Aberrant Transcripts Research Articles

Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts.

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.

Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience.

Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples. Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples FedericoII before shipment. Methodological and molecular data from reference specimens were annotated. Overall, a median DNA concentration of 3.3ng/µL (range 0.1-10.0ng/µL) and 13.4ng/µL (range 2.0-45.8ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7ng/µL (range 0.2-11.9ng/µL) and 9.3ng/µL (range 0.5-18.0ng/µL) were also detected. KRAS exon2 p.G12C, EGFR exon19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon14 skipping mutation. Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

Assessment of translation rate in leukemic cells and immune cells of the microenvironment by OPP protein synthesis assay.

Despite being tightly regulated, messenger RNA (mRNA) translation, a manner in which cells control expression of genes and rapidly respond to stimuli, is highly dysfunctional and plastic in pathologies including cancer. Conversely, the investigation of molecular mechanisms whereby mRNA translation becomes aberrant in cancer, as well as inhibition thereof, become critical in developing novel therapeutic approaches. More specifically, in malignancies such as chronic lymphocytic leukemia in which aberrant global and transcript specific translation has been linked with poorer patient outcomes, targeting translation is a relevant approach, with various translation inhibitors under development. Here we elaborate on a protein synthesis assay by flow cytometry, O-propargyl-puromycin, demonstrating global mRNA translation rate with a variety of different applications including cell lines, primary cells or co-culture systems in vitro. This method provides a comprehensive tool in quantifying the rate of global mRNA translation in cancer cells, as well as that of the tumor microenvironment cells, or in response to inhibitory therapeutic agents while offering the possibility to simultaneously assess other cellular markers.

Three exonic variants in the PHEX gene cause aberrant splicing in a minigene assay.

Background: X-linked hypophosphatemia (XLH, OMIM 307800) is a rare phosphorus metabolism disorder caused by PHEX gene variants. Many variants simply classified as missense or nonsense variants were only analyzed at the DNA level. However, growing evidence indicates that some of these variants may alter pre-mRNA splicing, causing diseases. Therefore, this study aimed to use bioinformatics tools and a minigene assay to ascertain the effects of PHEX variations on pre-mRNA splicing. Methods: We analyzed 174 variants in the PHEX gene described as missense or nonsense variants. Finally, we selected eight candidate variants using bioinformatics tools to evaluate their effects on pre-mRNA splicing using a minigene assay system. The complementary DNA (cDNA) sequence for the PHEX gene (RefSeq NM_000444.6) serves as the basis for DNA variant numbering. Results: Of the eight candidate variants, three were found to cause abnormal splicing. Variants c.617T>G p.(Leu206Trp) and c.621T>A p.(Tyr207*) in exon 5 altered the splicing of pre-mRNA, owing to the activation of a cryptic splice site in exon 5, which produced an aberrant transcript lacking a part of exon 5, whereas variant c.1700G>C p.(Arg567Pro) in exon 16 led to the activation of a cryptic splice site in intron 16, resulting in a partial inclusion of intron 16. Conclusion: Our study employed a minigene system, which has a great degree of flexibility to assess abnormal splicing patterns under the circumstances of patient mRNA samples that are not available, to explore the impact of the exonic variants on pre-mRNA splicing. Based on the aforementioned experimental findings, we demonstrated the importance of analyzing exonic variants at the mRNA level.

Clinical and functional characterization of rare compound heterozygous mutations in the SERPINC1 gene causing severe thrombophilia

