Temperature-induced changes in the wheat phosphoproteome reveal temperature-regulated interconversion of phosphoforms

Wheat (Triticum ssp.) is one of the most important human food sources. However, this crop is very sensitive to temperature changes. Specifically, processes during wheat leaf, flower, and seed development and photosynthesis, which all contribute to the yield of this crop, are affected by high temperature. While this has to some extent been investigated on physiological, developmental, and molecular levels, very little is known about early signalling events associated with an increase in temperature. Phosphorylation-mediated signalling mechanisms, which are quick and dynamic, are associated with plant growth and development, also under abiotic stress conditions. Therefore, we probed the impact of a short-term and mild increase in temperature on the wheat leaf and spikelet phosphoproteome. In total, 3822 (containing 5178 phosphosites) and 5581 phosphopeptides (containing 7023 phosphosites) were identified in leaf and spikelet samples, respectively. Following statistical analysis, the resulting data set provides the scientific community with a first large-scale plant phosphoproteome under the control of higher ambient temperature. This community resource on the high temperature-mediated wheat phosphoproteome will be valuable for future studies. Our analyses also revealed a core set of common proteins between leaf and spikelet, suggesting some level of conserved regulatory mechanisms. Furthermore, we observed temperature-regulated interconversion of phosphoforms, which probably impacts protein activity

The Role of Noncoding RNA Pseudouridylation in Nuclear Gene Expression Events

Pseudouridine is the most abundant internal RNA modification in stable noncoding RNAs (ncRNAs). It can be catalyzed by both RNA-dependent and RNA-independent mechanisms. Pseudouridylation impacts both the biochemical and biophysical properties of RNAs and thus influences RNA-mediated cellular processes. The investigation of nuclear-ncRNA pseudouridylation has demonstrated that it is critical for the proper control of multiple stages of gene expression regulation. Here, we review how nuclear-ncRNA pseudouridylation contributes to transcriptional regulation and pre-mRNA splicing

Converting nonsense codons into sense codons by targeted pseudouridylation. Nature. 2011; 474:395–398. [PubMed: 21677757

All three translation termination codons, or nonsense codons, contain a uridine residue at the first position of the codon Y, the C5-glycoside isomer of uridine, has many structural and biochemical differences from uridine 5 ( The in vitro results prompted us to investigate whether the pseudouridylation of a termination codon would elicit nonsense suppression in vivo. Taking advantage of the CUP1 reporter system 6 , we introduced a premature termination codon (PTC) at the second codon of the CUP1 gene, thus creating a new CUP1 reporter gene (termed cup1-PTC). Cup1p is a copper chelating protein that mediates resistance to copper sulphate (CuSO 4 ) To direct site-specific Y formation in vivo, we took advantage of the H/ACA ribonucleoprotein (RNP) family. H/ACA RNPs are primarily responsible for the post-transcriptional isomerization of uridine to Y within RNA To ensure that cup1-PTC was pseudouridylated in response to expression of snR81-1C, we measured Y formation within the PTC both in vitro and in vivo. To analyse Y formation in vitro, we monitored, by thin layer chromatography (TLC), Y formation on a 39-nucleotide fragment of RNA corresponding to the region of cup1-PTC containing the PTC To determine the pseudouridylation status of cup1-PTC in vivo, we analysed the PTC of cellularly derived cup1-PTC mRNA using a sitespecific and quantitative pseudouridylation assay, namely site-specific cleavage and radiolabelling followed by nuclease digestion an