Nonsense-mediated Decay Research Articles

Readon: a novel algorithm to identify read-through transcripts with long-read sequencing data.

There are many clustered transcriptionally active regions in the human genome, in which the transcription complex cannot immediately terminate transcription at the upstream gene termination site, but instead continues to transcribe intergenic regions and downstream genes, resulting in read-through transcripts. Several studies have demonstrated the regulatory roles of read-through transcripts in tumorigenesis and development. However, limited by the read length of next-generation sequencing, discovery of read-through transcripts has been slow. For long but also erroneous third-generation sequencing data, this study developed a novel minimizer sketch algorithm to accurately and quickly identify read-through transcripts. Readon initially splits the reference sequence into distinct active regions. It employs a sliding window approach within each region, calculates minimizers, and constructs the specialized structured arrays for query indexing. Following initial alignment anchor screening of candidate read-through transcripts, further confirmation steps are executed. Comparative assessments against existing software reveal Readon's superior performance on both simulated and validated real data. Additionally, two downstream tools are provided: one for predicting whether a read-through transcript is likely to undergo nonsense-mediated decay or encodes a protein, and another for visualizing splicing patterns. Readon is freely available on GitHub (https://github.com/Bulabula45/Readon).

Oncofetal SNRPE promotes HCC tumorigenesis by regulating the FGFR4 expression through alternative splicing.

Due to insufficient knowledge about key molecular events, Hepatocellular carcinoma (HCC) lacks effective treatment targets. Spliceosome-related genes were significantly altered in HCC. Oncofetal proteins are ideal tumor therapeutic targets. Screening of differentially expressed Spliceosome-related oncofetal protein in embryonic liver development and HCC helps discover effective therapeutic targets for HCC. Differentially expressed spliceosome genes were analysis in fetal liver and HCC through bioinformatics analysis. Small nuclear ribonucleoprotein polypeptide E (SNRPE) expression was detected in fetal liver, adult liver and HCC tissues. The role of SNRPE in HCC was performed multiple assays in vitro and in vivo. SNRPE-regulated alternative splicing was recognized by RNA-Seq and confirmed by multiple assays. We herein identified SNRPE as a crucial oncofetal splicing factor, significantly associated with the adverse prognosis of HCC. SOX2 was identified as the activator for SNRPE reactivation. Efficient knockdown of SNRPE resulted in the complete cessation of HCC tumorigenesis and progression. Mechanistically, SNRPE knockdown reduced FGFR4 mRNA expression by triggering nonsense-mediated RNA decay. A partial inhibition of SNRPE-induced malignant progression of HCC cells was observed upon FGFR4 knockdown. Our findings highlight SNRPE as a novel oncofetal splicing factor and shed light on the intricate relationship between oncofetal splicing factors, splicing events, and carcinogenesis. Consequently, SNRPE emerges as a potential therapeutic target for HCC treatment. Model of oncofetal SNRPE promotes HCC tumorigenesis by regulating the AS of FGFR4 pre-mRNA.

Fine-Tuning Amyloid Precursor Protein Expression through Nonsense-Mediated mRNA Decay.

Studies on genetic robustness recently revealed transcriptional adaptation (TA) as a mechanism by which an organism can compensate for genetic mutations through activation of homologous genes. Here, we discovered that genetic mutations, introducing a premature termination codon (PTC) in the amyloid precursor protein-b (appb) gene, activated TA of two other app family members, appa and amyloid precursor-like protein-2 (aplp2), in zebrafish. The observed transcriptional response of appa and aplp2 required degradation of mutant mRNA and did not depend on Appb protein level. Furthermore, TA between amyloid precursor protein (APP) family members was observed in human neuronal progenitor cells; however, compensation was only present during early neuronal differentiation and could not be detected in a more differentiated neuronal stage or adult zebrafish brain. Using knockdown and chemical inhibition, we showed that nonsense-mediated mRNA decay (NMD) is involved in degradation of mutant mRNA and that Upf1 and Upf2, key proteins in the NMD pathway, regulate the endogenous transcript levels of appa, appb, aplp1, and aplp2 In conclusion, our results suggest that the expression level of App family members is regulated by the NMD pathway and that mutations destabilizing app/APP mRNA can induce genetic compensation by other family members through TA in both zebrafish and human neuronal progenitors.

