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Root Transcriptomic Analysis Reveals Global Changes Induced by Systemic Infection of Solanum lycopersicum with Mild and Severe Variants of Potato Spindle Tuber Viroid
Get PDFPotato spindle tuber viroid (PSTVd) causes systemic infection in plant hosts. There are many studies on viroid-host plant interactions, but they have predominantly focused on the aboveground part of the plant. Here, we investigated transcriptomic profile changes in tomato roots systemically infected with mild or severe PSTVd variants using a combined microarray/RNA-seq approach. Analysis indicated differential expression of genes related to various Gene Ontology categories depending on the stage of infection and PSTVd variant. A majority of cell-wall-related genes were down-regulated at early infection stages, but at the late stage, the number of up-regulated genes increased significantly. Along with observed alterations of many lignin-related genes, performed lignin quantification indicated their disrupted level in PSTVd-infected roots. Altered expression of genes related to biosynthesis and signaling of auxin and cytokinin, which are crucial for lateral root development, was also identified. Comparison of both PSTVd infections showed that transcriptional changes induced by the severe variant were stronger than those caused by the mild variant, especially at the late infection stage. Taken together, we showed that similarly to aboveground plant parts, PSTVd infection in the underground tissues activates the plant immune response
Composition and processing activity of a semi-recombinant holo U7 snRNP
Get PDFIn animal cells, replication-dependent histone pre-mRNAs are cleaved at the 3' end by U7 snRNP consisting of two core components: a ∼60-nucleotide U7
snRNA and a ring of seven proteins, with Lsm10 and
Lsm11 replacing the spliceosomal SmD1 and SmD2.
Lsm11 interacts with FLASH and together they recruit the endonuclease CPSF73 and other polyadenylation factors, forming catalytically active holo U7
snRNP. Here, we assembled core U7 snRNP bound to
FLASH from recombinant components and analyzed
its appearance by electron microscopy and ability to
support histone pre-mRNA processing in the presence of polyadenylation factors from nuclear extracts. We demonstrate that semi-recombinant holo
U7 snRNP reconstituted in this manner has the same
composition and functional properties as endogenous U7 snRNP, and accurately cleaves histone pre-mRNAs in a reconstituted in vitro processing reaction. We also demonstrate that the U7-specific Sm
ring assembles efficiently in vitro on a spliceosomal
Sm site but the engineered U7 snRNP is functionally
impaired. This approach offers a unique opportunity
to study the importance of various regions in the Sm
proteins and U7 snRNA in 3' end processing of histone pre-mRNAs
Defects in the GINS complex increase the instability of repetitive sequences via a recombination-dependent mechanism
Get PDFFaithful replication and repair of DNA lesions ensure genome maintenance. During replication in eukaryotic cells, DNA is unwound by the CMG helicase complex, which is composed of three major components: the Cdc45 protein, Mcm2-7, and the GINS complex. The CMG in complex with DNA polymerase epsilon (CMG-E) participates in the establishment and progression of the replisome. Impaired functioning of the CMG-E was shown to induce genomic instability and promote the development of various diseases. Therefore, CMG-E components play important roles as caretakers of the genome. In Saccharomyces cerevisiae, the GINS complex is composed of the Psf1, Psf2, Psf3, and Sld5 essential subunits. The Psf1-1 mutant form fails to interact with Psf3, resulting in impaired replisome assembly and chromosome replication. Here, we show increased instability of repeat tracts (mononucleotide, dinucleotide, trinucleotide and longer) in yeast psf1-1 mutants. To identify the mechanisms underlying this effect, we analyzed repeated sequence instability using derivatives of psf1-1 strains lacking genes involved in translesion synthesis, recombination, or mismatch repair. Among these derivatives, deletion of RAD52, RAD51, MMS2, POL32, or PIF1 significantly decreased DNA repeat instability. These results, together with the observed increased amounts of single-stranded DNA regions and Rfa1 foci suggest that recombinational mechanisms make important contributions to repeat tract instability in psf1-1 cells. We propose that defective functioning of the CMG-E complex in psf1-1 cells impairs
the progression of DNA replication what increases the contribution of repair mechanisms such as template switch and break-induced replication. These processes require sequence homology search which in case of a repeated DNA tract may result in misalignment leading to its expansion or contraction
Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes.
Get PDFTo accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of
plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were
inserted into genes and transferred together with flanking genomic sequences of desired size by recombination
into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified
DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLINDEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription
factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red
fluorescent protein) tags, and a PIPL (His18-StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs
is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three
known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification
and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their
association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively.
The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies
Nest characteristics determine nest microclimate and affect breeding output in an Antarctic seabird, the Wilson’s storm-petrel.
