In advanced prostate cancer (PC), in particular after acquisition of resistance to androgen receptor (AR) signaling inhibitors (ARSI), upregulation of AR splice variants compromises endocrine therapy efficiency. Androgen receptor splice variant-7 (ARV7) is clinically the most relevant and has a distinct 3' untranslated region (3'UTR) compared to the AR full-length variant, suggesting a unique post-transcriptional regulation. Here, we set out to evaluate the applicability of the ARV7 3'UTR as a therapy target. A common single nucleotide polymorphism, rs5918762, was found to affect the splicing rate and thus the expression of ARV7 in cellular models and patient specimens. Serine/arginine-rich splicing factor 9 (SRSF9) was found to bind to and increase the inclusion of the cryptic exon 3 of ARV7 during the splicing process in the alternative C allele of rs5918762. The dual specificity protein kinase CLK2 interferes with the activity of SRSF9 by regulating its expression. Inhibition of the Cdc2-like kinase (CLK) family by the small molecules cirtuvivint or lorecivivint results in the decreased expression of ARV7. Both inhibitors show potent anti-proliferative effects in enzalutamide-treated or -naive PC models. Thus, targeting aberrant alternative splicing at the 3'UTR of ARV7 by disturbing the CLK2/SRSF9 axis might be a valuable therapeutic approach in late stage, ARSI-resistant PC.

• MeSH
• 3' Untranslated Regions genetics   MeSH
• Alternative Splicing genetics   drug effects   MeSH
• Receptors, Androgen * metabolism   genetics   MeSH
• Polymorphism, Single Nucleotide genetics   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms * genetics   metabolism   pathology   drug therapy   MeSH
• Protein Isoforms genetics   metabolism   MeSH
• Protein Serine-Threonine Kinases genetics   metabolism   antagonists & inhibitors   MeSH
• Gene Expression Regulation, Neoplastic * drug effects   MeSH
• Serine-Arginine Splicing Factors * metabolism   genetics   MeSH
• RNA Splicing genetics   MeSH
• Protein-Tyrosine Kinases * genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Through the agnostic screening of patients with uncharacterised disease phenotypes for an upregulation of type I interferon (IFN) signalling, we identified a cohort of individuals heterozygous for mutations in PTPN1, encoding the protein-tyrosine phosphatase 1B (PTP1B). We aimed to describe the clinical phenotype and molecular and cellular pathology of this new disease. METHODS: In this case series, we identified patients and collected clinical and neuroradiological data through collaboration with paediatric neurology and clinical genetics colleagues across Europe (Czechia, France, Germany, Italy, Slovenia, and the UK) and Israel. Variants in PTPN1 were identified by exome and directed Sanger sequencing. The expression of IFN-stimulated genes was determined by quantitative (q) PCR or NanoString technology. Experiments to assess RNA and protein expression and to investigate type 1 IFN signalling were undertaken in patient fibroblasts, hTERT-immortalised BJ-5ta fibroblasts, and RPE-1 cells using CRISPR-Cas9 editing and standard cell biology techniques. FINDINGS: Between Dec 20, 2013, and Jan 11, 2023, we identified 12 patients from 11 families who were heterozygous for mutations in PTPN1. We found ten novel or very rare variants in PTPN1 (frequency on gnomAD version 4.1.0 of <1·25 × 10:sup>-6). Six variants were predicted as STOP mutations, two involved canonical splice-site nucleotides, and two were missense substitutions. In three patients, the variant occurred de novo, whereas in nine affected individuals, the variant was inherited from an asymptomatic parent. The clinical phenotype was characterised by the subacute onset (age range 1-8 years) of loss of motor and language skills in the absence of seizures after initially normal development, leading to spastic dystonia and bulbar involvement. Neuroimaging variably demonstrated cerebral atrophy (sometimes unilateral initially) or high T2 white matter signal. Neopterin in CSF was elevated in all ten patients who were tested, and all probands demonstrated an upregulation of IFN-stimulated genes in whole blood. Although clinical stabilisation and neuroradiological improvement was seen in both treated and untreated patients, in six of eight treated patients, high-dose corticosteroids were judged clinically to result in an improvement in neurological status. Of the four asymptomatic parents tested, IFN signalling in blood was normal (three patients) or minimally elevated (one patient). Analysis of patient blood and fibroblasts showed that tested PTPN1 variants led to reduced levels of PTPN1 mRNA and PTP1B protein, and in-vitro assays demonstrated that loss of PTP1B function was associated with impaired negative regulation of type 1 IFN signalling. INTERPRETATION: PTPN1 haploinsufficiency causes a type 1 IFN-driven autoinflammatory encephalopathy. Notably, some patients demonstrated stabilisation, and even recovery, of neurological function in the absence of treatment, whereas in others, the disease appeared to be responsive to immune suppression. Prospective studies are needed to investigate the safety and efficacy of specific immune suppression approaches in this disease population. FUNDING: The UK Medical Research Council, the European Research Council, and the Agence Nationale de la Recherche.