A Novel Frameshift Variant of the ELF4 Gene in a Patient with Autoinflammatory Disease: Clinical Features, Transcriptomic Profiling and Functional Studies.

We described the diagnosis and treatment of a patient with autoinflammatory disease, named "Deficiency in ELF4, X-linked (DEX)". A novel ELF4 variant was discovered and its pathogenic mechanism was elucidated. The data about clinical, laboratory and endoscopic features, treatment, and follow-up of a patient with DEX were analyzed. Whole exome sequencing and Sanger sequencing were performed to identify potential pathogenic variants. The mRNA and protein levels of ELF4 were analyzed by qPCR and Western blotting, respectively. The association of ELF4 frameshift variant with nonsense-mediated mRNA decay (NMD) in the pathogenesis DEX was examined. Moreover, RNA-seq was performed to identify the key molecular events triggered by ELF4 variant. The relationship between ELF4 and IFN-β activity was validated using a dual-luciferase reporter assay and a ChIP-qPCR assay. An 11-year-old boy presented with a Behçet's-like phenotype. The laboratory abnormality was the most obvious in elevated inflammatory indicators. Endoscopy revealed multiple ileocecal ulcers. Intestinal histopathology showed inflammatory cell infiltrations. The patient was treated with long-term immunosuppressant and TNF-α blocker (adalimumab), which reaped an excellent response over 16 months of follow-up. Genetic analysis identified a maternal hemizygote frameshift variant (c.1022del, p.Q341Rfs*30) in ELF4 gene in the proband. The novel variant decreased the mRNA level of ELF4 via the NMD pathway. Mechanistically, insufficient expression of ELF4 disturbed the immune system, leading to immunological disorders and pathogen susceptibility, and disrupted ELF4-activating IFN-βresponses. This analysis detailed the clinical characteristics of a Chinese patient with DEX who harbored a novel ELF4 frameshift variant. For the first time, we used patient-derived cells and carried out transcriptomic analysis to delve into the mechanism of ELF4 variant in DEX.

Physiological Consequences of Nonsense-Mediated Decay and Its Role in Adaptive Responses.

The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway is a quality control mechanism that degrades aberrant mRNA containing one or more premature termination codons (PTCs). Recent discoveries indicate that NMD also differentially regulates mRNA from wild-type protein-coding genes despite lacking PTCs. Together with studies showing that NMD is involved in development and adaptive responses that influence health and longevity, these findings point to an expanded role of NMD that adds a new layer of complexity in the post-transcriptional regulation of gene expression. However, the extent of its control, whether different types of NMD play different roles, and the resulting physiological outcomes remain unclear and need further elucidation. Here, we review different branches of NMD and what is known of the physiological outcomes associated with this type of regulation. We identify significant gaps in the understanding of this process and the utility of genetic tools in accelerating progress in this area.

The NM_033380.2 transcript of the COL4A5 gene contains a variable splice site c.4822-10T>C, which has been identified as a causative factor for Alport syndrome.

