Normal synthesis, transport and decay of mRNA in the absence of its translation.
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Adenosine↗
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Results for “mRNA decay”
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Molecular Determinants and Therapeutic Targeting of Stop Codon Readthrough in Eukaryotic Translation.
Accurate translation termination is essential for proteome integrity and in eukaryotes is primarily governed by the release factors eRF1 and eRF3, which ensure precise recognition of stop codons and efficient release of nascent polypeptides. However, proteome integrity is challenged by mutations that generate premature termination codons (PTCs), leading to truncated, nonfunctional proteins and degradation of the aberrant transcript via nonsense-mediated mRNA decay (NMD). Collectively, these events account for ∼1800 human genetic diseases. Translational readthrough, the process by which near-cognate tRNAs decode stop codons and allow ribosomes to continue elongation beyond the stop codon, represents a possibility to suppress PTCs and restore full-length protein synthesis. Initially discovered in viruses as a mechanism to expand coding capacity, readthrough is now recognized as a regulated feature of eukaryotic gene expression influenced by both cis-acting sequence elements and trans-acting factors. Recent evidence highlights the remarkable context dependence of readthrough, revealing variation across transcripts, tissues, and developmental stages. In this review, we examine the molecular determinants that define stop codon recognition and readthrough efficiency, with particular emphasis on nucleotide context. We further discuss the mechanisms and binding sites of small molecules that promote PTC readthrough, and summarize the clinical development landscape of readthrough-inducing compounds for the treatment of diseases caused by nonsense mutations.
Humans↗
SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.
Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.
Humans↗
YIPFα1A expression is regulated by multilayered molecular mechanisms.
Yip domain family (YIPF) proteins are five-pass transmembrane proteins that localize primarily to the Golgi apparatus. These proteins assemble into higher-order complexes with each α-subunit pairing specifically with a β-subunit to form a dimer which then assemble into complexes with two to four dimers. Notably, β-subunit expression depends on the corresponding α-subunit partner, and conventional transient overexpression of α-subunits has been extremely inefficient, hindering deeper analysis of YIPF complexes. To identify the cause of poor exogenous expression, we examined YIPF gene features and found two properties correlated with low expression: (i) rare-codon enrichment in the CDS and (ii) extended 3' UTRs. Experimental analyses focusing on YIPFα1A revealed that rare-codon enrichment suppresses expression mainly at the mRNA level, consistent with translation-coupled mRNA decay, whereas inclusion of the native 3' UTR enhances expression by increasing mRNA abundance. Deletion mapping further showed that a proximal 3' UTR segment (51-150) is necessary and sufficient for mRNA stabilization, thereby elevating both mRNA and protein levels. Conversely, a distal 3' UTR fragment (1116-2230) increased mRNA but not protein levels, suggesting translational repression resulting in a reduced protein-to-mRNA ratio. Together, these findings explain the discrepancy between endogenous and exogenous YIPFα1A expression and propose a multilayered regulatory model in which rare codons decrease mRNA, the proximal 3' UTR stabilizes mRNA, and the distal 3' UTR reduces translation. Impact statement Our work advances YIPF biology and identifies post‑transcriptional mechanisms governing multi‑pass membrane proteins. We show rare‑codon and 3' UTR‑based control of trafficking proteins-an area largely unexplored-and introduce a new paradigm for membrane‑traffic regulation that will guide future studies of complex assembly, localization, and homeostasis.
3' Untranslated Regions↗
Long-range mRNA folding shapes expression and sequence of bacterial genes.
Bacterial gene expression is strongly influenced by local mRNA secondary structure, yet the impact of long-range folding remains poorly understood. Here, we show that sequences hundreds of nucleotides from the mRNA 5' end can act as potent repressors of gene expression through long-range base pairing to the ribosome binding site (RBS), subjecting anti-RBS sequences to negative selection. Using massively parallel reporter assays in Bacillus subtilis, we identify anti-RBS sequences as among the strongest determinants of reduced mRNA abundance across the transcript body. We demonstrate that distal anti-RBS elements engage in long-range folding with the Shine-Dalgarno sequence, blocking ribosome entry and promoting mRNA decay. Consistent with these repressive effects, anti-RBS-like sequences are depleted throughout diverse bacterial coding sequences but not from leaderless transcripts, and introducing distal anti-RBS to native genes reduces expression. Our findings establish that long-range mRNA folding is a conserved force shaping gene expression and constrains coding sequence evolution.
Bacillus subtilis↗
Differential stability of mRNAs coding for alpha and gonadotropin beta subunits in cultured rat pituitary cells.
Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) result from the assembly of a common subunit alpha and a unique subunit beta, expressed in the same cell by single, structurally-related genes. In order to compare the intrinsic stability of the alpha, LH beta and FSH beta mRNA transcripts, we used cultured rat pituitary cells incubated in presence of actinomycin D. Hybridization with 32P-labelled rat cDNA probes showed that the cell content of all three mRNAs decreased with time, but at different rates. Apparent half-lives, estimated as the time necessary to observe a 50% mRNA decay, were 1.0 +/- 0.13 h for FSH beta, 6.5 +/- 0.25 h for alpha and 44 +/- 0.5 h for LH beta, stability thus exhibiting an inverse relation to the sizes of the corresponding mRNAs (approximately 1700, 800 and 700 nucleotides, respectively). Northern analysis revealed that the decline in mRNA abundance was associated with a progressive decrease in the length of mRNAs, most clearly visible for alpha and LH beta. For the most stable LH beta mRNA, shortening was apparent as early as 2 h after exposure to actinomycin D thus preceding neatly the decrease in amount starting at about 10-12 h. In vitro RNase H digestion demonstrated that shortening resulted from a reduction of the length of the poly(A) tract. These data establish that the three mRNAs coding for gonadotropin subunits have different stabilities although they share substantial homology. Diversity in size and sequence essentially resides in untranslated regions in which, we suggest, specific motifs and protein factors may interact to determine mRNA stability.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Proximity labeling puts ZFP36L1 as central hub for posttranscriptional regulation networks in T cells.
Effective T cell responses against pathogens require a rapid yet tightly controlled remodeling of the proteome, and RNA binding proteins (RBPs) are key in this process. For instance, the RBP ZFP36L1 prevents excessive protein production and thereby limits immunopathology. ZFP36L1 is primarily known to mediate mRNA decay, but it can also regulate other processes. How its mode of action relates to its interaction partners is, however, not well-understood. Here, we mapped the ZFP36L1 interactome in primary human T cells. Using proximity labeling, we identified known and new interactors that regulate 3'UTR-mediated RNA degradation, deadenylation, stress granule/p-body formation, as well as 5'UTR-mediated translation repression and mRNA decapping. Snapshot analysis uncovered the ZFP36L1 interactome dynamics and RNA (in)dependency throughout T cell activation. Intriguingly, proximity labeling also uncovered regulators of ZFP36L1 protein expression. This included the helicase UPF1, which not only interacts with ZFP36L1 protein but that may also promote its protein expression. Altogether, this comprehensive interactome map underlines the versatility of interactions with ZFP36L1 and their possible role in cellular function.
Humans↗
Discordant neoplasms in monozygotic twins with a germline RECQL5 variant.
RECQL5 is a member of the RecQ helicase family involved in DNA replication, homologous recombination, and maintenance of genomic stability. While germline pathogenic variants in other RecQ helicases cause established cancer predisposition syndromes, the role of RECQL5 in human cancer susceptibility remains uncertain. We report monozygotic adolescent twins with distinct tumors: dysembryoplastic neuroepithelial tumor in one twin and Burkitt lymphoma in the other. Clinical genome sequencing was initially nondiagnostic, but reanalysis identified a rare heterozygous nonsense variant in RECQL5 (NM_004259.7:c.2698C>T, p.(Gln900Ter)), present in both twins and their unaffected mother. The variant is predicted to undergo nonsense-mediated mRNA decay or produce a truncated protein lacking the C-terminal SRI (Set2-Rpb1 interacting) domain, which mediates interaction with RNA polymerase II. However, tumor sequencing data were not available to evaluate loss of heterozygosity or second somatic events. Given the unaffected carrier parent, lack of tumor molecular confirmation, and the biological heterogeneity of the tumors, a causal relationship for this variant cannot be established. This case highlights the challenges of interpreting rare germline variants in genes with emerging but incompletely characterized disease associations. Although the available evidence is insufficient to establish a definitive causal relationship, the identification of a shared loss-of-function RECQL5 variant in monozygotic twins with distinct tumors is noteworthy and adds to the limited clinical evidence suggesting a potential role for RECQL5 in cancer susceptibility. Additional functional studies, tumor-based analyses and the accumulation of well-characterized clinical cases will be essential to determine whether RECQL5 contributes to hereditary cancer predisposition.
Adolescent↗
A synonymous NPR2 variant causes acromesomelic dysplasia through aberrant pre-mRNA splicing.