Get PDFThe importance of nest characteristics for birds breeding in the extreme climate conditions of polar regions, has been greatly understudied. Nest parameters, like nest orientation, exposure and insulation, could strongly influence microclimate and protection against precipitation of the nest, thereby affecting breeding success. A burrow nesting seabird, the Wilson's storm-petrel (Oceanites oceanicus) is an excellent model species to investigate the importance of nest characteristics, as it is the smallest endotherm breeding in the Antarctic. Here, we investigated the effects of nest parameters such as internal nest dimensions, nest micro-topography and thermal properties of the nest burrow and the influence of weather conditions on breeding output, measured as hatching success, chick survival, and chick growth. We collected data during the austral summers of 2017 and 2018, on King George Island, maritime Antarctica. Our results showed that the thermal microclimate of the nest burrow was significantly improved by a small entrance size, a low nest height, and insulation and tended to be enhanced by a low wind exposition index and an eastern nest site orientation. In addition, an eastern nest site orientation significantly reduced the chance of snow blocking. However, the relationships between nest characteristics and breeding output were complex and might be affected by other parameters like food availability and parental quality. The relation between chick growth and nest air temperature remained especially indistinct. Nevertheless, our results indicate that nest characteristics that enhance the thermal microclimate and reduce the risk of snow blocking favoured both hatching success and chick survival. Due to climate change in the Antarctic, snowfall is expected to increase in the future, which will likely enhance the importance of nest characteristics that determine snow blocking. Additionally, despite global warming, thermally favourable nest burrows will likely still be advantageous in the highly variable and challenging Antarctic climate
Quantitative proteomics revealed C6orf203/MTRES1 as a factor preventing stress-induced transcription deficiency in human mitochondria
Get PDFMaintenance of mitochondrial gene expression is
crucial for cellular homeostasis. Stress conditions
may lead to a temporary reduction of mitochondrial
genome copy number, raising the risk of insufficient
expression of mitochondrial encoded genes. Little
is known how compensatory mechanisms operate
to maintain proper mitochondrial transcripts levels
upon disturbed transcription and which proteins are
involved in them. Here we performed a quantitative
proteomic screen to search for proteins that sustain
expression of mtDNA under stress conditions. Analysis
of stress-induced changes of the human mitochondrial
proteome led to the identification of several
proteins with poorly defined functions among which
we focused on C6orf203, which we named MTRES1
(Mitochondrial Transcription Rescue Factor 1). We
found that the level of MTRES1 is elevated in cells
under stress and we show that this upregulation of
MTRES1 prevents mitochondrial transcript loss under
perturbed mitochondrial gene expression. This
protective effect depends on the RNA binding activity
of MTRES1. Functional analysis revealed that
MTRES1 associates with mitochondrial RNA polymerase
POLRMT and acts by increasing mitochondrial
transcription, without changing the stability of
mitochondrial RNAs. We propose that MTRES1 is an
example of a protein that protects the cell from mitochondrial
RNA loss during stress
Repression of yeast RNA polymerase III by stress leads to ubiquitylation and proteasomal degradation of its largest subunit, C160.
Get PDFRespiratory growth and various stress conditions repress RNA polymerase III (Pol III) transcription in Saccharomyces cerevisiae. Here we report a degradation of the largest Pol III catalytic subunit, C160 as a consequence of Pol III transcription repression. We observed C160 degradation in response to transfer of yeast from fermentation to respiration conditions, as well as treatment with rapamycin or inhibition of nucleotide biosynthesis. We also detected ubiquitylated forms of C160 and demonstrated that C160 protein degradation is dependent on proteasome activity. A comparable time-course study of Pol III repression upon metabolic shift from fermentation to respiration shows that the transcription inhibition is correlated with Pol III dissociation from chromatin but that the degradation of C160 subunit is a downstream event. Despite blocking degradation of C160 by proteasome, Pol III-transcribed genes are under proper regulation. We postulate that the degradation of C160 is activated under stress conditions to reduce the amount of existing Pol III complex and prevent its de novo assembly
Trehalose, mannitol and arabitol as indicators of fungal metabolism in late Cretaceous and Miocene deposits
Get PDFPhosphoproteomic Analysis Reveals that Dehydrins ERD10 and ERD14 are Phosphorylated by SNF1-related Protein Kinase 2.10 in Response to Osmotic Stress
Get PDFSNF1-related protein kinases 2 (SnRK2s) regulate the plant responses to abiotic stresses, especially water deficits. They are activated in plants subjected to osmotic stress, and some of them are additionally activated in response to enhanced concentrations of abscisic acid (ABA) in plant cells. The SnRK2s that are activated in response to ABA are key elements of ABA signaling that regulate plant acclimation to environmental stresses and ABA-dependent development. Much less is known about the SnRK2s that are not activated by ABA, albeit several studies have shown that these kinases are also involved in response to osmotic stress. Here, we show that one of the Arabidopsis thaliana ABA-non-activated SnRK2s, SnRK2.10, regulates not only the response to salinity but also the plant sensitivity to dehydration. Several potential SnRK2.10 targets phosphorylated in response to stress were identified by a phosphoproteomic approach, including the dehydrins ERD10 and ERD14. Their phosphorylation by SnRK2.10 was confirmed in vitro. Our data suggest that the phosphorylation of ERD14 within the S-segment is involved in the regulation of dehydrin subcellular localization in response to stress
Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7
Get PDFNuclear and mitochondrial genome mutations lead to various mitochondrial diseases, many of which affect the mitochondrial respiratory chain. The proteome of the intermembrane space (IMS) of mitochondria consists of several important assembly factors that participate in the biogenesis of mitochondrial respiratory chain complexes. The present study comprehensively analyzed a recently identified IMS protein cytochrome c oxidase assembly factor 7 (COA7), or RESpiratory chain Assembly 1 (RESA1) factor that is associated with a rare form of mitochondrial leukoencephalopathy and complex IV deficiency. We found that COA7 requires the mitochondrial IMS import and assembly (MIA) pathway for efficient accumulation in the IMS We also found that pathogenic mutant versions of COA7 are imported slower than the wild-type protein, and mislocalized proteins are degraded in the cytosol by the proteasome. Interestingly, proteasome inhibition rescued both the mitochondrial localization of COA7 and complex IV activity in patient-derived fibroblasts. We propose proteasome inhibition as a novel therapeutic approach for a broad range of mitochondrial pathologies associated with the decreased levels of mitochondrial proteins