Alport Syndrome (AS) is a genetic kidney disorder characterized by progressive hearing loss and atypical eye symptoms, resulting in a poor prognosis and lack of effective targeted therapy. The primary mode of inheritance is X-linked dominant (XLAS) due to variants in the COL4A5 gene. This study revealed a previously unidentified alternative form of the COL4A5 gene, namely, the c.4822-10T>C variant, which was confirmed through in vitro experiments. To investigate the impact of a splicing variant on COL4A5 mRNA production, an in vitro minigene splicing assay was utilized. Additionally, molecular dynamics was employed to predict the ability of α5(IV) to form a triple helix. Results from the experiment revealed that the wild-type (WT) plasmid produced two distinct mRNA products simultaneously. Sequence analysis using the BLAST database revealed a 173-bp deletion in the mRNA sequence of the first product, indicating a potential similarity to the XM_016942897.2 transcript of Pan troglodytes. The second mRNA product of the WT plasmid contained the full sequence of exons 51, 52, and 53, as anticipated. Conversely, the mutant (MT) plasmid generated a single mRNA product with a 173-bp deletion in exon 52, leading to the identification of the mature mRNA expression as NM_033380.2: COL4A5: c.4822_4994del. In the context of nonsense-mediated mRNA decay (NMD), the deletion c.4822_4994 results in the production of a truncated protein, p.His1608*, that terminates prematurely. This truncated protein may disrupt the secondary structure of α5(IV) and potentially cause an abnormal conformation of α345(IV). This study examines the relationship between the variable splicing pattern in the NM_033380.2 transcript of the COL4A5 gene in XLAS patients and the presence of the COL4A5 gene splice variant c.4822-10T>C. Our findings indicate that the c.4822-10T>C splice variant leads to activation of nonsense-mediated mRNA degradation (NMD) and reduced COL4A5 mRNA expression, resulting in inadequate synthesis of the corresponding proteins. This aligns with the patient's immunofluorescence results showing negative α5(IV) chain presence at the glomerular basement membrane, bursa, and tubular basement membrane, confirming the pathogenic nature of c.4822-10T>C.

Human disease-causing mutations result in loss of leiomodin 2 through nonsense-mediated mRNA decay.

The leiomodin (Lmod) family of actin-binding proteins play a critical role in muscle function, highlighted by the fact that mutations in all three family members (LMOD1-3) result in human myopathies. Mutations in the cardiac predominant isoform, LMOD2 lead to severe neonatal dilated cardiomyopathy. Most of the disease-causing mutations in the LMOD gene family are nonsense, or frameshift, mutations predicted to result in expression of truncated proteins. However, in nearly all cases of disease, little to no LMOD protein is expressed. We show here that nonsense-mediated mRNA decay, a cellular mechanism which eliminates mRNAs with premature termination codons, underlies loss of mutant protein from two independent LMOD2 disease-causing mutations. Furthermore, we generated steric-blocking oligonucleotides that obstruct deposition of the exon junction complex, preventing nonsense-mediated mRNA decay of mutant LMOD2 transcripts, thereby restoring mutant protein expression. Our investigation lays the initial groundwork for potential therapeutic intervention in LMOD-linked myopathies.

A novel PSMB8 isoform associated with multiple sclerosis lesions induces P-body formation.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Current therapies primarily target the inflammatory component of the disease and are highly effective in early stages of MS while limited therapies have an effect in the more chronic progressive stages of MS where resident glia have a larger role. MS lesions tend to be inflammatory even after the initial peripheral immune cell invasion has subsided and this inflammation is known to cause alternative splicing events. We used qPCR of normal-appearing white matter and white matter lesions from postmortem MS tissue, in vitro studies, and immunostaining in MS tissue to investigate the alternative splicing of one gene known to be important during recovery in an animal model of MS, PSMB8. We found a novel, intron-retained isoform which has not been annotated, upregulated specifically in MS patient white matter lesions. We found that this novel isoform activates the nonsense-mediated decay pathway in primary human astrocytes, the most populous glial cell in the CNS, and is then degraded. Overexpression of this isoform in astrocytes leads to an increased number of processing bodies in vitro, the primary site of mRNA decay. Finally, we demonstrated that MS white matter lesions have a higher burden of processing bodies compared to normal-appearing white matter, predominantly in GFAP-positive astrocytes. The increase in alternative splicing of the PSMB8 gene, the stress that this alternative splicing causes, and the observation that processing bodies are increased in white matter lesions suggests that the lesion microenvironment may lead to increased alternative splicing of many genes. This alternative splicing may blunt the protective or reparative responses of resident glia in and around white matter lesions in MS patients.