IntroductionHereditary antithrombin (AT) deficiency is a rare autosomal dominant disorder with significant clinical heterogeneity. In the study, we identified a patient with AT deficiency caused by compound heterozygous mutations in the SERPINC1 gene. MethodsA total of 9 individuals from three generations were investigated. The mutations were identified by direct sequencing of SERPINC1. Multiple in silico tools were programmed to predict the conservation of mutations and the effect on the AT structure. The coagulation state was evaluated by the thrombin generation assay. Recombinant AT was overexpressed in HEK293T cells; the mRNA level was determined using RT-qPCR. Western blotting, ELISA, and immunocytofluorescence were applied to characterize the recombinant AT protein. ResultsThe proband was a 26-year-old male who experienced recurrent venous thrombosis. He presented the type I deficiency with 33 % AT activity and a synchronized decrease in AT antigen. Genetic screening revealed that he carried a heterozygous c.318_319insT (p.Asn107*) in exon 2 and a heterozygous c.922G > T (p.Gly308Cys) in exon 5, both of which were completely conserved in homologous species and resulted in enhanced thrombin generation capability. Hydrophobicity analysis suggested that the p.Gly308Cys mutation may interfere with the hydrophobic state of residues 307–313. In vitro expression studies indicated that the levels of the recombinant protein AT-G308C decreased to 46.98 % ± 2.94 % and 41.35 % ± 1.48 % in transfected cell lysates and media, respectively. After treatment with a proteasome inhibitor (MG132), the quantity of AT-G308C protein in the cytoplasm was replenished to a level comparable to that of the wild type. The mRNA level of AT-N107* was significantly reduced and the recombinant protein AT-N107* was not detected in either the lysate or the culture media. ConclusionThese two mutations were responsible for the AT defects and clinical phenotypes of the proband. The p.Gly308Cys mutation could lead to proteasome-dependent degradation of the AT protein in the cytoplasm by altering local residue hydrophobicity. The c.318_319insT could eliminate aberrant transcripts by triggering nonsense-mediated mRNA degradation. Both mutations resulted in type I AT deficiency.

A Mild DBA Phenotype Associated with a Non-Canonical Splice Site Mutation in RPS26

A 37-years-old Italian female came to the observation of an adult hematology unit for the first time because of an undefined form of congenital anemia. The patient's unrelated parents and her sister were apparently healthy. Hypochromic anemia was observed for the first time at the age of 11 months and treated with one blood transfusion. Oral iron supplementation was started but was ineffective (Hb around to 7 g/dL). At that time, Fanconi's anemia was excluded by cytogenetic analysis, as also DBA The patient showed normal growth and somatopsychic development. Hematology showed moderate macrocytic anemia (MCV ranging from 115-121 fl, Hb levels ranging from 7 to 9 g/dL) with anisocytosis and hypochromia, inadequate reticulocytosis, normal bilirubin and haptoglobin, increased serum ferritin, increased HbF (7-8%), slightly increased erythropoietin, but proportional to anemia, and erythroblastic hyperplasia with variable dyserythropoiesis on bone marrow aspiration and bone marrow biopsy. From the age of 20 onwards, Hb levels ranged between 6.8 and 7.5 g/dL, without transfusion support and allowing a normal working activity. The patient underwent cycles of steroid treatment with moderate benefit. Abdomen ultrasound analysis showed mild steatosis with presence of gallstones, and no signs of focal lesions. At the age of 38 years a first spontaneous pregnancy resulted in intrauterine loss of a female fetus at 21 weeks of gestation because of umbilical cord strangulation. During pregnancy worsening of anemia (Hb 5.8 g/dL) required four blood transfusions. A second spontaneous pregnancy resulted in embryonic loss. Subsequently, she was subjected to several IVF attempts: at 47 years a pregnancy resulted in embryonic loss at 8 weeks of gestation, whereas at 49 years a further pregnancy resulted in a healthy male now 9 years old. Hb levels around 8g/dL were maintained by 20 transfusions, starting at 4 months of gestation. The patient is now 59 and has not developed any tumors, so far. To ascertain the nature of anemia, the patient underwent several investigations: RBC morphology showed anysopoikilocytosis with 10% elliptocytes and 6% spherocytes. Osmotic fragility tests, Eosin-5' maleimide (EMA)-binding test, unstable hemoglobins, SDS-PAGE electrophoresis of red cell membrane proteins as well as the assay of RBC metabolism were normal, except for a decrease of pyruvate kinase/hexokinase (PK/HK) ratio (as sometime happens in presence of dyserythropoiesis). Targeted-NGS sequencing using a panel of 48 genes associated with congenital hemolytic anemias, including RBC membrane defects, RBC metabolism and congenital dyserythropoietic anemias was performed without finding any pathogenic variant. Clinical Exome Sequencing analysis showed the presence of a heterozygous mutation in RPS26 gene: c.3+5G>C. Reverse transcription PCR on total RNA extracted from the patient's PBMC showed that the variant is associated with activation of a cryptic splice site. The aberrant transcript, which is not present in controls, lacks the last 18bp of exon 2 including the ATG (hg19). The fact that the RPS26: c. 3+5G>C mutation does not alter the canonical GT suggests that the mutation may be leaky. This would explain the patient's mild phenotype. A minigene assay is needed to ascertain this hypothesis. In conclusion, if not identified in infancy, milder forms of DBA may be under- or misdiagnosed, further underlying the role of NGS in diagnosis of atypical inherited anemias. The present case further supports the hypothesis that mutations in RPS26 may be associated to a lower cancer risk in DBA.

