Browse
Submit Data
Databases
API
Help

Dataset Information

14 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

AUG sequences are required to sustain nonsense-codon-mediated suppression of splicing.

ABSTRACT: More than 90% of human genes are rich in intronic latent 5' splice sites whose utilization in pre-mRNA splicing would introduce in-frame stop codons into the resultant mRNAs. We have therefore hypothesized that suppression of splicing (SOS) at latent 5' splice sites regulates alternative 5' splice site selection in a way that prevents the production of toxic nonsense mRNAs and verified this idea by showing that the removal of such in-frame stop codons is sufficient to activate latent splicing. Splicing control by SOS requires recognition of the mRNA reading frame, presumably recognizing the start codon sequence. Here we show that AUG sequences are indeed essential for SOS. Although protein translation does not seem to be required for SOS, the first AUG is shown here to be necessary but not sufficient. We further show that latent splicing can be elicited upon treatment with pactamycin-a drug known to block translation by its ability to recognize an RNA fold-but not by treatment with other drugs that inhibit translation through other mechanisms. The effect of pactamycin on SOS is dependent neither on steady-state translation nor on the pioneer round of translation. This effect is found for both transfected and endogenous genes, indicating that SOS is a natural mechanism.

SUBMITTER: Kamhi E

PROVIDER: S-EPMC1524910 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Attenuation of nonsense-mediated mRNA decay enhances in vivo nonsense suppression.

Project description:Nonsense suppression therapy is an approach to treat genetic diseases caused by nonsense mutations. This therapeutic strategy pharmacologically suppresses translation termination at Premature Termination Codons (PTCs) in order to restore expression of functional protein. However, the process of Nonsense-Mediated mRNA Decay (NMD), which reduces the abundance of mRNAs containing PTCs, frequently limits this approach. Here, we used a mouse model of the lysosomal storage disease mucopolysaccharidosis I-Hurler (MPS I-H) that carries a PTC in the Idua locus to test whether NMD attenuation can enhance PTC suppression in vivo. Idua encodes alpha-L-iduronidase, an enzyme required for degradation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. We found that the NMD attenuator NMDI-1 increased the abundance of the PTC-containing Idua transcript. Furthermore, co-administration of NMDI-1 with the PTC suppression drug gentamicin enhanced alpha-L-iduronidase activity compared to gentamicin alone, leading to a greater reduction of GAG storage in mouse tissues, including the brain. These results demonstrate that NMD attenuation significantly enhances suppression therapy in vivo.

| S-EPMC3622682 | biostudies-literature

Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity.

Project description:Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). The level of sensitivity of a PTC-containing mRNA to NMD is multifactorial. We have previously shown that human β-globin mRNAs carrying PTCs in close proximity to the translation initiation AUG codon escape NMD. This was called the 'AUG-proximity effect'. The present analysis of nonsense codons in the human α-globin mRNA illustrates that the determinants of the AUG-proximity effect are in fact quite complex, reflecting the ability of the ribosome to re-initiate translation 3' to the PTC and the specific sequence and secondary structure of the translated ORF. These data support a model in which the time taken to translate the short ORF, impacted by distance, sequence, and structure, not only modulates translation re-initiation, but also impacts on the exact boundary of AUG-proximity protection from NMD.

| S-EPMC4513866 | biostudies-literature

Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression.

Project description:High-throughput screening (HTS) assays used in drug discovery frequently use reporter enzymes such as firefly luciferase (FLuc) as indicators of target activity. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay signal modulation. In rare cases, this activity appears counterintuitive; for example, some FLuc inhibitors, acting through posttranslational Fluc reporter stabilization, appear to activate gene expression. Previous efforts to characterize molecules that influence luciferase activity identified a subset of 3,5-diaryl-oxadiazole-containing compounds as FLuc inhibitors. Here, we evaluate a number of compounds with this structural motif for activity against FLuc. One such compound is PTC124 {3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid}, a molecule originally identified in a cell-based FLuc assay as having nonsense codon suppression activity [Welch EM, et al., Nature (2007) 447:87-91]. We find that the potency of FLuc inhibition for the tested compounds strictly correlates with their activity in a FLuc reporter cell-based nonsense codon assay, with PTC124 emerging as the most potent FLuc inhibitor (IC(50) = 7 +/- 1 nM). However, these compounds, including PTC124, fail to show nonsense codon suppression activity when Renilla reniformis luciferase (RLuc) is used as a reporter and are inactive against the RLuc enzyme. This suggests that the initial discovery of PTC124 may have been biased by its direct effect on the FLuc reporter, implicating firefly luciferase as a molecular target of PTC124. Our results demonstrate the value of understanding potential interactions between reporter enzymes and chemical compounds and emphasize the importance of implementing the appropriate control assays before interpreting HTS results.

| S-EPMC2638738 | biostudies-literature

Smg1 is required for embryogenesis and regulates diverse genes via alternative splicing coupled to nonsense-mediated mRNA decay.

Project description:Smg1 is a PI3K-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbor premature termination codons (PTCs) as a result of events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. By using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5. High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from approximately 9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signaling, membrane dynamics, cell death, and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.

| S-EPMC2901484 | biostudies-literature

Combining nonsense mutation suppression therapy with nonsense-mediated decay inhibition in neurofibromatosis type 1.

