Project description:5-methylcytosine (m5C) is emerging as an important epi-transcriptome modification involving RNA stability and translation efficiency in various biological processes. However, it remains unclear how m5C contributes to the dynamic regulation of transcriptome during the development of Plasmodium. Here, we identified the presence of 5-methylcytosine (m5C) modification in rodent (P. yoelii) and human (P. falciparum) malaria parasites transcriptome and depicted a comprehensive characterization landscape of m5C mRNA modification at single-nucleotide resolution (RNA-BisSeq) from asexual replicating stage to gametocyte development. Through transcriptome-wide profiling of mRNA m5C modification, we found that m5C modified mRNA displayed higher stability than non-m5C modified mRNA during the development of Plasmodium. We identified Plasmodium ortholog of NSUN2 as an mRNA m5C methyltransferase in malaria parasites. LC–MS/MS and RNA-BisSeq analysis revealed a large decrease in mRNA m5C modification at transcriptome-wide level upon Nsun2 knockout. Absence of Nsun2 severely reduced gametocyte production in either rodent (P. yoelii) or human (P. falciparum) malaria parasites. Meanwhile, some genes related to gametocytogenesis displayed a great reduction of m5C modification. Together, our data provides comprehensive mRNA m5C profiles in Plasmodium genus and reveals m5C modification-mediated mRNA stability as a novel mechanism regulating sexual differentiation of a unicellular eukaryote.
Project description:5-methylcytosine (m5C) is emerging as an important epi-transcriptome modification involving RNA stability and translation efficiency in various biological processes. However, it remains unclear how m5C contributes to the dynamic regulation of transcriptome during the development of Plasmodium. Here, we identified the presence of 5-methylcytosine (m5C) modification in rodent (P. yoelii) and human (P. falciparum) malaria parasites transcriptome and depicted a comprehensive characterization landscape of m5C mRNA modification at single-nucleotide resolution (RNA-BisSeq) from asexual replicating stage to gametocyte development. Through transcriptome-wide profiling of mRNA m5C modification, we found that m5C modified mRNA displayed higher stability than non-m5C modified mRNA during the development of Plasmodium. We identified Plasmodium ortholog of NSUN2 as an mRNA m5C methyltransferase in malaria parasites. LC–MS/MS and RNA-BisSeq analysis revealed a large decrease in mRNA m5C modification at transcriptome-wide level upon Nsun2 knockout. Absence of Nsun2 severely reduced gametocyte production in either rodent (P. yoelii) or human (P. falciparum) malaria parasites. Meanwhile, some genes related to gametocytogenesis displayed a great reduction of m5C modification. Together, our data provides comprehensive mRNA m5C profiles in Plasmodium genus and reveals m5C modification-mediated mRNA stability as a novel mechanism regulating sexual differentiation of a unicellular eukaryote.
Project description:5-methylcytosine (m5C) is emerging as an important epi-transcriptome modification involving RNA stability and translation efficiency in various biological processes. However, it remains unclear how m5C contributes to the dynamic regulation of transcriptome during the development of Plasmodium. Here, we identified the presence of 5-methylcytosine (m5C) modification in rodent (P. yoelii) and human (P. falciparum) malaria parasites transcriptome and depicted a comprehensive characterization landscape of m5C mRNA modification at single-nucleotide resolution (RNA-BisSeq) from asexual replicating stage to gametocyte development. Through transcriptome-wide profiling of mRNA m5C modification, we found that m5C modified mRNA displayed higher stability than non-m5C modified mRNA during the development of Plasmodium. We identified Plasmodium ortholog of NSUN2 as an mRNA m5C methyltransferase in malaria parasites. LC–MS/MS and RNA-BisSeq analysis revealed a large decrease in mRNA m5C modification at transcriptome-wide level upon Nsun2 knockout. Absence of Nsun2 severely reduced gametocyte production in either rodent (P. yoelii) or human (P. falciparum) malaria parasites. Meanwhile, some genes related to gametocytogenesis displayed a great reduction of m5C modification. Together, our data provides comprehensive mRNA m5C profiles in Plasmodium genus and reveals m5C modification-mediated mRNA stability as a novel mechanism regulating sexual differentiation of a unicellular eukaryote.
Project description:Identification of targets of the protein disulfide reductase thioredoxin using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and thiol specific differential labeling with isotope-coded affinity tags (ICAT). Reduction of specific target disulfides is quantified by measuring ratios of cysteine residues labeled with the heavy (13C) and light (12C) ICAT reagents in peptides derived from tryptic digests of Trx-treated and non-treated samples. Keywords: protein, LC-MS/MS, ICAT
Project description:skin tissue of LCG was taken as the research object in this study, and quantitative proteomics strategy of LC-MS/MS mass spectrometry was used to identify and quantitatively analyze differential proteins. Combined with bioinformatics analysis, the formation mechanism of cashmere in LCG was discussed in depth, in order to find the internal relationship between phosphorylation modification and hair follicle development in cashmere goat, and to comprehend the regulation of protein phosphorylation modification on fiber growth in LCG.
Project description:Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. We report that protein arginine phosphorylation plays a physiological significant role for the regulation of protein activity. We detected 121 arginine phospho-sites for 87 proteins in the Gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidences that arginine phosphorylations are involved in the fine-tuned signal transduction of many critical cellular processes, such as protein degradation, motility, competence, stringent and stress response. Our results suggest that in B. subtilis the activity of a protein arginine phosphatase allows a fast regulation of protein activity by protein arginine kinases and that protein arginine phosphorylations play an important role as a reversible post-translational modification in bacteria. Cells were grown under vigorous agitation at 37 M-BM-0C in a defined medium (StM-CM-<lke et al., 1993, J Gen Microbiol 139, 2041-2045). Samples were taken at OD500 0.4 and 1h upon entry into stationary phase. Microarray hybridizations were performed with RNA from three biological replicates. The individual samples were labeled with Cy5; a reference pool containing equal amounts of RNA from all 10 samples was labeled with Cy3.
Project description:Phosphorylation of Ribosomal Protein S6 (RPS6) was the first post-translational modification of the ribosome to be identified and is a commonly-used readout for mTORC1 activity. Although the cellular and organismal functions of RPS6 phosphorylation are known, its molecular consequences on translation are less well understood. Here we use selective ribosome footprinting to analyze the location of ribosomes containing phosphorylated RPS6 on endogenous mRNAs in cells. We find that RPS6 becomes progressively dephosphorylated on ribosomes as they translate an mRNA. As a consequence, average RPS6 phosphorylation is higher on mRNAs with short coding sequences (CDSs) compared to mRNAs with long CDSs. Loss of RPS6 phosphorylation causes a correspondingly larger drop in translation efficiency of mRNAs with short CDSs than long CDSs. Interestingly, mRNAs with 5’ TOP motifs are translated well also in the absence of RPS6 phosphorylation despite short CDS lengths, suggesting they are translated via a different mode. In sum this provides a dynamic view of RPS6 phosphorylation on ribosomes as they translate mRNAs and the functional consequence on translation.