Accelerated Progression in Lower-Risk Myelodysplastic Neoplasms: Attenuated Cell Cycle, DNA Damage Response, and Augmented Oncogenic Transcriptome Signatures

Patients with myelodysplastic neoplasms (MDS) face the risk of transformation to acute myeloid leukemia (AML). Although prognostic scoring systems exist for risk stratification and treatment decision making in MDS patients (Garcia-Manero G. Am J Hematol. 2023;98(8):1307-1325), disease management remains challenging due to the heterogeneity of clinical courses and long-term outcomes. The natural history of patients with lower-risk MDS (LR-MDS) is very heterogeneous and some LR-MDS patients experience rapid progression despite a generally favorable prognosis. The aim of our study was to uncover the molecular mechanisms underlying accelerated progression in hematopoietic stem/progenitor cells of patients with LR-MDS regardless of driver mutations. We focused on the transcriptome of bone marrow (BM) CD34 + cells from diagnostic samples using RNA-seq and used various bioinformatic pipelines to examine differentially expressed protein-coding genes, and long non-coding RNAs (lncRNAs) as well as differential alternative splicing events. RNA-seq dataset of CD34 + ribodepleted RNA samples from 53 LR-MDS patients without accelerated progression (stMDS) and 8 who progressed within 20 months (prMDS) showed 845 differentially expressed genes (ІlogFCІ > 1, FDR < 0.01) between these groups. CD34 + cells of prMDS exhibited a transcriptional pattern of quiescent-like cell state with overall decreased metabolism signatures and significantly reduced lineage differentiation compared to stMDS CD34 + cells. Gene set enrichment analysis (GSEA) showed that cell cycle- and cell cycle checkpoint- associated genes and activation of ATR in response to replication stress were all significantly under-represented in prMDS CD34 + BM (Fig. 1A). Cellular pathways from GO Biological Processes associated with cellular responses to stress and DNA damage responses (DDR) were downregulated in prMDS compared to stMDS. The only GO terms that were significantly upregulated in prMDS were processes related to cell-matrix adhesion or cell-cell adhesion. Indeed, key transcription factor controlling changes in cell-cell adhesion, ZEB1, was significantly overexpressed in prMDS BM CD34 + cells, whereas E-cadherin expression ( CDH1) was decreased; the expression levels of CDH1 and ZEB1 showed a significant moderate negative correlation (Fig. 1B). Additionally, prMDS samples exhibited high levels of aberrant splicing and global lncRNA expression. Analysis of aberrant transcripts, deregulated lncRNAs and their targets suggested their contribution to the attenuation of DDR pathways in prMDS. Given that the downregulation of DDR gene expression was the major feature distinguishing the prMDS CD34 + cell transcriptome from that of stMDS in our cohort, we selected - from the transcriptional profile of the top 50 downregulated genes in GSEA - a prognostic DDR-associated gene signature consisting of 19 genes. Using regularized Cox regression analysis of the expression profile of 19 DDR-associated genes, the probability of a progression-free disease was calculated in our cohort. High DDR gene expressors had a significantly higher probability of progression-free disease (p < 0.001) compared to low DDR gene expressors (Fig. 2A). Among the top 50 upregulated genes in the prMDS vs. stMDS samples, ranked by GSEA, overexpression of previously identified markers of leukemic progression and/or poor MDS/AML survival ( SPNS2, MN1, DOCK1 and others) was demonstrated. Interestingly, overexpression of NEK3, a gene not previously associated with MDS/AML progression, showed a predictive power for accelerated progression in LR-MDS with the best significance of curve separation among the genes tested (Fig. 2B). Furthermore, NEK3 expression was an independent prognostic factor (p<0.001) for progression-free disease in a multivariate analysis of important clinical and genetic factors. To conclude, our data suggest that DDR gene expression signature and NEK3 expression appear to be a significant predictor of LR-MDS progression and may be applicable at the time of diagnosis for the prognostic stratification of patients with LR-MDS, regardless of their mutational status. Supported by AZV (NU21-03-00565) and (NU23-03-00401); MH CZ-DRO (UHKT, 00023736); Program EXCELES, ID Project No. LX22NPO5102, and UP Young Researcher Grant Competition, ID Project JG_2023_016.

