Though RNAi and RNA-splicing machineries are involved in regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, their precise roles in coronavirus disease 2019 (COVID-19) pathogenesis remain unclear. Herein, we show that decreased RNAi component (Dicer and XPO5) and splicing factor (SRSF3 and hnRNPA3) expression correlate with increased COVID-19 severity. SARS-CoV-2 N protein induces the autophagic degradation of Dicer, XPO5, SRSF3, and hnRNPA3, inhibiting miRNA biogenesis and RNA splicing and triggering DNA damage, proteotoxic stress, and pneumonia. Dicer, XPO5, SRSF3, and hnRNPA3 knockdown increases, while their overexpression decreases, N protein-induced pneumonia\'s severity. Older mice show lower expression of Dicer, XPO5, SRSF3, and hnRNPA3 in their lung tissues and exhibit more severe N protein-induced pneumonia than younger mice. PJ34, a poly(ADP-ribose) polymerase inhibitor, or anastrozole, an aromatase inhibitor, ameliorates N protein- or SARS-CoV-2-induced pneumonia by restoring Dicer, XPO5, SRSF3, and hnRNPA3 expression. These findings will aid in developing improved treatments for SARS-CoV-2-associated pneumonia.

BACKGROUND: Hereditary spherocytosis (HS, MIM#612641) is one of the most common hereditary hemolytic disorders. This study aimed to confirm a novel variant\'s pathogenicity and reveal a patient\'s genetic etiology.
METHODS: The clinical data of a patient with HS who underwent genetic sequencing at the Children\'s Hospital of Chongqing Medical University were reviewed retrospectively. In silico prediction and in vitro minigene splicing reporter system were then conducted on the detected variant to analyze its intramolecular impact. A summary of the literature related to HS due to SPTB gene variants was also presented.
RESULTS: A novel variant (c.301-2 A > G) in the SPTB gene (NM_001024858.4) was identified in the proband. Using Sanger sequencing, we conclusively confirmed that the inheritance of the variant could not be traced to the biological parents. The in vitro minigene assay revealed three different transcripts derived from the c.301-2 A > G variant: r.301_474del, r.301_306delCCAAAG, and r.301-1_301-57ins. Through a literature review, patients with HS who had been genotypically validated were summarized and the SPTB gene variant profile was mapped.
CONCLUSIONS: We identified a splicing variant of the SPTB gene, thus confirming its aberrant translation. The novel variant was the probable genetic etiology of the proband with HS. Our findings expanded the variant spectrum of the SPTB gene, thus improving the understanding of the associated hereditary hemolytic disorders from a clinical and molecular perspective and contributing to the foundation of genetic counseling and diagnosis.

BACKGROUND: In recent years, covalent modifications on RNA nucleotides have emerged as pivotal moieties influencing the structure, function, and regulatory processes of RNA Polymerase II transcripts such as mRNAs and lncRNAs. However, our understanding of their biological roles and whether these roles are conserved across eukaryotes remains limited.
RESULTS: In this study, we leveraged standard polyadenylation-enriched RNA-sequencing data to identify and characterize RNA modifications that introduce base-pairing errors into cDNA reads. Our investigation incorporated data from three Poaceae (Zea mays, Sorghum bicolor, and Setaria italica), as well as publicly available data from a range of stress and genetic contexts in Sorghum and Arabidopsis thaliana. We uncovered a strong enrichment of RNA covalent modifications (RCMs) deposited on a conserved core set of nuclear mRNAs involved in photosynthesis and translation across these species. However, the cohort of modified transcripts changed based on environmental context and developmental program, a pattern that was also conserved across flowering plants. We determined that RCMs can partly explain accession-level differences in drought tolerance in Sorghum, with stress-associated genes receiving a higher level of RCMs in a drought tolerant accession. To address function, we determined that RCMs are significantly enriched near exon junctions within coding regions, suggesting an association with splicing. Intriguingly, we found that these base-pair disrupting RCMs are associated with stable mRNAs, are highly correlated with protein abundance, and thus likely associated with facilitating translation.
CONCLUSIONS: Our data point to a conserved role for RCMs in mRNA stability and translation across the flowering plant lineage.