Glutathione peroxidase (GPX1) - Selenocysteine metabolism preserves the follicular fluid's (FF) redox homeostasis via IGF-1- NMD cascade in follicular ovarian cysts (FOCs)

Follicular ovarian cysts (FOCs) are characterized by follicles in the ovaries that are >20 mm in diameter and persist for >10 days without the corpus luteum, leading to anovulation, dysregulation of folliculogenesis and subfertility in humans and livestock species. Despite their clinical significance, the precise impact of FOCs on oocyte reserve, maturation, and quality still needs to be explored. While FOCs are observed in both human and livestock populations, they are notably prevalent in livestock species. Consequently, livestock species serve as valuable models for investigating the molecular intricacies of FOCs. Thus, in this study, using goat FOCs, we performed integrated proteomic, metabolomic and functional analyses to demonstrate that oocyte maturation is hampered due to increased reactive oxygen species (ROS) in FOCs follicular fluid (FF) via downregulation of glutathione peroxidase (GPX1), a critical antioxidant seleno enzyme required to negate oxidative stress. Notably, GPX1 reduction was positively correlated with the FF's decline of free selenium and selenocysteine metabolic enzymes, O-phosphoryl-tRNA (Sec) selenium transferase (SEPSECS) and selenocysteine lyase (SCLY) levels. Adding GPX1, selenocysteine, or selenium to the culture media rescued the oocyte maturation abnormalities caused by FOCs FF by down-regulating the ROS. Additionally, we demonstrate that substituting GPX1 regulator, Insulin-like growth factor-I (IGF-1) in the in vitro maturation media improved the oocyte maturation in the cystic FF by down-regulating the ROS activity via suppressing Non-sense-mediated decay (NMD) of GPX1. In contrast, inhibition of IGF-1R and the target of rapamycin complex 1 (mTORC1) hampered the oocyte maturation via NMD up-regulation. These findings imply that the GPX1 regulation via selenocysteine metabolism and the IGF-1-mediated NMD may be critical for the redox homeostasis of FF. We propose that GPX1 enhancers hold promise as therapeutics for enhancing the competence of FOCs oocytes. However, further in vivo studies are necessary to validate these findings observed in vitro.

Post-transcriptional Regulation of Gene Expression via Unproductive Splicing.

Unproductive splicing is a mechanism of post-transcriptional gene expression control in which premature stop codons are inserted into protein-coding transcripts as a result of regulated alternative splicing, leading to their degradation via the nonsense-mediated decay pathway. This mechanism is especially characteristic of RNA-binding proteins, which regulate each other's expression levels and those of other genes in multiple auto- and cross-regulatory loops. Deregulation of unproductive splicing is a cause of serious human diseases, including cancers, and is increasingly being considered as a prominent therapeutic target. This review discusses the types of unproductive splicing events, the mechanisms of auto- and cross-regulation, nonsense-mediated decay escape, and problems in identifying unproductive splice isoforms. It also provides examples of deregulation of unproductive splicing in human diseases and discusses therapeutic strategies for its correction using antisense oligonucleotides and small molecules.

Caspases compromise SLU7 and UPF1 stability and NMD activity during hepatocarcinogenesis