Sensing of H2O2-induced oxidative stress by the UPF factor complex is crucial for activation of catalase-3 expression in Neurospora.

UPF-1-UPF-2-UPF-3 complex-orchestrated nonsense-mediated mRNA decay (NMD) is a well-characterized eukaryotic cellular surveillance mechanism that not only degrades aberrant transcripts to protect the integrity of the transcriptome but also eliminates normal transcripts to facilitate appropriate cellular responses to physiological and environmental changes. Here, we describe the multifaceted regulatory roles of the Neurospora crassa UPF complex in catalase-3 (cat-3) gene expression, which is essential for scavenging H2O2-induced oxidative stress. First, losing UPF proteins markedly slowed down the decay rate of cat-3 mRNA. Second, UPF proteins indirectly attenuated the transcriptional activity of cat-3 gene by boosting the decay of cpc-1 and ngf-1 mRNAs, which encode a well-studied transcription factor and a histone acetyltransferase, respectively. Further study showed that under oxidative stress condition, UPF proteins were degraded, followed by increased CPC-1 and NGF-1 activity, finally activating cat-3 expression to resist oxidative stress. Together, our data illustrate a sophisticated regulatory network of the cat-3 gene mediated by the UPF complex under physiological and H2O2-induced oxidative stress conditions.

A deregulated m6A writer complex axis driven by BRD4 confers an epitranscriptomic vulnerability in combined DNA repair–targeted therapy

Aberrant transcripts expression of the m6A methyltransferase complex (MTC) is widely found across human cancers, suggesting a dysregulated signaling cascade which integrates m6A epitranscriptome to drive tumorigenesis. However, the responsible transcriptional machinery directing the expression of distinct MTC subunits remains unclear. Here, we identified an unappreciated interplay between the histone acetyl-lysine reader BRD4 and the m6A writer complex across human cancers. BRD4 directly stimulates transcripts expression of seven MTC subunits, allowing the maintenance of the nuclear writer complex integrity. Upon BET inhibition, this BRD4-MTC signaling cascade accounts for global m6A reduction and the subsequent dynamic alteration of BRD4-dependent transcriptome, resulting in impaired DNA damage response that involves activation of homologous recombination (HR) repair and repression of apoptosis. We further demonstrated that the combined synergy upon BET/PARP inhibition largely relies on disrupted m6A modification of HR and apoptotic genes, counteracting PARP inhibitor (PARPi) resistance in patient-derived xenograft models. Our study revealed a widespread active cross-talk between BRD4-dependent epigenetic and MTC-mediated epitranscriptomic networks, which provides a unique therapeutic vulnerability that can be leveraged in combined DNA repair-targeted therapy.

