overall survival. In conclusion, SF3B1 emerges as both a potential prognostic factor and therapeutic target in PDAC, impacting cell proliferation, migration, and apoptosis. These findings warrant future studies on this new therapeutic strategy against PDAC.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Fatores de Processamento de RNA , Humanos , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de RNA/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Camundongos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Compostos de Epóxi/farmacologia , Compostos de Epóxi/uso terapêutico , Prognóstico , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Macrolídeos/uso terapêutico , Macrolídeos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Splicing de RNA , Processamento Alternativo , Feminino , Movimento Celular/genética
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