scholarly journals Reversion of the Arabidopsis rpn12a-1 exon-trap mutation by an intragenic suppressor that weakens the chimeric 5’ splice site

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 60 ◽  
Author(s):  
Jasmina Kurepa ◽  
Yan Li ◽  
Jan A Smalle
Keyword(s):  
Splice Site ◽  
Plant Hormone ◽  
Stop Codon ◽  
Proteasome Activity ◽  
26S Proteasome ◽  
Critical Threshold ◽  
Wild Type ◽  
Base Pairs ◽  
Previous Conclusion ◽  
Proteasome Subunits

Background: In the Arabidopsis 26S proteasome mutant rpn12a-1, an exon-trap T-DNA is inserted 531 base pairs downstream of the RPN12a STOP codon. We have previously shown that this insertion activates a STOP codon-associated latent 5' splice site that competes with the polyadenylation signal during processing of the pre-mRNA. As a result of this dual input from splicing and polyadenylation in the rpn12a-1 mutant, two RPN12a transcripts are produced and they encode the wild-type RPN12a and a chimeric RPN12a-NPTII protein. Both proteins form complexes with other proteasome subunits leading to the formation of wild-type and mutant proteasome versions. The net result of this heterogeneity of proteasome particles is a reduction of total cellular proteasome activity. One of the consequences of reduced proteasomal activity is decreased sensitivity to the major plant hormone cytokinin.Methods: We performed ethyl methanesulfonate mutagenesis of rpn12a-1 and isolated revertants with wild-type cytokinin sensitivity.Results: We describe the isolation and analyses of suppressor of rpn12a-1 (sor1). The sor1 mutation is intragenic and located at the fifth position of the chimeric intron. This mutation weakens the activated 5' splice site associated with the STOP codon and tilts the processing of the RPN12a mRNA back towards polyadenylation.Conclusions: These results validate our earlier interpretation of the unusual nature of the rpn12a-1 mutation. Furthermore, the data show that optimal 26S proteasome activity requires RPN12a accumulation beyond a critical threshold. Finally, this finding reinforces our previous conclusion that proteasome function is critical for the cytokinin-dependent regulation of plant growth.

scholarly journals Multi-omic analysis reveals the biochemical changes underpinning the varied phenotypes of the Arabidopsis long-period mutant rve 4 6 8

2021 ◽  
Author(s):  
Sabine Scandola ◽  
Devang Mehta ◽  
Maria C Rodriguez ◽  
Qiaomu Li ◽  
Richard Glen Uhrig
Keyword(s):  
Plant Cell ◽  
Protein Function ◽  
Cell Function ◽  
26S Proteasome ◽  
Circadian Oscillation ◽  
Wild Type ◽  
Leaf Surface Area ◽  
Proteasome Subunits ◽  
Cell Processes ◽  
Delayed Flowering

Plants are able to sense changes in their light environments, such as the onset of day and night, as well as anticipate these changes in order to adapt and survive. Central to this ability is the plant circadian clock, a molecular circuit that precisely orchestrates plant cell processes over the course of a day. REVEILLE proteins (RVEs) are recently discovered members of the plant circadian circuitry that activate the evening complex and PRR genes to maintain regular circadian oscillation. The RVE 8 protein and its two homologs, RVE 4 and 6, have been shown to limit the length of the circadian period, with rve 4 6 8 triple-knockout plants possessing an elongated period along with increased leaf surface area, biomass and delayed flowering relative to wild-type Col-0 plants. Here, using a multi-omics approach consisting of phenomics, transcriptomics, proteomics, and metabolomics we demonstrate how RVE8-like proteins impact diel plant cell function and draw novel connections to a number of plant cell processes that underpin the growth and development phenotypes observed in rve 4 6 8 plants. In particular, we reveal that loss of RVE8-like proteins results in altered carbohydrate, organic acid and lipid metabolism, including a starch excess phenotype at ZT0. We further demonstrate that RVE8-like proteins have a unique impact on the abundance and phosphorylation of 26S proteasome subunits, in addition to impacting the abundance and phosphorylation status of a number of protein kinases. Overall, this robust, multi-omic dataset, provides substantial new insights into RVE8-like protein function and the far reaching impact RVE8-like proteins have on the diel plant cell environment.

scholarly journals The proteasome activity reporter GFP-Cl1 is degraded by autophagy in the aging model Podospora anserina