A murine model of BSCL2-associated Celia's encephalopathy

Celia's encephalopathy or progressive encephalopathy with/without lipodystrophy is a neurodegenerative disease with a fatal prognosis in childhood. It is generally caused by the c.985C > T variant in the BSCL2 gene, leading to the skipping of exon 7 and resulting in an aberrant seipin protein (Celia-seipin).To precisely define the temporal evolution and the mechanisms involved in neurodegeneration, lipodystrophy and fatty liver in Celia's encephalopathy, our group has generated the first global knock-in murine model for the aberrant human transcript of BSCL2 (Bscl2Celia/Celia) using a strategy based on the Cre/loxP recombination system. In order to carry out a characterization at the neurological, adipose tissue and hepatic level, behavioral studies, brain PET, metabolic, histological and molecular studies were performed.Around 12% of homozygous and 5.4% of heterozygous knock-in mice showed severe neurological symptoms early in life, and their life expectancy was dramatically reduced. Severe generalized lipodystrophy and mild hepatic steatosis were present in these affected animals, while serum triglycerides and glucose metabolism were normal, with no insulin resistance. Furthermore, the study revealed a reduction in brain glucose uptake, along with patchy loss of Purkinje cells and the presence of intranuclear inclusions in cerebellar cortex cells. Homozygous, non-severely-affected knock-in mice showed a decrease in locomotor activity and greater anxiety compared with their wild type littermates.Bscl2Celia/Celia is the first murine model of Celia's encephalopathy which partially recapitulates the phenotype and severe neurodegenerative picture suffered by these patients. This model will provide a helpful tool to investigate both the progressive encephalopathy with/without lipodystrophy and congenital generalized lipodystrophy.

TEQUILA-seq: a versatile and low-cost method for targeted long-read RNA sequencing

Long-read RNA sequencing (RNA-seq) is a powerful technology for transcriptome analysis, but the relatively low throughput of current long-read sequencing platforms limits transcript coverage. One strategy for overcoming this bottleneck is targeted long-read RNA-seq for preselected gene panels. We present TEQUILA-seq, a versatile, easy-to-implement, and low-cost method for targeted long-read RNA-seq utilizing isothermally linear-amplified capture probes. When performed on the Oxford nanopore platform with multiple gene panels of varying sizes, TEQUILA-seq consistently and substantially enriches transcript coverage while preserving transcript quantification. We profile full-length transcript isoforms of 468 actionable cancer genes across 40 representative breast cancer cell lines. We identify transcript isoforms enriched in specific subtypes and discover novel transcript isoforms in extensively studied cancer genes such as TP53. Among cancer genes, tumor suppressor genes (TSGs) are significantly enriched for aberrant transcript isoforms targeted for degradation via mRNA nonsense-mediated decay, revealing a common RNA-associated mechanism for TSG inactivation. TEQUILA-seq reduces the per-reaction cost of targeted capture by 2-3 orders of magnitude, as compared to a standard commercial solution. TEQUILA-seq can be broadly used for targeted sequencing of full-length transcripts in diverse biomedical research settings.

Detection of Hybrid Fusion Transcripts, Aberrant Transcript Expression, and Specific Single Nucleotide Variants in Acute Leukemia and Myeloid Disorders with Recurrent Gene Rearrangements

Introduction: A variety of gene rearrangements and molecular alterations are key drivers in the pathobiology of acute leukemia and myeloid disorders; current classification systems increasingly incorporate these findings in diagnostic algorithms. Therefore, clinical laboratories require versatile tools, which can detect an increasing number and variety of molecular and cytogenetic alterations of clinical significance. Methods: We validated an RNA-based next-generation sequencing (NGS) assay that enables the detection of: (i) numerous hybrid fusion transcripts (including rare/novel gene partners), (ii) aberrantly expressed EVI1 (MECOM) and IKZF1 (Del exons 4–7) transcripts, and (iii) hotspot variants in KIT, ABL1, NPM1 (relevant in the context of gene rearrangement status). Results: For hybrid fusion transcripts, the assay showed 98–100% concordance for known positive and negative samples, with an analytical sensitivity (i.e., limit of detection) of approximately 0.8% cells. Samples with underlying EVI1 (MECOM) translocations demonstrated increased EVI1 (MECOM) expression. Aberrant IKZF1 (Del exons 4–7) transcripts detectable with the assay were also present on orthogonal reverse transcription PCR. Specific hotspot mutations in KIT, ABL1, and NPM1 detected with the assay showed 100% concordance with orthogonal testing. Lastly, several illustrative samples are included to highlight the assay’s clinically relevant contributions to patient workup. Conclusion: Through its ability to simultaneously detect various gene rearrangements, aberrantly expressed transcripts, and hotspot mutations, this RNA-based NGS assay is a valuable tool for clinical laboratories to supplement other molecular and cytogenetic methods used in the diagnostic workup and in clinical research for patients with acute leukemia and myeloid disorders.

The novel compound heterozygous variants identified in a Chinese family with glucose phosphate isomerase deficiency and pathogenicity analysis

Background and Aims:Glucose phosphate isomerase (GPI) deficiency is an extremely rare autosomal recessive disorder caused by mutations in the GPI gene. In this research, the proband displaying typical manifestations of haemolytic anaemia and his family members were recruited to analyse the pathogenicity of the detected variants.Methods:Peripheral blood samples were collected from the family members and genomic DNA was extracted and targeted for capture and sequencing. The effect of the candidate pathogenic variants on splicing was further investigated using the minigene splicing system. The computer simulation was also used for further analysis of the detected data.Results:The proband carried the compound heterozygous variants c.633 + 3 A > G and c.295G > T in the GPI gene, which have never been reported before. In the genealogy, co-segregation of the mutant genotype with the phenotype was established. The minigene study showed that intronic mutations resulted in abnormal pre-mRNA splicing. Specifically, the two aberrant transcripts: r.546_633del and r.633 + 1_633 + 2insGT were transcribed by the minigene plasmid expressing the c.633 + 3 A > G variant. The missense mutation c.295G > T in exon 3 resulted in altering glycine at codon 87 to cysteine which was predicted to be pathogenic in an in silico analysis. Deeper analyses revealed that the Gly87Cys missense mutation led to steric hindrance. Compared to the wild-type, the mutation G87C led to a significant increase in intermolecular forces.Conclusion:Overall, the novel compound heterozygous variants in the GPI gene contributed to the etiology of the disease. Genetic testing can assist in the diagnosis. The novel gene variants identified in the present study has further expanded the mutational spectrum of GPI deficiency, which can better guide family counselling.

SKI complex: A multifaceted cytoplasmic RNA exosome cofactor in mRNA metabolism with links to disease, developmental processes, and antiviral responses.

RNA stability and quality control are integral parts of gene expression regulation. A key factor shaping eukaryotic transcriptomes, mainly via 3'-5' exoribonucleolytic trimming or degradation of diverse transcripts in nuclear and cytoplasmic compartments, is the RNA exosome. Precise exosome targeting to various RNA molecules requires strict collaboration with specialized auxiliary factors, which facilitate interactions with its substrates. The predominant class of cytoplasmic RNA targeted by the exosome are protein-coding transcripts, which are carefully scrutinized for errors during translation. Normal, functional mRNAs are turned over following protein synthesis by the exosome or by Xrn1 5'-3'-exonuclease, acting in concert with Dcp1/2 decapping complex. In turn, aberrant transcripts are eliminated by dedicated surveillance pathways, triggered whenever ribosome translocation is impaired. Cytoplasmic 3'-5' mRNA decay and surveillance are dependent on the tight cooperation between the exosome and its evolutionary conserved co-factor-the SKI (superkiller)complex (SKIc). Here, we summarize recent findings from structural, biochemical, and functional studies of SKIc roles in controlling cytoplasmic RNA metabolism, including links to various cellular processes. Mechanism of SKIc action is illuminated by presentation of its spatial structure and details of its interactions with exosome and ribosome. Furthermore, contribution of SKIc and exosome to various mRNA decay pathways, usually converging on recycling of ribosomal subunits, is delineated. A crucial physiological role of SKIc is emphasized by describing association between its dysfunction and devastating human disease-a trichohepatoenteric syndrome (THES). Eventually, we discuss SKIc functions in the regulation of antiviral defense systems, cell signaling and developmental transitions, emerging from interdisciplinary investigations. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

The endosomal escape vehicle platform enhances delivery of oligonucleotides in preclinical models of neuromuscular disorders

Biological therapeutic agents are highly targeted and potent but limited in their ability to reach intracellular targets. These limitations often necessitate high therapeutic doses and can be associated with less-than-optimal therapeutic activity. One promising solution for therapeutic agent delivery is use of cell-penetrating peptides. Canonical cell-penetrating peptides, however, are limited by low efficiencies of cellular uptake and endosomal escape, minimal proteolytic stability, and toxicity. To overcome these limitations, we designed a family of proprietary cyclic cell-penetrating peptides that form the core of our endosomal escape vehicle technology capable of delivering therapeutic agent-conjugated cargo intracellularly. We demonstrated the therapeutic potential of this endosomal escape vehicle platform in preclinical models of muscular dystrophy with distinct disease etiology. An endosomal escape vehicle-conjugated, splice-modulating oligonucleotide restored dystrophin protein expression in striated muscles in the mdx mouse, amodel for Duchenne muscular dystrophy. Furthermore, another endosomal escape vehicle-conjugated, sterically blocking oligonucleotide led to knockdown of aberrant transcript expression levels in facioscapulohumeral muscular dystrophy patient-derived skeletal muscle cells. These findings suggest a significant therapeutic potential of our endosomal escape vehicle conjugated oligonucleotides for targeted upregulation and downregulation of gene expression in neuromuscular diseases, with possible broader application of this platform for delivery of intracellular biological agents.

Balanced Translocation Disrupting JAG1 Identified by Optical Genomic Mapping in Suspected Alagille Syndrome

We report the clinical and genetic features of a Han Chinese boy who presented with disease suspect for Alagille syndrome (ALGS). Multiple genetic analyses (panel sequencing, multiplex-ligation-dependent probe amplification, and whole genome sequencing) failed to uncover a causative variant. Optical genomic mapping detected a reciprocal translocation between chromosomes 4 and 20, interrupting JAG1. Long-range polymerase chain reaction and targeted sequencing identified the exact breakpoints. Sanger sequencing and reanalysis of genome sequencing raw data further confirmed the result. This translocation is expected to generate aberrant JAG1 transcripts that lead to complete loss of JAG1 expression. This is the first t(4;20)(q22.1;p12.2) balanced translocation detected by optical genomic mapping and characterized at base-pair resolution in ALGS. Our approach permitted precise diagnosis and genetic counseling.

Detecting ALK, ROS1, and RET fusions and the METΔex14 splicing variant in liquid biopsies of non-small-cell lung cancer patients using RNA-based techniques.

ALK, ROS1, and RET fusions and MET∆ex14 variant associate with response to targeted therapies in non-small-cell lung cancer (NSCLC). Technologies for fusion testing in tissue must be adapted to liquid biopsies, which are often the only material available. In this study, circulating-free RNA (cfRNA) and extracellular vesicle RNA (EV-RNA) were purified from liquid biopsies. Fusion and MET∆ex14 transcripts were analyzed by nCounter (Nanostring) and digital PCR (dPCR) using the QuantStudio® System (Applied Biosystems). We found that nCounter detected ALK, ROS1, RET, or MET∆ex14 aberrant transcripts in 28/40 cfRNA samples from positive patients and 0/16 of control individuals (70% sensitivity). Regarding dPCR, aberrant transcripts were detected in the cfRNA of 25/40 positive patients. Concordance between the two techniques was 58%. Inferior results were obtained when analyzing EV-RNA, where nCounter often failed due to a low amount of input RNA. Finally, results of dPCR testing in serial liquid biopsies of five patients correlated with response to targeted therapy. We conclude that nCounter can be used for multiplex detection of fusion and MET∆ex14 transcripts in liquid biopsies, showing a performance comparable with next-generation sequencing platforms. dPCR could be employed for disease follow-up in patients with a known alteration. cfRNA should be preferred over EV-RNA for these analyses.