Precise regulation of pre-mRNA splicing is essential for normal development, and its disruption represents an important but frequently underrecognized mechanism of human disease. The C-type natriuretic peptide (CNP) receptor NPR2 is a critical regulator of growth plate chondrocyte proliferation and differentiation, and loss-of-function variants in NPR2 cause acromesomelic dysplasia, Maroteaux type (AMDM). Here, we identify a homozygous synonymous NPR2 variant (NM_003995.4:c.2484C > T) in an individual with AMDM and demonstrate its pathogenic mechanism at the RNA level. Although predicted to be silent at the protein level, in silico analysis suggested splice donor gain. Functional analysis using patient-derived leukocyte RNA revealed aberrant splicing leading to partial exon truncation, frameshift, and premature termination of NPR2 which is predicted to trigger nonsense-mediated mRNA decay given its position upstream of multiple downstream exon-exon junctions. Heterozygous family members expressed both normal and aberrant transcripts, whereas the affected individual showed exclusive expression of the aberrant isoform, consistent with a dosage-dependent loss-of-function mechanism. These findings establish aberrant splicing induced by a synonymous variant as a disease-causing mechanism affecting a core developmental signaling pathway. Our study highlights the importance of transcript-level functional analysis in the interpretation of rare variants and underscores the central role of precise RNA processing in skeletal development and human disease.
Humans↗
Control of RNase E-mediated RNA degradation by 5'-terminal base pairing in E. coli.
Despite the variety of messenger RNA half-lives in bacteria (0.5-30 min in Escherichia coli) and their importance in controlling gene expression, their molecular basis remains obscure. The lifetime of an entire mRNA molecule can be determined by features near its 5' end, but no 5' exoribonuclease has been identified in any prokaryotic organism. A mutation that inactivates E. coli RNase E also increases the average lifetime of bulk E. coli mRNA and of many individual messages, suggesting that cleavage by this endonuclease may be the rate-determining step in the degradation of most mRNAs in E. coli. We have investigated the substrate preference of RNase E in E. coli by using variants of RNA I, a small untranslated RNA whose swift degradation in vivo is initiated by RNase E cleavage at an internal site. We report here that RNase E has an unprecedented substrate specificity for an endoribonuclease, as it preferentially cleaves RNAs that have several unpaired nucleotides at the 5' end. The sensitivity of RNase E to 5'-terminal base pairing may explain how determinants near the 5' end can control rates of mRNA decay in bacteria.
Bacterial Outer Membrane Proteins↗
A Novel Homozygous Mutation in ARL2BP Causes Multiple Morphological Abnormalities of the Flagella and Primary Ciliary Dyskinesia.
Primary ciliary dyskinesia (PCD) and multiple morphological abnormalities of the sperm flagella (MMAF) frequently co-occur in male infertility. However, the genetic basis of this syndromic presentation remains unclear. Using whole-exome sequencing, we identified a novel homozygous ARL2BP splice-site mutation (c.294-2A>G) in a 23-year-old infertile male from a consanguineous family who presented with syndromic PCD and MMAF. This variant causes aberrant pre-mRNA splicing and triggers nonsense-mediated mRNA decay, resulting in the complete absence of ARL2BP protein expression. Transmission electron microscopy revealed extensive disorganization of flagellar axonemes with consistent central pair (CP) microtubule depletion and disorganization of peripheral doublets. Immunofluorescence confirmed a severe deficiency of the CP protein SPAG6 in the sperm flagella. Notably, the patient presented without retinal symptoms. Given that ARL2BP-related retinitis pigmentosa generally emerges during the third decade, long-term ophthalmological follow-up is essential to detect delayed-onset retinal degeneration. In conclusion, these findings confirm ARL2BP as a causative gene for both PCD and MMAF, expanding the genotypic and phenotypic spectrum of ciliopathies.
Humans↗
A novel frameshift variant leads to familial osteopetrosis with variable phenotypes in a Chinese Han consanguineous family.
Osteopetrosis, a group of highly heterogeneous genetic bone disorders, is characterized by deafness, increased bone density, hepatosplenomegaly, pancytopenia and intellectual disability. Osteopetrosis can be divided into three subtypes: autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IARO), and autosomal dominant osteopetrosis (ADO). CLCN7 has been reported to be the most common gene responsible for the ADO-II subtype. In this study, a novel variant, c.175dupA (p.Met59Asnfs*8), of CLCN7 was identified in a Chinese Han consanguineous family with suspected ADO-II. The proband was homozygous for the p.Met59Asnfs*8 variant and exhibited multiple severe phenotypes, including deafness, short stature, brittle bones, optic atrophy, hepatosplenomegaly, intellectual disability, cleft palate and recurrent infection. However, except for the mother of the proband, who presented a series of clinical phenotypes caused by bone marrow failure, all the other family members who were heterozygous had no obvious abnormal phenotypes. Our study suggested that the novel variant p.Met59Asnfs*8 in CLCN7 was very likely pathogenic factor in our suspected ADO-II family. The phenotypes of heterozygous carriers may be affected by incomplete penetrance. Loss of function of CLCN7 caused by nonsense-mediated mRNA decay (NMD) due to the frameshift variant was likely the underlying pathogenic mechanism. This study broadened the mutation spectrum of CLCN7, provided a foundation for timely and effective clinical intervention for related diseases, and demonstrates the importance of genetic counselling.