• MeSH
• Child MeSH
• Haploinsufficiency * genetics   MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Mutation genetics   MeSH
• Brain Diseases genetics   MeSH
• Neuroinflammatory Diseases genetics   MeSH
• Child, Preschool MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 1 * genetics   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

The exon junction complex (EJC) is a key player in metazoan mRNA quality control and is placed upstream of the exon-exon junction after splicing. Its inner core is composed of Magoh, Y14, eIF4AIII and BTZ and the outer core of proteins involved in mRNA splicing (CWC22), export (Yra1), translation (PYM) and nonsense mediated decay (NMD, UPF1/2/3). Trypanosoma brucei encodes only two genes with introns, but all mRNAs are processed by trans-splicing. The presence of three core EJC proteins and a potential BTZ homologue (Rbp25) in trypanosomes has been suggested to adapt of the EJC function to mark trans-spliced mRNAs. We analysed trypanosome EJC components and noticed major differences between eIF4AIII and Magoh/Y14: (i) whilst eIF4AIII is essential, knocking out both Magoh and Y14 elicits only a mild growth phenotype (ii) eIF4AIII localization is mostly nucleolar, while Magoh and Y14 are nucleolar and nucleoplasmic but excluded from the cytoplasm (iii) eIF4AIII associates with nucleolar proteins and the splicing factor CWC22, but not with Y14 or Magoh, while Magoh and Y14 associate with each other, but not with eIF4AIII, CWC22 or nucleolar proteins. Our data argue against the presence of a functional EJC in trypanosomes, but indicate that eIF4AIII adopted non-EJC related, essential functions, while Magoh and Y14 became redundant. Trypanosomes also possess homologues to the NMD proteins UPF1 and UPF2. Depletion of UPF1 causes only a minor reduction in growth and phylogenetic analyses show several independent losses of UPF1 and UPF2, as well as complete loss of UPF3 in the Kinetoplastida group, indicating that UPF1-dependent NMD is not essential. Regardless, we demonstrate that UPF1 depletion restores the mRNA levels of a PTC reporter. Altogether, we show that the almost intron-less trypanosomes are in the process of losing the canonical EJC/NMD pathways: Y14 and Magoh have become redundant and the still-functional UPF1-dependent NMD pathway is not essential.

• MeSH
• Eukaryotic Initiation Factor-4A metabolism   genetics   MeSH
• Exons genetics   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Nonsense Mediated mRNA Decay * MeSH
• Protozoan Proteins * metabolism   genetics   MeSH
• RNA Splicing MeSH
• Trypanosoma brucei brucei * metabolism   genetics   MeSH
• Publication type
• Journal Article MeSH

Retinitis pigmentosa (RP) is a hereditary disorder caused by mutations in more than 70 different genes including those that encode proteins important for pre-mRNA splicing. Most RP-associated mutations in splicing factors reduce either their expression, stability or incorporation into functional splicing complexes. However, we have previously shown that two RP mutations in PRPF8 (F2314L and Y2334N) and two in SNRNP200 (S1087L and R1090L) behaved differently, and it was still unclear how these mutations affect the functions of both proteins. To investigate this in the context of functional spliceosomes, we used iCLIP in HeLa and retinal pigment epithelial (RPE) cells. We found that both mutations in the RNA helicase SNRNP200 change its interaction with U4 and U6 snRNAs. The significantly broader binding profile of mutated SNRNP200 within the U4 region upstream of the U4/U6 stem I strongly suggests that its activity to unwind snRNAs is impaired. This was confirmed by FRAP measurements and helicase activity assays comparing mutant and WT protein. The RP variants of PRPF8 did not affect snRNAs, but showed a reduced binding to pre-mRNAs, which resulted in the slower splicing of introns and altered expression of hundreds of genes in RPE cells. This suggests that changes in the expression and splicing of specific genes are the main driver of retinal degeneration in PRPF8-linked RP.