Project description:Neurofibromatosis type 1 (NF1) results from germline mutations in the tumor-suppressor gene NF1 and predisposes patients to developing nervous system tumors. Twenty percent of NF1 patients harbor nonsense mutations resulting in premature termination codons (PTCs). Nonsense suppression therapies can facilitate ribosomal readthrough of PTCs to restore full-length protein, but their potential in NF1 is underexplored. We developed a minipig model of NF1 carrying a PTC to test whether nonsense suppression could restore expression of the NF1-encoded protein neurofibromin in vitro and in vivo. Nonsense suppression did not reliably increase neurofibromin in primary NF1-/- Schwann cells isolated from minipig neurofibromas but could reduce phosphorylated ERK. Gentamicin in vivo produced a similar plasma pharmacokinetic profile to humans and was detectable in clinically relevant tissues, including cerebral cortex, sciatic nerve, optic nerve, and skin. In gentamicin-treated animals, increased neurofibromin expression was seen in the optic nerve. Nonsense-mediated decay (NMD) causes degradation of transcripts with PTCs, which could impede nonsense suppression therapies. Nonsense suppression in combination with NMD inhibition restored neurofibromin protein expression in primary NF1-/- Schwann cells isolated from minipig neurofibromas. Thus, the effectiveness of nonsense suppression therapies can be improved in NF1 by the concurrent use of NMD inhibitors.

| S-EPMC10384610 | biostudies-literature

Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression.

Project description:The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression.

| S-EPMC4811451 | biostudies-literature

Nonsense suppression by near-cognate tRNAs employs alternative base pairing at codon positions 1 and 3.

Project description:Premature termination codons (PTCs) in an mRNA ORF inactivate gene function by causing production of a truncated protein and destabilization of the mRNA. Readthrough of a PTC allows ribosomal A-site insertion of a near-cognate tRNA, leading to synthesis of a full-length protein from otherwise defective mRNA. To understand the mechanism of such nonsense suppression, we developed a yeast system that allows purification and sequence analysis of full-length readthrough products arising as a consequence of endogenous readthrough or the compromised termination fidelity attributable to the loss of Upf (up-frameshift) factors, defective release factors, or the presence of the aminoglycoside gentamicin. Unlike classical "wobble" models, our analyses showed that three of four possible near-cognate tRNAs could mispair at position 1 or 3 of nonsense codons and that, irrespective of whether readthrough is endogenous or induced, the same sets of amino acids are inserted. We identified the insertion of Gln, Tyr, and Lys at UAA and UAG, whereas Trp, Arg, and Cys were inserted at UGA, and the frequency of insertion of individual amino acids was distinct for specific nonsense codons and readthrough-inducing agents. Our analysis suggests that the use of genetic or chemical means to increase readthrough does not promote novel or alternative mispairing events; rather, readthrough effectors cause quantitative enhancement of endogenous mistranslation events. Knowledge of the amino acids incorporated during readthrough not only elucidates the decoding process but also may allow predictions of the functionality of readthrough protein products.

| S-EPMC4364220 | biostudies-literature

Mechanism of Nonsense-Mediated mRNA Decay Stimulation by Splicing Factor SRSF1.

Project description:The splicing factor SRSF1 promotes nonsense-mediated mRNA decay (NMD), a quality control mechanism that degrades mRNAs with premature termination codons (PTCs). Here we show that transcript-bound SRSF1 increases the binding of NMD factor UPF1 to mRNAs while in, or associated with, the nucleus, bypassing UPF2 recruitment and promoting NMD. SRSF1 promotes NMD when positioned downstream of a PTC, which resembles the mode of action of exon junction complex (EJC) and NMD factors. Moreover, splicing and/or EJC deposition increase the effect of SRSF1 on NMD. Lastly, SRSF1 enhances NMD of PTC-containing endogenous transcripts that result from various events. Our findings reveal an alternative mechanism for UPF1 recruitment, uncovering an additional connection between splicing and NMD. SRSF1's role in the mRNA's journey from splicing to decay has broad implications for gene expression regulation and genetic diseases.

| S-EPMC5999336 | biostudies-literature

ICE1 promotes the link between splicing and nonsense-mediated mRNA decay.

Project description:The nonsense-mediated mRNA decay (NMD) pathway detects aberrant transcripts containing premature termination codons (PTCs) and regulates expression of 5-10% of non-aberrant human mRNAs. To date, most proteins involved in NMD have been identified by genetic screens in model organisms; however, the increased complexity of gene expression regulation in human cells suggests that additional proteins may participate in the human NMD pathway. To identify proteins required for NMD, we performed a genome-wide RNAi screen against >21,000 genes. Canonical members of the NMD pathway were highly enriched as top hits in the siRNA screen, along with numerous candidate NMD factors, including the conserved ICE1/KIAA0947 protein. RNAseq studies reveal that depletion of ICE1 globally enhances accumulation and stability of NMD-target mRNAs. Further, our data suggest that ICE1 uses a putative MIF4G domain to interact with exon junction complex (EJC) proteins and promotes the association of the NMD protein UPF3B with the EJC.

| S-EPMC5896957 | biostudies-literature

Microbiota-dependent signals are required to sustain TLR-mediated immune responses.

Project description:Host-commensal interactions are critical for the generation of robust inflammatory responses, yet the mechanisms leading to this effect remain poorly understood. Using a murine model of cytokine storm, we identified that host microbiota are required to sustain systemic TLR-driven immune responses. Mice treated with broad-spectrum antibiotics or raised in germ-free conditions responded normally to an initial TLR signal but failed to sustain production of proinflammatory cytokines following administration of repeated TLR signals in vivo. Mechanistically, host microbiota primed JAK signaling in myeloid progenitors to promote TLR-enhanced myelopoiesis, which is required for the accumulation of TLR-responsive monocytes. In the absence of TLR-enhanced monocytopoiesis, antibiotic-treated mice lost their ability to respond to repeated TLR stimuli and were protected from cytokine storm-induced immunopathology. These data reveal priming of TLR-enhanced myelopoiesis as a microbiota-dependent mechanism that regulates systemic inflammatory responses and highlight a role for host commensals in the pathogenesis of cytokine storm syndromes.

| S-EPMC6485364 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data