Adult↗
Clinical and molecular characterization of TCF12 variants in an Asian pediatric cohort with craniosynostosis.
BACKGROUND: Craniosynostosis is a genetically heterogeneous craniofacial disorder caused by the premature fusion of one or more cranial sutures. Pathogenic variants in TCF12, encoding a basic helix-loop-helix (bHLH) transcription factor, represent a major cause of autosomal dominant coronal craniosynostosis and are characterized by incomplete penetrance and marked phenotypic variability. However, clinical and molecular data from Asian pediatric populations remain limited. METHODS: Trio-based whole-exome sequencing was performed on ten pediatric patients with cranial deformities and their parents. The identified TCF12 variants were classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines and validated by Sanger sequencing. Detailed clinical and radiological data were collected. In addition, a comprehensive literature review was conducted to summarize previously reported TCF12 variants and associated phenotypes. RESULTS: Ten distinct heterozygous TCF12 variants were identified in ten unrelated pediatric patients, all of which were classified as pathogenic or likely pathogenic according to ACMG criteria. Six variants were inherited, and four occurred de novo. Seven patients had imaging-confirmed craniosynostosis, predominantly involving the coronal sutures (five bilateral and one unilateral), while one patient presented with multisuture craniosynostosis (left coronal and sagittal sutures). Three patients showed cranial deformities without radiographic evidence of suture fusion. Phenotypic heterogeneity and incomplete penetrance were observed, including a mildly affected parent. Most pathogenic variants were truncating variants distributed mainly across exons 14-19 and predicted to induce loss of function, either through nonsense-mediated mRNA decay or the production of truncated proteins lacking the entire C-terminal bHLH domain. Structural modeling analysis further indicated that the bHLH-domain-located missense variant p.Arg603Trp alters the local DNA-binding conformation of TCF12 and impairs its binding affinity to the E-box DNA motif. CONCLUSIONS: This study provides additional clinical and molecular data on TCF12-related craniosynostosis in a pediatric cohort from an Asian population. Our findings support haploinsufficiency as the central pathogenic mechanism, primarily driven by truncating variants affecting the C-terminal bHLH domain. The marked clinical heterogeneity, the presence of mild or evolving phenotypes, and incomplete penetrance observed in our cohort underscore the importance of early diagnosis and longitudinal clinical surveillance in affected families.
Humans↗
hnRNPC facilitates coronavirus replication by directly binding the frameshift-stimulatory element of viral genomic RNA.
Translation of key viral replicative proteins in coronaviruses requires a programmed -1 ribosomal frameshifting (-1 PRF) event controlled by the viral frameshift-stimulatory element (FSE). Although previous studies have analyzed host factor dependencies of coronaviruses, how host cellular factors alter -1 PRF efficiency and affect viral replication remains poorly understood. Here, using RNA pull-down combined with LC-MS/MS analysis, we identified heterogeneous nuclear ribonucleoprotein C (hnRNPC) as a major interacting protein of FSE RNA. Coronavirus infection triggers hnRNPC mRNA decay, alters hnRNPC protein levels, and induces its cytoplasmic relocalization, where it appears to bind directly to FSE RNA through residues Asn7 and Asn83. This binding is associated with increased -1 PRF efficiency and may facilitate coronavirus replication. Deletion mapping analysis shows that hnRNPC preferentially binds U-rich regions of the FSE RNA. Finally, we demonstrated that the small molecule Elbasvir directly binds hnRNPC, disrupting the interaction between hnRNPC and FSE RNA and inhibiting coronavirus replication by decreasing -1 PRF efficiency. Collectively, our study identifies hnRNPC as a key host cofactor for coronaviruses and provides a novel target for broad-spectrum antiviral drug development.
RNA, Viral↗
Identification of a novel and a recurrent CDC45 variant in a Chinese family with Meier-Gorlin syndrome 7 and a literature review.