• MeSH
• HeLa Cells MeSH
• Humans MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear metabolism   genetics   MeSH
• Mutation * MeSH
• Eye Proteins genetics   metabolism   MeSH
• RNA Precursors * metabolism   genetics   MeSH
• RNA-Binding Proteins metabolism   genetics   MeSH
• Retinal Pigment Epithelium metabolism   pathology   MeSH
• Retinitis Pigmentosa * genetics   metabolism   pathology   MeSH
• Ribonucleoproteins, Small Nuclear metabolism   genetics   MeSH
• RNA, Small Nuclear genetics   metabolism   MeSH
• RNA Splicing * genetics   MeSH
• Spliceosomes metabolism   genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Dual reporters encoding two distinct proteins within the same mRNA have had a crucial role in identifying and characterizing unconventional mechanisms of eukaryotic translation. These mechanisms include initiation via internal ribosomal entry sites (IRESs), ribosomal frameshifting, stop codon readthrough and reinitiation. This design enables the expression of one reporter to be influenced by the specific mechanism under investigation, while the other reporter serves as an internal control. However, challenges arise when intervening test sequences are placed between these two reporters. Such sequences can inadvertently impact the expression or function of either reporter, independent of translation-related changes, potentially biasing the results. These effects may occur due to cryptic regulatory elements inducing or affecting transcription initiation, splicing, polyadenylation and antisense transcription as well as unpredictable effects of the translated test sequences on the stability and activity of the reporters. Unfortunately, these unintended effects may lead to misinterpretation of data and the publication of incorrect conclusions in the scientific literature. To address this issue and to assist the scientific community in accurately interpreting dual-reporter experiments, we have developed comprehensive guidelines. These guidelines cover experimental design, interpretation and the minimal requirements for reporting results. They are designed to aid researchers conducting these experiments as well as reviewers, editors and other investigators who seek to evaluate published data.

• MeSH
• Eukaryota genetics   MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Protein Biosynthesis genetics   MeSH
• Genes, Reporter * MeSH
• Guidelines as Topic MeSH
• Research Design standards   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Familial dysautonomia is a debilitating congenital neurodegenerative disorder with no causative therapy. It is caused by a homozygous mutation in ELP1 gene, resulting in the production of the transcript lacking exon 20. The compounds studied as potential treatments include the clinical candidate kinetin, a plant hormone from the cytokinin family. We explored the relationship between the structure of a set of kinetin derivatives (N = 72) and their ability to correct aberrant splicing of the ELP1 gene. Active compounds can be obtained by the substitution of the purine ring with chlorine and fluorine at the C2 atom, with a small alkyl group at the N7 atom, or with diverse groups at the C8 atom. On the other hand, a substitution at the N3 or N9 atoms resulted in a loss of activity. We successfully tested a hypothesis inspired by the remarkable tolerance of the position C8 to substitution, postulating that the imidazole of the purine moiety is not required for the activity. We also evaluated the activity of phytohormones from other families, but none of them corrected ELP1 mRNA aberrant splicing. A panel of in vitro ADME assays, including evaluation of transport across model barriers, stability in plasma and in the presence of liver microsomal fraction as well as plasma protein binding, was used for an initial estimation of the potential bioavailability of the active compounds. Finally, a RNA-seq data suggest that 8-aminokinetin modulates expression spliceosome components.

• MeSH
• Kinetin * pharmacology   chemistry   MeSH
• Humans MeSH
• Molecular Structure MeSH
• RNA Precursors * genetics   metabolism   MeSH
• RNA Splicing * drug effects   MeSH
• Transcriptional Elongation Factors metabolism   genetics   MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