Background & AimsThe homeostasis of the cellular transcriptome depends on transcription and splicing mechanisms. Moreover, the fidelity of gene expression, essential to preserve cellular identity and function is secured by different quality control mechanisms including nonsense-mediated RNA decay (NMD). In this context, alternative splicing is coupled to NMD, and several alterations in these mechanisms leading to the accumulation of aberrant gene isoforms have been involved in human disease including cancer. MethodsRNAseq, Westen blot, qPCR, co-IP in multiple silenced culture cell lines (replicates n≥4), primary hepatocytes and samples of animal models (Jo2, APAP, Mdr2-/- mice, n≥3). ResultsHere we describe that in animal models of liver injury and in human HCC (TCGA, NT =50 vs HCC =374), the process of NMD is inhibited. Moreover, we demonstrate that the splicing factor SLU7 interacts with and preserves the levels of the NMD effector UPF1, and that SLU7 is required for a correct NMD. Our previous findings demonstrated that SLU7 expression is reduced in the diseased liver, contributing to hepatocellular de-differentiation and genome instability during disease progression. However, little was known about the mechanisms underlying SLU7 downregulation. Here we provide evidence showing that caspases activated during liver damage would be responsible of the cleavage and degradation of SLU7. ConclusionsHere we identify the downregulation of UPF1 and the inhibition of NMD as a new molecular pathway contributing to the malignant reshaping of the liver transcriptome. Moreover, and importantly, we uncover caspases activation as the mechanism responsible for the downregulation of SLU7 expression during liver disease progression, which is a new link between apoptosis an hepatocarcinogenesis. Impact and implicationsThe mechanisms involved in the reshaping of hepatocellular transcriptome driving the progressive loss of cell identity and function in liver disease are not completely understood. In this context, we provide evidence on the impairment of a key mRNA surveillance mechanism known as nonsense-mediated mRNA decay (NMD). Mechanistically, we uncover a novel role for the splicing factor SLU7 in the regulation of NMD, including its ability to interact and preserve the levels the key NMD factor UPF1. Moreover, we demonstrate that the activation of caspases during liver damage mediates SLU7 and UPF1 proteins degradation and NMD inhibition. Our findings identify potential new markers of liver disease progression, and SLU7 as a novel therapeutic target to prevent the functional decay of the chronically injured organ.

Functional transcriptome analysis revealed upregulation of MAPK-SMAD signalling pathways in chronic heat stress in crossbred cattle.

Animal geneticists and breeders have the impending challenge of enhancing the resilience of Indian livestock to heat stress through better selection strategies. Climate change's impact on livestock is more intense in tropical countries like India where dairy cattle crossbreeds are more sensitive to heat stress. The main reason for this study was to find the missing relative changes in transcript levels in thermo-neutral and heat stress conditions in crossbred cattle through whole-transcriptome analysis of RNA-Seq data. Differentially expressed genes (DEGs) identified based on the minimum log twofold change value and false discovery rate 0.05 revealed 468 up-regulated genes and 2273 down-regulated significant genes. Functional annotation and pathway analysis of these significant DEGs were compared based on Gene Ontology (Biological process), Kyoto Encyclopedia of Genes and Genome (KEGG), and Reactome pathways using g: Profiler, ShinyGO v0.76, and iDEP.951 web tools. On finding network visualization, the most over-represented and correlated pathways were neuronal and sensory organ development, calcium signalling pathway, Mitogen-activated protein kinase (MAPK) and Smad signalling pathway, Ras-proximate-1, or Ras-related protein 1 (Rap 1) signalling pathway, apoptosis, and oxidative stress. Similarly, down-regulated genes were most expressed in mRNA processing, immune system, B-cell receptor signalling pathway, Nucleotide oligomerization domain(NOD)-like receptors (NLRs) signalling pathway and nonsense-mediated decay (NMD) pathway. The heat stress-responsive genes identified in this study will facilitate our understanding of the molecular basis for climate resilience and heat tolerance in Indian dairy crossbreeds.

Nonsense-mediated mRNA decay of metal-binding activator MAC1 is dependent on copper levels and 3'-UTR length in Saccharomyces cerevisiae.