A wave of deep intronic mutations in X-linked Alport syndrome

X-linked Alport syndrome (XLAS) is an inherited kidney disease caused exclusively by pathogenic variants in the COL4A5 gene. In 10-20% of cases, DNA sequencing of COL4A5 exons or flanking regions cannot identify molecular causes. Here, our objective was to use a transcriptomic approach to identify causative events in a group of 19 patients with XLAS without identified mutation by Alport gene panel sequencing. Bulk RNAseq and/or targeted RNAseq using a capture panel of kidney genes was performed. Alternative splicing events were compared to those of 15 controls by a developed bioinformatic score. When using targeted RNAseq, COL4A5 coverage was found to be 23-fold higher than with bulk RNASeq and revealed 30 significant alternative splicing events in 17 of the 19 patients. After computational scoring, a pathogenic transcript was found in all patients. A causative variant affecting COL4A5 splicing and absent in the general population was identified in all cases. Altogether, we developed a simple and robust method for identification of aberrant transcripts due to pathogenic deep-intronic COL4A5 variants. Thus, these variants, potentially targetable by specific antisense oligonucleotide therapies, were found in a high percentage of patients with XLAS in whom pathogenic variants were missed by conventional DNA sequencing.

The Effect of Glucocorticoids on TAF1 Gene Transcription in X-linked Dystonia Parkinsonism.

X-linked Dystonia Parkinsonism (XDP) is associated with a SINE-VNTR- Alu (SVA) retrotransposon insertion in an intron of the TAF1 gene that alters gene transcription and splicing. In this study, we determined if the SVA insertion introduces glucocorticoid (GC)-responsive cis-regulatory elements that may contribute to dysregulated TAF1 transcription and XDP disease progression. We performed in silico analysis to identify potential GC receptor (GR) binding sites within the XDP-SVA. We also conducted promoter-reporter assays on HeLa and HEK293T cells to assess the intrinsic promoter activity of three XDP-SVA variants representing different hexameric repeat lengths associated with differences in disease onset. We treated XDP fibroblast cell models with GR agonist (CORT) or antagonist (RU486), then subjected TAF1 and the XDP-associated aberrant transcript, TAF1-32i to gene expression analysis. A transcription factor binding site search revealed three binding sites for GR within the XDP-SVA-two within the SINE region and one in the Alu region. Promoter-reporter assays showed induction of XDP-SVA promoter activity upon CORT treatment that was dependent on the cell line and XDP-SVA hexamer repeat length. Gene expression analysis showed that baseline TAF1 levels differed between control and patient fibroblast cell lines, and treatment with CORT led to an increasing trend in the expression of the aberrant TAF1-32i transcript but did not reach statistical significance. Treatment with RU486 increased TAF1 mRNA expression only in the control cell lines. Using reporter assays, the XDP-SVA was shown to exhibit CORT-dependent transcriptional activation. Gene expression analysis also showed that GC signaling may influence TAF1 and TAF1-32i expression, possibly through interaction with the XDP-SVA. Our data provide a potential link between stress and XDP progression.

Oncogenic CDK13 mutations impede nuclear RNA surveillance.

RNA surveillance pathways detect and degrade defective transcripts to ensure RNA fidelity. We found that disrupted nuclear RNA surveillance is oncogenic. Cyclin-dependent kinase 13 (CDK13) is mutated in melanoma, and patient-mutated CDK13 accelerates zebrafish melanoma. CDK13 mutation causes aberrant RNA stabilization. CDK13 is required for ZC3H14 phosphorylation, which is necessary and sufficient to promote nuclear RNA degradation. Mutant CDK13 fails to activate nuclear RNA surveillance, causing aberrant protein-coding transcripts to be stabilized and translated. Forced aberrant RNA expression accelerates melanoma in zebrafish. We found recurrent mutations in genes encoding nuclear RNA surveillance components in many malignancies, establishing nuclear RNA surveillance as a tumor-suppressive pathway. Activating nuclear RNA surveillance is crucial to avoid accumulation of aberrant RNAs and their ensuing consequences in development and disease.