BACKGROUND: Mucopolysaccharidosis type I (MPS-I) is a rare autosomal recessive genetic lysosomal storage disorder that is caused by pathogenic variants of the α-L-iduronidase (IDUA) gene. This study aimed to identify the genetic causes of MPS-I in a Chinese patient and construct a minigene of IDUA to analyze its variants upon splicing.
METHODS: Whole-exome sequencing (WES) and Sanger sequencing were used to confirm the potential causative variants. Single-nucleotide polymorphism (SNP) array was subsequently performed to confirm uniparental disomy (UPD). Minigene assay was performed to analyze the effect on splicing of mRNA. We meanwhile explored the conservative analysis and protein homology simulation.
RESULTS: A novel homozygous splicing mutation of IDUA, c.159-9T>A, was identified in an individual presenting with overlapping features of MPS-I. Interestingly, only the father and sisters, but not the mother, carried the variant in a heterozygous state. WES and SNP array analyses validated paternal UPD on chromosome 4. Minigene splicing revealed two aberrant splicing events: exon 2 skipping and intron 1 retention. Moreover, the specific structure of the mutant protein obviously changed according to the results of the homologous model.
CONCLUSIONS: This study describes a rare autosomal recessive disorder with paternal UPD of chromosome 4 leading to the homozygosity of the IDUA splicing variant in patients with MPS-I for the first time. This study expands the variant spectrum of IDUA and provides insights into the splicing system, facilitating its enhanced diagnosis and treatment.

暂无摘要。

Biallelic variants in USH2A are associated with retinitis pigmentosa (RP) and Type 2 Usher Syndrome (USH2), leading to impaired vision and, additionally, hearing loss in the latter. Although the introduction of next-generation sequencing into clinical diagnostics has led to a significant uplift in molecular diagnostic rates, many patients remain molecularly unsolved. It is thought that non-coding variants or variants of uncertain significance contribute significantly to this diagnostic gap. This study aims to demonstrate the clinical utility of the reverse transcription-polymerase chain reaction (RT-PCR)-Oxford Nanopore Technology (ONT) sequencing of USH2A mRNA transcripts from nasal epithelial cells to determine the splice-altering effect of candidate variants. Five affected individuals with USH2 or non-syndromic RP who had undergone whole genome sequencing were recruited for further investigation. All individuals had uncertain genotypes in USH2A, including deep intronic rare variants, c.8682-654C>G, c.9055+389G>A, and c.9959-2971C>T; a synonymous variant of uncertain significance, c.2139C>T; p.(Gly713=); and a predicted loss of function duplication spanning an intron/exon boundary, c.3812-3_3837dup p.(Met1280Ter). In silico assessment using SpliceAI provided splice-altering predictions for all candidate variants which were investigated using ONT sequencing. All predictions were found to be accurate; however, in the case of c.3812-3_3837dup, the outcome was a complex cryptic splicing pattern with predominant in-frame exon 18 skipping and a low level of exon 18 inclusion leading to the predicted stop gain. This study detected and functionally characterised simple and complex mis-splicing patterns in USH2A arising from previously unknown deep intronic variants and previously reported variants of uncertain significance, confirming the pathogenicity of the variants.

Gene co-expression networks may encode hitherto inadequately recognized vulnerabilities for adult gliomas. By identifying evolutionally conserved gene co-expression modules around EGFR (EM) or PDGFRA (PM), we recently proposed an EM/PM classification scheme, which assigns IDH-wildtype glioblastomas (GBM) into the EM subtype committed in neural stem cell compartment, IDH-mutant astrocytomas and oligodendrogliomas into the PM subtype committed in early oligodendrocyte lineage. Here, we report the identification of EM/PM subtype-specific gene co-expression networks and the characterization of hub gene polypyrimidine tract-binding protein 1 (PTBP1) as a genomic alteration-independent vulnerability in IDH-wildtype GBM. Supervised by the EM/PM classification scheme, we applied weighted gene co-expression network analysis to identify subtype-specific global gene co-expression modules. These gene co-expression modules were characterized for their clinical relevance, cellular origin and conserved expression pattern during brain development. Using lentiviral vector-mediated constitutive or inducible knockdown, we characterized the effects of PTBP1 on the survival of IDH-wildtype GBM cells, which was complemented with the analysis of PTBP1-depedent splicing pattern and overexpression of splicing target neuron-specific CDC42 (CDC42-N) isoform.  Transcriptomes of adult gliomas can be robustly assigned into 4 large gene co-expression modules that are prognostically relevant and are derived from either malignant cells of the EM/PM subtypes or tumor microenvironment. The EM subtype is associated with a malignant cell-intrinsic gene module involved in pre-mRNA splicing, DNA replication and damage response, and chromosome segregation, and a microenvironment-derived gene module predominantly involved in extracellular matrix organization and infiltrating immune cells. The PM subtype is associated with two malignant cell-intrinsic gene modules predominantly involved in transcriptional regulation and mRNA translation, respectively. Expression levels of these gene modules are independent prognostic factors and malignant cell-intrinsic gene modules are conserved during brain development. Focusing on the EM subtype, we identified PTBP1 as the most significant hub for the malignant cell-intrinsic gene module. PTBP1 is not altered in most glioma genomes. PTBP1 represses the conserved splicing of CDC42-N. PTBP1 knockdown or CDC42-N overexpression disrupts actin cytoskeleton dynamics, causing accumulation of reactive oxygen species and cell apoptosis. PTBP1-mediated repression of CDC42-N splicing represents a potential genomic alteration-independent, developmentally conserved vulnerability in IDH-wildtype GBM.