The RNA editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) is essential for correct functioning of innate immune responses. The ADAR1p110 isoform is mainly nuclear and ADAR1p150, which is interferon (IFN) inducible, is predominately cytoplasmic. Using three different methods - co-immunoprecipitation (co-IP) of endogenous ADAR1, Strep-tag co-IP and BioID with individual ADAR1 isoforms - a comprehensive interactome was generated during both homeostasis and the IFN response. Both known and novel interactors as well as editing regulators were identified. Nuclear proteins were detected as stable interactors with both ADAR1 isoforms. In contrast, BioID identified distinct protein networks for each ADAR1 isoform, with nuclear components observed with ADAR1p110 and components of cytoplasmic cellular condensates with ADAR1p150. RNase A digestion distinguished between distal and proximal interactors, as did a double-stranded RNA (dsRNA)-binding mutant of ADAR1 which demonstrated the importance of dsRNA binding for ADAR1 interactions. IFN treatment did not affect the core ADAR1 interactomes but resulted in novel interactions, the majority of which are proximal interactions retained after RNase A treatment. Short treatment with high molecular weight poly(I:C) during the IFN response resulted in dsRNA-binding-dependent changes in the proximal protein network of ADAR1p110 and association of the ADAR1p150 proximal protein network with some components of antiviral stress granules.

• MeSH
• Adenosine Deaminase * metabolism   genetics   MeSH
• Cell Nucleus * metabolism   MeSH
• Cytoplasm * metabolism   MeSH
• RNA, Double-Stranded metabolism   genetics   MeSH
• RNA Editing MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Interferons metabolism   genetics   MeSH
• Humans MeSH
• Protein Interaction Maps MeSH
• Poly I-C pharmacology   MeSH
• Protein Isoforms * metabolism   genetics   MeSH
• RNA-Binding Proteins * metabolism   genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

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.

• MeSH
• Alternative Splicing * MeSH
• Humans MeSH
• RNA, Messenger metabolism   genetics   MeSH
• Evolution, Molecular MeSH
• Tumor Suppressor Protein p53 * metabolism   genetics   MeSH
• Transcription Initiation Site MeSH
• Protein Isoforms * genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Single-cell RNA sequencing (scRNA-seq) methods are widely used in life sciences, including immunology. Typical scRNA-seq analysis pipelines quantify the abundance of particular transcripts without accounting for alternative splicing. However, a well-established pan-leukocyte surface marker, CD45, encoded by the PTPRC gene, presents alternatively spliced variants that define different immune cell subsets. Information about some of the splicing patterns in particular cells in the scRNA-seq data can be obtained using isotype-specific DNA oligo-tagged anti-CD45 antibodies. However, this requires generation of an additional sequencing DNA library. Here, we present IDEIS, an easy-to-use software for CD45 isoform quantification that uses single-cell transcriptomic data as the input. We showed that IDEIS accurately identifies canonical human CD45 isoforms in datasets generated by 10× Genomics 5' sequencing assays. Moreover, we used IDEIS to determine the specificity of the Ptprc splicing pattern in mouse leukocyte subsets.

• MeSH
• Alternative Splicing MeSH
• Single-Cell Analysis methods   MeSH
• Leukocyte Common Antigens * genetics   metabolism   MeSH
• Leukocytes metabolism   immunology   MeSH
• Humans MeSH
• Mice MeSH
• Protein Isoforms genetics   MeSH
• Sequence Analysis, RNA methods   MeSH
• Software * MeSH
• Gene Expression Profiling methods   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Effective translation of rare disease diagnosis knowledge into therapeutic applications is achievable within a reasonable timeframe; where mutations are amenable to current antisense oligonucleotide technology. In our study, we identified five distinct types of abnormal splice-causing mutations in patients with rare genetic disorders and developed a tailored antisense oligonucleotide for each mutation type using phosphorodiamidate morpholino oligomers with or without octa-guanidine dendrimers and 2'-O-methoxyethyl phosphorothioate. We observed variations in treatment effects and efficiencies, influenced by both the chosen chemistry and the specific nature of the aberrant splicing patterns targeted for correction. Our study demonstrated the successful correction of all five different types of aberrant splicing. Our findings reveal that effective correction of aberrant splicing can depend on altering the chemical composition of oligonucleotides and suggest a fast, efficient, and feasible approach for developing personalized therapeutic interventions for genetic disorders within short time frames.

• MeSH
• Oligonucleotides, Antisense * therapeutic use   genetics   MeSH
• Genetic Diseases, Inborn genetics   therapy   MeSH
• Humans MeSH
• Morpholinos therapeutic use   genetics   MeSH
• Mutation * MeSH
• RNA Splicing * MeSH
• Rare Diseases * genetics   drug therapy   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH