Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. Overall, we monitored the stability of 1,941 human N-terminal proteoforms, including 147 N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. Ribosome profiling of lactimidomycin and cycloheximide treated human Jurkat T-lymphocytes
Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. Overall, we monitored the stability of 1,941 human N-terminal proteoforms, including 147 N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:<p>Mitochondria execute essential metabolic, biosynthetic, and signaling functions, yet the regulatory principles governing their proteome remain incompletely understood. Here, we develop a thermal proteome profiling (TPP) workflow applied directly to purified mitochondria, enabling systematic analysis of organelle-specific protein thermal stability. This approach substantially increases mitochondrial proteome and sequence coverage compared with whole-cell TPP, enhances detection of protein–ligand interactions, and provides high-resolution access to proteoform- and protein–protein interaction (PPI)–level organization. Using complex III and I inhibitors, we show that disrupting electron transport chain (ETC) activity broadly destabilizes mitochondrial matrix proteins, revealing a previously unrecognized requirement for ETC function in maintaining the structural integrity of the mitochondrial gene expression machinery and supporting mitochondrial translation. Deep proteoform analysis identifies extensive mitochondrial proteoform diversity arising from phosphorylation, alternative splicing, and proteolytic processing, including previously uncharacterized C-terminal cleavage of SLC25A24. Co-melting network analysis improves recovery of known mitochondrial PPIs and uncovers novel interactions, leading to the discovery of AK4 as a negative regulator of mitochondrial protein synthesis and L2HGDH as a factor required for coenzyme Q biosynthesis. Finally, applying mitochondrial TPP to Mgme1-deficient mice reveals early, abundance-independent remodeling of mtDNA replication and translation factors, including sequestration of Ssbp1 into condensate-like assemblies driven by accumulation of aberrant mtDNA fragments. Together, this work establishes mitochondrial TPP as a powerful and versatile platform for resolving multilayered regulation of the mitochondrial proteome in vitro and in vivo.</p>