BACKGROUND: Global developmental delay with speech and behavioral abnormalities (OMIM: 619243) is an autosomal dominant disease caused by variants in TNRC6B gene.
METHODS: We reviewed and summarized clinical manifestations and genotypes in patients previously reported with TNRC6B gene variants. We used several prediction tools to predict pathogenicity and performed minigene assays to verify the function of the synonymous variant affecting RNA splicing.
RESULTS: The patient presented with convulsive seizures and developmental delay. WES combined with functional studies diagnosed a child with a synonymous variant in TNRC6B gene. Through minigene assay and Sanger sequencing, we demonstrated that c.3141G > A variant induced exon 7 skipping and the synonymous variant was pathogenic.
CONCLUSIONS: Synonymous variants do not change the amino acids encoded by the codon, so we usually consider synonymous variants to be benign and ignore their pathogenicity. Minigene assay is a valuable tool to identify the effect of variation on RNA splicing and identify synonymous variants\' benign or pathogenic. We showed that the synonymous variant was pathogenic by minigene assay. WES combined with minigene assay establishes a robust basis for genetic counseling and diagnosing diseases.

Interactions between double-stranded RNA (dsRNA) and proteins play an important role in cellular homeostasis by regulating the editing, stability, and splicing of intracellular RNA. The identification of dsRNA-binding proteins (dsRBPs) is key; however, it has long been challenging to purify dsRBPs from cells. In this study, we developed a novel method, dsRBPC (dsRNA-binding protein capture), to purify cellular dsRBPs based on classic phase separation purification procedures. A global dsRNA-binding proteome of LLC-PK1 cells was obtained, and we identified 1326 dsRBPs, including 1303 putative novel dsRBPs. Functional analyses suggested that these enriched dsRBPs are mainly associated with rRNA processing, RNA splicing, transcriptional regulation, and nucleocytoplasmic transport. We also found that the ARM (armadillo/beta-catenin-like repeats) motif is a previously unknown dsRNA-binding domain, as demonstrated by biochemical experiments. Collectively, this study provides a useful approach for dsRBP identification and the discovery of a global dsRNA-binding proteome to comprehensively map the dsRNA - protein interaction network.

BACKGROUND: Hemorrhagic enteritis, caused by Turkey Hemorrhagic Enteritis Virus (THEV), is a disease affecting turkey poults characterized by immunosuppression and bloody diarrhea. An avirulent THEV strain that retains the immunosuppressive ability is used as a live vaccine. Characterizing the splice map of THEV is an essential step that would allow studies of individual genes mediating its immunosuppressive functions. We used RNA sequencing to characterize the splice map of THEV for the first time, providing key insights into the THEV gene expression and mRNA structures.
METHODS: After infecting a turkey B-cell line with the vaccine strain, samples in triplicates were collected at 4-, 12-, 24-, and 72-hours post-infection. Total RNA was extracted, and poly-A-tailed mRNA sequenced. Reads were mapped to the THEV genome after trimming and transcripts assembled with StringTie. We performed PCR of THEV cDNA, cloned the PCR products, and used Sanger sequencing to validate all identified splice junctions.
RESULTS: Researchers previously annotated the THEV genome as encoding 23 open reading frames (ORFs). We identified 29 spliced transcripts from our RNA sequencing data, all containing novel exons although some exons matched some previously annotated ORFs. The three annotated splice junctions were also corroborated by our data. During validation we identified five additional unique transcripts, a subset of which were further validated by 3\' rapid amplification of cDNA ends (3\' RACE). Thus, we report that the genome of THEV contains 34 transcripts with the coding capacity for all annotated ORFs. However, we found six of the previously annotated ORFs to be truncated ORFs on the basis of the identification of an in-frame upstream start codon or the detection of additional coding exons. We also identified three of the annotated ORFs with longer or shorter isoforms, and seven novel unannotated ORFs that could potentially be translated; although it is beyond the scope of this manuscript to investigate whether they are translated.
CONCLUSIONS: Similar to human adenoviruses, all THEV transcripts are spliced and organized into five transcription units under the control of their cognate promoters. The genes are expressed under temporal regulation and THEV also produces multiple distinctly spliced transcripts that code for the same protein. Studies of the newly identified potential proteins should be urgently performed as these proteins may have roles in THEV-induced immunosuppression. Also, knowing the splicing of THEV genes should be invaluable to future research focusing on studying THEV genes, as this will allow accurate cloning of the mRNAs.