The nonsense-mediated mRNA decay (NMD) pathway was initially identified as a surveillance pathway that degrades mRNAs containing premature termination codons (PTCs). NMD is now also recognized as a post-transcriptional regulatory pathway that regulates the expression of natural mRNAs. Earlier studies demonstrated that regulation of functionally related natural mRNAs by NMD can be differential and condition-specific in Saccharomyces cerevisiae. Here, we investigated the regulation of MAC1 mRNAs by NMD in response to copper as well as the role the MAC1 3'-UTR plays in this regulation. MAC1 is a copper-sensing transcription factor that regulates the high-affinity copper uptake system. MAC1 expression is activated upon copper deprivation. We found that MAC1 mRNAs are regulated by NMD under complete minimal (CM) but escaped NMD under low and high copper conditions. Mac1 protein regulated gene, CTR1 is not regulated by NMD in conditions where MAC1 mRNAs are NMD sensitive. We also found that the MAC1 3'-UTR is the NMD targeting feature on the mRNAs, and that MAC1 mRNAs lacking 3'-UTRs were stabilized during copper deprivation. Our results demonstrate a mechanism of regulation for a metal-sensing transcription factor, at both the post-transcriptional and post-translational levels, where MAC1 mRNA levels are regulated by NMD and copper, while the activity of Mac1p is controlled by copper levels.

UPF1 helicase orchestrates mutually exclusive interactions with the SMG6 endonuclease and UPF2.

Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. Here, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by different conformational states connected to the RNA-binding status of UPF1. Our findings rationalize a key event in metazoan NMD and advance our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.

CRISPR/Cas9 screen reveals that targeting TRIM34 enhances ferroptosis sensitivity and augments immunotherapy efficacy in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a prevalent malignancy characterized by complex heterogeneity and drug resistance. Resistance to ferroptosis is closely related to the progression of HCC. While HCC tumors vary in their sensitivity to ferroptosis, the precise factors underlying this heterogeneity remain unclear. In this study, we sought to elucidate the mechanisms that contribute to ferroptosis resistance in HCC. Whole-genome CRISPR/Cas9 screen using a subtoxic concentration (IC20) of ferroptosis inducer erastin in the HCC cell line Huh7 revealed TRIM34 as a critical driver of ferroptosis resistance in HCC. Further investigation revealed that TRIM34 suppresses ferroptosis in HCC cells, promoting their proliferation, migration, and invasion both in vitro and in vivo. Furthermore, TRIM34 expression is elevated in HCC tumor tissues, correlating with a poor prognosis. Mechanistically, TRIM34 directly interacts with Up-frameshift 1 (UPF1), a core component of the nonsense-mediated mRNA decay (NMD) pathway, to promote its ubiquitination and degradation. This interaction suppresses GPX4 transcript degradation, thus promoting the protein levels of this critical ferroptosis suppressor in HCC. In light of the close crosstalk between ferroptosis and the adaptive immune response in cancer, HCC cells with targeting knockdown of TRIM34 exhibited an improved response to anti-PD-1 treatment. Taken together, the TRIM34/UPF1/GPX4 axis mediates ferroptosis resistance in HCC, thereby promoting malignant phenotypes. Targeting TRIM34 may thus represent a promising new strategy for HCC treatment.

SPAST Intragenic CNVs Lead to Hereditary Spastic Paraplegia via a Haploinsufficiency Mechanism.

The most common form of hereditary spastic paraplegia (HSP), SPG4 is caused by single nucleotide variants and microrearrangements in the SPAST gene. The high percentage of multi-exonic deletions or duplications observed in SPG4 patients is predisposed by the presence of a high frequency of Alu sequences in the gene sequence. In the present study, we analyzed DNA and RNA samples collected from patients with different microrearrangements in SPAST to map gene breakpoints and evaluate the mutation mechanism. The study group consisted of 69 individuals, including 50 SPG4 patients and 19 healthy relatives from 18 families. Affected family members from 17 families carried varying ranges of microrearrangements in the SPAST gene, while one individual had a single nucleotide variant in the 5'UTR of SPAST. To detect the breakpoints of the SPAST gene, long-range PCR followed by sequencing was performed. The breakpoint sequence was detected for five different intragenic SPAST deletions and one duplication, revealing Alu-mediated microhomology at breakpoint junctions resulting from non-allelic homologous recombination in these patients. Furthermore, SPAST gene expression analysis was performed using patient RNA samples extracted from whole blood. Quantitative real-time PCR tests performed in 14 patients suggest no expression of transcripts with microrearrangements in 5 of them. The obtained data indicate that nonsense-mediated decay degradation is not the only mechanism of hereditary spastic paraplegia in patients with SPAST microrearrangements.

Abstract PO2-03-08: Uncovering Novel Potential Prognostic Biomarkers in Basal-Like Breast Cancer using Transcriptomic Data of 1,899 Patients

Abstract Introduction: Patients with basal-like breast cancer (BLBC) predominantly represented by triple-negative breast cancer have shown a high recurrence rate and are characterized by poor prognosis. There is an urgent need to undercover reliable prognostic biomarkers that can help in the clinical management of such patients and identify additional therapeutic targets. The objective of this study was to create a comprehensive transcriptomic database on a large scale and leverage it to identify and prioritize cancer-related genes associated with BLBC patients’ outcomes. Methods: We identified breast cancer cohorts from public repositories that contained gene expression data at the transcriptome level, along with clinical follow-up information. BLBC were identified using the PAM50 signature. All samples were standardized using a standard array normalization coupled with scaling to have a mean expression across all genes of 1000 in each sample and incorporated into a unified database. Redundant samples were removed. For each gene, Cox univariate survival analysis was conducted, to account for multiple hypothesis testing, the false discovery rate was computed, and a significant cutoff of 1% was employed to determine the highest statistical significance. Association with RFS and OS was performed. Multivariate analysis was performed for selected genes involving clinical and pathological variables. To uncover higher-level functions related to altered RFS, gene ontology analysis was performed using the enrichGO function in the TNM plotter (http://www.tnmplot.com). Results: The complete integrated database comprises 1,899 samples from 52 breast cancer datasets. Altogether, 2,342 genes were correlated with relapse-free survival (RFS), and 1,149 genes were correlated with overall survival (OS). 619 genes were statistically significant for both RFS and OS. The most significant genes were ANGPTL4 (p=4.25E-08, HR=2.02), NHP2 (p=5.98E-10, HR=1.93), STK3 (p=4.86E-10, HR=1.93), GBE1 (p=2.77E-09, HR=1.86), and PMVK (p=3.65E-09, HR=1.85) for RFS and PINK1 (p=1.64E-05 , HR=3.31), CAMK2N1 (p=1.06E-07 , HR=2.93), CACFD1 (p=4.79E-04 , HR=2.61), SCAP (p=3.29E-04 , HR=2.6), SDC1 (p=2.81E-04 , HR=2.57), for OS. The most significant gene ontology biological processes upregulated in tumors with a worse prognosis include GO:0000184, nuclear-transcribed mRNA catabolic process, nonsense-mediated decay (p=6.64E-18); GO:0045047 , protein targeting to ER (p=6.64E-18); GO:0006614, SRP-dependent cotranslational protein targeting to membrane (p=9.08E-18); GO:0072599, establishment of protein localization to endoplasmic reticulum (p=1.30E-17); and GO:0006613, cotranslational protein targeting to membrane (p=1.90E-17). Conclusions: Our results help to prioritize genes and to neglect those which are most likely to fail in studies aiming to establish new clinically useful biomarkers and therapeutic targets in BLBC. Citation Format: Balazs Gyorffy, Libero Santarpia. Uncovering Novel Potential Prognostic Biomarkers in Basal-Like Breast Cancer using Transcriptomic Data of 1,899 Patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-03-08.

Rules and impacts of nonsense-mediated mRNA decay in the degradation of long noncoding RNAs.

Nonsense-mediated mRNA decay (NMD) is a quality-control process that selectively degrades mRNAs having premature termination codon, upstream open reading frame, or unusually long 3'UTR. NMD detects such mRNAs and rapidly degrades them during initial rounds of translation in the eukaryotic cells. Since NMD is a translation-dependent cytoplasmic mRNA surveillance process, the noncoding RNAs were initially believed to be NMD-resistant. The sequence feature-based analysis has revealed that many putative long noncoding RNAs (lncRNAs) have short open reading frames, most of which have translation potential. Subsequent transcriptome-based molecular studies showed an association of a large set of such putative lncRNAs with translating ribosomes, and some of them produce stable and functionally active micropeptides. The translationally active lncRNAs typically have relatively longer and unprotected 3'UTR, which can induce their NMD-dependent degradation. This review defines the mechanism and regulation of NMD-dependent degradation of lncRNAs and its impact on biological processes related to the functions of lncRNAs or their encoded micropeptides. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability RNA in Disease and Development > RNA in Disease.