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 230 ◽  
Author(s):  
Matthias Wiemer ◽  
Heinz D. Osiewacz
Keyword(s):  
Protein Quality ◽  
Ubiquitin Proteasome System ◽  
Substantial Effect ◽  
Podospora Anserina ◽  
Proteasome Activity ◽  
Protein Abundance ◽  
Wild Type ◽  
Vital Function ◽  
Proteasome Subunits ◽  
Ubiquitin Proteasome

The degradation of damaged proteins is an important vital function especially during aging and stress. The ubiquitin proteasome system is one of the major cellular machineries for protein degradation. Health and longevity are associated with high proteasome activity. To demonstrate such a role in aging of Podospora anserina, we first analyzed the transcript and protein abundance of selected proteasome components in wild-type cultures of different age. No significant differences were observed. Next, in order to increase the overall proteasome abundance we generated strains overexpressing the catalytic proteasome subunits PaPRE2 and PaPRE3. Although transcript levels were strongly increased, no substantial effect on the abundance of the corresponding proteins was observed. Finally, the analysis of the P. anserina strains expressing the sequence coding for the CL1 degron fused to the Gfp gene revealed no evidence for degradation of the GFP-CL1 fusion protein by the proteasome. Instead, our results demonstrate the degradation of the CL1-degron sequence via autophagy, indicating that basal autophagy appears to be a very effective protein quality control pathway in P. anserina.

Abstract 205: Dysfunction of the 19S Proteosome Contributes to Adverse Cardiac Remodeling in Mice With Cardiac Restricted Overexpression of Tumor Necrosis Factor

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Wei Wang ◽  
Gabor Szalai ◽  
Natarajan Sivasubramanian ◽  
Douglas L Mann
Keyword(s):  
Tumor Necrosis Factor ◽  
Cardiac Remodeling ◽  
Tumor Necrosis ◽  
Left Ventricular ◽  
Proteasome Activity ◽  
26S Proteasome ◽  
Wild Type ◽  
Wild Type Littermate ◽  
Littermate Control ◽  
Necrosis Factor

We have shown previously that mice with cardiac restricted overexression of tumor necrosis factor (sTNF mice) develop progressive cardiac remodeling. Given that the ubiquitin/ proteasome system regulates protein turnover in eukaryotic cells and is thereby a major factor in controlling cell cycle, apoptosis, signal transduction pathways, which are important in cardiac remodeling, the purpose of this study was to assess the function of 19S, 20S and 26S proteasome in sTNF mice and littermate control mice. Results : Activity of the 26S proteasome was significantly suppressed in the hearts of the sTNF mice compared with the wild type littermates, and was detectable as early as 4-weeks of age. To explore the mechanism for the dysfunction of the 26S proteosome, we examined the activity of the 19S and 20S subunits. We observed cleavage of two subunits of the 19S regulatory complex of the proteasome, namely S1 and S10b, in the hearts of the sTNF mice. S1 is involved in the linking of the lid and the base of the 19S complex, while S10b is one of the six ATPase subunits in the base of the 19S complex. Interestingly, we observed that the expression of six subunits of the 20S core complex, β2, β2i, β3, β4, β5, and β7 were up-regulated in the hearts of the sTNF mice compared with the wild type littermate control mice. Despite the higher expression of the six β subunits in the 20S complex, we observed no difference in the 20S proteasome activity in hearts between the sTNF mice and the wild type littermate controls. Conclusion : Taken together, these results suggest that cleavage of S1 and S10b might contribute to the low 26S proteasome activity in the hearts of the sTNF mice. Given that the 26S proteasome is responsible for degrading pro-apoptotic pathways, as well as turning off pro-fibrotic signaling (TGF-beta), these results suggest that dysfunction of the 26S proteasome might be one of the mechanisms underlying left ventricular remodeling in the setting of sustained inflammation.