INTRODUCTION: Meier-Gorlin syndrome 7 (MGORS7) is a rare autosomal recessive disorder characterized by primordial dwarfism, craniosynostosis, and patellar aplasia, caused by pathogenic variants of CDC45. Here, we report a Chinese patient presenting with classic hallmarks of MGORS7 alongside atypical clinical features, including hearing and visual impairments. METHODS: Clinical and radiological data were collected. Whole-genome sequencing and Sanger sequencing were performed to identify and validate the causative variants. Their functional effects were investigated using an exon-trapping assay, and a literature review of previously reported MGORS7 cases was conducted. RESULTS: Genetic analysis identified two compound heterozygous CDC45 variants: c.1416C>T (p.H472=) and c.1559+2T>A, which are a recurrent variant in the East Asian population and a novel variant, respectively. Our exon-trapping assay indicated that c.1559+2T>A induced aberrant splicing, generating transcripts predicted to undergo nonsense-mediated mRNA decay. Additionally, growth hormone therapy was initiated in our patient, with a noted improvement in growth parameters in the initial assessment and without immediate complications. The literature review identified a total of 32 CDC45 variants in 29 patients with MGORS7, who showed high heterogeneity in clinical phenotypes. DISCUSSION: Our study further expanded the mutational spectrum of CDC45 and provided a preliminary clinical observation suggesting that growth hormone therapy may be beneficial for growth retardation in patients with MGORS7.
CDC45↗
Expanding the Genomic Spectrum of NHLRC2-Associated FINCA Disease: Integrated Bioinformatic Characterization of a Novel Deep Intronic Variant Predicted to Activate a Pseudoexon.
NHLRC2-associated FINCA disease is an ultra-rare autosomal recessive multisystem disorder caused by biallelic pathogenic variants in NHLRC2. Its mutational spectrum and genotype-phenotype correlations remain incompletely defined, and the contribution of non-coding variants is poorly understood. Here, we report a male infant with a severe FINCA-like phenotype, including early-onset hemolytic anemia, pulmonary involvement, neurodevelopmental impairment, growth failure, recurrent infections, and fatal progression at 8.5 months. Whole-genome sequencing identified a compound heterozygous NHLRC2 genotype comprising the previously reported pathogenic missense variant c.442G>T (p.Asp148Tyr) and a novel deep intronic variant, c.331+6863A>G. Segregation analysis confirmed inheritance from different parents. Integrated genomic and splicing analysis predicted that c.331+6863A>G creates a strong cryptic donor splice site and supports pseudoexon inclusion. Reconstruction of the predicted aberrant transcript indicated premature termination and potential susceptibility to nonsense-mediated mRNA decay. To our knowledge, this is the first reported deep intronic NHLRC2 variant predicted to activate pseudoexon inclusion. Although experimental validation was unavailable, convergent clinical, segregation, population, and computational evidence supports c.331+6863A>G as the most plausible second disease-associated allele. This case expands the genomic spectrum of NHLRC2-associated FINCA disease and highlights the diagnostic value of phenotype-driven whole-genome sequencing.
Humans↗
[The origin of mitochondrial RNA in animal tissues. 4. Post-transcriptional RNA modification and polyribosome formation in isolated mitochondria].
The kinetics of synthesis of various mitochondrial RNA classes and their contribution to polyribosome formation was studied in isolated rat liver mitochondria and in inner membrane fraction obrained from the latter. In pulse labelling experiments it was shown that mitochondrial mRNA synthesis occurred via heavy precursor (30S) not bound to polysomes followed by maturation of polysome-bound mRNA. The time course of polysomal mRNA decay in isolated mitochondria and of protein synthesis inactivation were studied under actinomycin D blockage of mitochondrial DNA trascription. The functional organization of transcriptional units of the mitochondrial genome is discussed in relation to the data obtained.
Animals↗
Stability of alpha and beta globin messenger RNA during induced differentiation of mouse erythroleukemia cells.
Murine erythroleukemia cells (MELC) are induced to express erythroid differentiation when cultured with hexamethylene bisacetamide (HMBA). Newly synthesized alpha and beta globin mRNA are both relatively stable, half-life (t1/2) greater than 50 hr, early in the course of induced differentiation. In fully induced cells there is a decrease in stability of both newly synthesized alpha and beta globin mRNA. The decay of alpha mRNA is faster, (t 1/2, 10--12 hr) than beta globin mRNA (t1/2, 20--22 hr). Thus, differences in stability of alpha and beta globin mRNA plays a role in determining the ratio of alpha to beta mRNA content in differentiated erythroid cells.
Acetamides↗