A c.726C>G (p.Tyr242Ter) nonsense mutation-associated with splicing alteration (NASA) of WDR45 gene underlies β-propeller protein-associated neurodegeneration (BPAN)

Neurodegeneration with brain iron accumulation (NBIA) is a clinically and genetically heterogeneous disease characterized by increased iron deposition in the basal ganglia and progressive degeneration of the nervous system in adulthood. However, in early childhood, there were no characteristic features to perform early diagnosis. In our study, a female child exhibited global developmental delay, intellectual disability, and febrile seizure without other distinct clinical phenotypes. Through whole exome sequencing (WES), a de novo nonsense mutation (c.726C > G, p. Tyr242Ter) of WDR45 gene was identified in this child. She was finally diagnosed as β‐propeller protein-associated neurodegeneration (BPAN), one of the recently identified subtypes of NBIA. This mutation could act as a premature stop codon (PSC) which rendered the mutated transcripts to be degraded by nonsense-mediated mRNA decay (NMD), leading to decreased levels of PSC-containing mRNAs. Additionally, through mini-gene splicing assays, this mutation could result in an unprecedented novel transcript with the exon 9 of WDR45 excluded by nonsense-associated splicing alteration (NASA). Transcriptome sequencing (RNA-seq) on total RNAs from PBMCs of the trio revealed three types of alternative splicing events in the patient. Further research implied that downregulation of iron transport genes (TFRC, TFR2, SCARA5) might be the underlying mechanism for the iron accumulation in patients with deficient WDR45. This is the first report about NASA happening in WDR45. It implies that nonsense mutations approximal to splicing sites could affect the disease pathogenesis through more than one molecular mechanism and should be taken into consideration when conducting genetic counseling.

Readthrough-induced misincorporated amino acid ratios guide mutant-specific therapeutic approaches for two CFTR nonsense mutations.

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature termination codons (PTCs) represent ∼9% of CF mutations that typically cause severe expression defects of the CFTR anion channel. Despite the prevalence of PTCs as the underlying cause of genetic diseases, understanding the therapeutic susceptibilities of their molecular defects, both at the transcript and protein levels remains partially elucidated. Given that the molecular pathologies depend on the PTC positions in CF, multiple pharmacological interventions are required to suppress the accelerated nonsense-mediated mRNA decay (NMD), to correct the CFTR conformational defect caused by misincorporated amino acids, and to enhance the inefficient stop codon readthrough. The G418-induced readthrough outcome was previously investigated only in reporter models that mimic the impact of the local sequence context on PTC mutations in CFTR. To identify the misincorporated amino acids and their ratios for PTCs in the context of full-length CFTR readthrough, we developed an affinity purification (AP)-tandem mass spectrometry (AP-MS/MS) pipeline. We confirmed the incorporation of Cys, Arg, and Trp residues at the UGA stop codons of G542X, R1162X, and S1196X in CFTR. Notably, we observed that the Cys and Arg incorporation was favored over that of Trp into these CFTR PTCs, suggesting that the transcript sequence beyond the proximity of PTCs and/or other factors can impact the amino acid incorporation and full-length CFTR functional expression. Additionally, establishing the misincorporated amino acid ratios in the readthrough CFTR PTCs aided in maximizing the functional rescue efficiency of PTCs by optimizing CFTR modulator combinations. Collectively, our findings contribute to the understanding of molecular defects underlying various CFTR nonsense mutations and provide a foundation to refine mutation-dependent therapeutic strategies for various CF-causing nonsense mutations.