Ectopic Transcript Analysis Indicates that Allelic Exclusion is an Important Cause of Type I Protein C Deficiency in Patients with Nonsense and Frameshift Mutations in the PROC Gene

1996 ◽  
Vol 75 (06) ◽  
pp. 870-876 ◽  
Author(s):  
José Manuel Soria ◽  
Lutz-Peter Berg ◽  
Jordi Fontcuberta ◽  
Vijay V Kakkar ◽  
Xavier Estivill ◽  
...  
Keyword(s):  
Splice Site ◽  
Protein C ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Allelic Exclusion ◽  
Polymorphism Analysis ◽  
Type I ◽  
Protein C Deficiency ◽  
Nonsense Mutations ◽  
Stop Codons

SummaryNonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these' lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through “allelic exclusion” at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3’ to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons

scholarly journals The NALCN Channel Regulator UNC-80 Functions in a Subset of Interneurons To Regulate Caenorhabditis elegans Reversal Behavior

2019 ◽  
Vol 10 (1) ◽  
pp. 199-210 ◽  
Author(s):  
Chuanman Zhou ◽  
Jintao Luo ◽  
Xiaohui He ◽  
Qian Zhou ◽  
Yunxia He ◽  
...  
Keyword(s):  
Plant Hormone ◽  
Neuronal Excitability ◽  
Resting Membrane Potential ◽  
Loss Of Function ◽  
Wild Type ◽  
C Elegans ◽  
Link Type ◽  
Complex Functions ◽  
And Function ◽  
Multiple Loss

NALCN (Na+leak channel, non-selective) is a conserved, voltage-insensitive cation channel that regulates resting membrane potential and neuronal excitability. UNC79 and UNC80 are key regulators of the channel function. However, the behavioral effects of the channel complex are not entirely clear and the neurons in which the channel functions remain to be identified. In a forward genetic screen for C. elegans mutants with defective avoidance response to the plant hormone methyl salicylate (MeSa), we isolated multiple loss-of-function mutations in unc-80 and unc-79. C. elegans NALCN mutants exhibited similarly defective MeSa avoidance. Interestingly, NALCN, unc-80 and unc-79 mutants all showed wild type-like responses to other attractive or repelling odorants, suggesting that NALCN does not broadly affect odor detection or related forward and reversal behaviors. To understand in which neurons the channel functions, we determined the identities of a subset of unc-80-expressing neurons. We found that unc-79 and unc-80 are expressed and function in overlapping neurons, which verified previous assumptions. Neuron-specific transgene rescue and knockdown experiments suggest that the command interneurons AVA and AVE and the anterior guidepost neuron AVG can play a sufficient role in mediating unc-80 regulation of the MeSa avoidance. Though primarily based on genetic analyses, our results further imply that MeSa might activate NALCN by direct or indirect actions. Altogether, we provide an initial look into the key neurons in which the NALCN channel complex functions and identify a novel function of the channel in regulating C. elegans reversal behavior through command interneurons.

Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 661-668
Author(s):  
Mandy Kim ◽  
Erika Wolff ◽  
Tiffany Huang ◽  
Lilit Garibyan ◽  
Ashlee M Earl ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Deinococcus Radiodurans ◽  
Genetic System ◽  
Base Change ◽  
Base Substitution ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
Radiation Induced ◽  
Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

scholarly journals Nonrecombinant meiosis I nondisjunction in Saccharomyces cerevisiae induced by tRNA ochre suppressors.

Genetics ◽  
1989 ◽  
Vol 123 (1) ◽  
pp. 81-95 ◽  
Author(s):  
E J Louis ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Stop Codon ◽  
Fold Increase ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nonsense Mutations ◽  
Chromosome Iii ◽  
Meiosis I ◽  
Chromosome Nondisjunction

Abstract The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.

Molecular Markers for Rapidly Identifying Candidate Genes in Chlamydomonas reinhardtii: ERY1 and ERY2 Encode Chloroplast Ribosomal Proteins

Genetics ◽  
2003 ◽  
Vol 164 (4) ◽  
pp. 1345-1353
Author(s):  
Amber K Bowers ◽  
Jennifer A Keller ◽  
Susan K Dutcher
Keyword(s):  
Molecular Markers ◽  
Chlamydomonas Reinhardtii ◽  
Candidate Genes ◽  
Splice Site ◽  
Genomic Dna ◽  
Ribosomal Proteins ◽  
Genomic Sequence ◽  
Point Mutations ◽  
Acceptor Site ◽  
Wild Type

Abstract To take advantage of available expressed sequence tags and genomic sequence, we have developed 64 PCR-based molecular markers in Chlamydomonas reinhardtii that map to the 17 linkage groups. These markers will allow the rapid association of a candidate gene sequence with previously identified mutations. As proof of principle, we have identified the genes encoded by the ERY1 and ERY2 loci. Mendelian mutations that confer resistance to erythromycin define three unlinked nuclear loci in C. reinhardtii. Candidate genes ribosomal protein L4 (RPL4) and L22 (RPL22) are tightly linked to the ERY1 locus and ERY2 locus, respectively. Genomic DNA for RPL4 from wild type and five mutant ery1 alleles was amplified and sequenced and three different point mutations were found. Two different glycine residues (G102 and G112) are replaced by aspartic acid and both are in the unstructured region of RPL4 that lines the peptide exit tunnel of the chloroplast ribosome. The other two alleles change a splice site acceptor site. Genomic DNA for RPL22 from wild type and three mutant ery2 alleles was amplified and sequenced and revealed three different point mutations. Two alleles have premature stop codons and one allele changes a splice site acceptor site.

scholarly journals Elimination of Introns at the Drosophila suppressor-of-forked Locus by P-Element-Mediated Gene Conversion Shows That an RNA Lacking a Stop Codon is Dispensable

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 345-351
Author(s):  
Carol J Williams ◽  
Kevin O'Hare
Keyword(s):  
X Chromosome ◽  
Stop Codon ◽  
Alternative Polyadenylation ◽  
Gene Replacement ◽  
P Element ◽  
Wild Type ◽  
Genomic Location ◽  
Chromosomal Breaks ◽  
White Locus ◽  
Cleavage Stimulation Factor

Abstract The suppressor of forked [su(f)] locus affects the phenotype of mutations caused by transposable element insertions at unlinked loci. It encodes a putative 84-kD protein with homology to two proteins involved in mRNA 3′ end processing; the product of the yeast RNA14 gene and the 77-kD subunit of human cleavage stimulation factor. Three su(f) mRNAs are produced by alternative polyadenylation. The 2. 6 and 2.9-kb mRNAs encode the same 84-kD protein while a 1.3-kb RNA, which terminates within the fourth intron, is unusual in having no stop codon. Using P-element-mediated gene replacement we have copied sequences from a transformation construct into the su(f) gene creating a su(f) allele at the normal genomic location that lacks the first five introns. This allele is viable and appears wild type for su(f) function, demonstrating that the 1.3-kb RNA and the sequences contained within the deleted introns are dispensable for su(f) function. Compared with studies on gene replacement at the white locus, chromosomal breaks at su(f) appear to be less efficiently repaired from ectopic sites, perhaps because of the location of su(f) at the euchromatin/heterochromatin boundary on the X chromosome.

scholarly journals A rare large duplication of MLH1 identified in Lynch syndrome

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Abhishek Kumar ◽  
Nagarajan Paramasivam ◽  
Obul Reddy Bandapalli ◽  
Matthias Schlesner ◽  
Tianhui Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lynch Syndrome ◽  
Splice Site ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Splice Donor Site ◽  
Laboratory Diagnostics ◽  
Mmr Genes ◽  
Base Pair Deletion ◽  
Splice Site Variant

Abstract Background The most frequently identified strong cancer predisposition mutations for colorectal cancer (CRC) are those in the mismatch repair (MMR) genes in Lynch syndrome. Laboratory diagnostics include testing tumors for immunohistochemical staining (IHC) of the Lynch syndrome-associated DNA MMR proteins and/or for microsatellite instability (MSI) followed by sequencing or other techniques, such as denaturing high performance liquid chromatography (DHPLC), to identify the mutation. Methods In an ongoing project focusing on finding Mendelian cancer syndromes we applied whole-exome/whole-genome sequencing (WES/WGS) to 19 CRC families. Results Three families were identified with a pathogenic/likely pathogenic germline variant in a MMR gene that had previously tested negative in DHPLC gene variant screening. All families had a history of CRC in several family members across multiple generations. Tumor analysis showed loss of the MMR protein IHC staining corresponding to the mutated genes, as well as MSI. In family A, a structural variant, a duplication of exons 4 to 13, was identified in MLH1. The duplication was predicted to lead to a frameshift at amino acid 520 and a premature stop codon at amino acid 539. In family B, a 1 base pair deletion was found in MLH1, resulting in a frameshift and a stop codon at amino acid 491. In family C, we identified a splice site variant in MSH2, which was predicted to lead loss of a splice donor site. Conclusions We identified altogether three pathogenic/likely pathogenic variants in the MMR genes in three of the 19 sequenced families. The MLH1 variants, a duplication of exons 4 to 13 and a frameshift variant, were novel, based on the InSiGHT and ClinVar databases; the MSH2 splice site variant was reported by a single submitter in ClinVar. As a variant class, duplications have rarely been reported in the MMR gene literature, particularly those covering several exons.

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