Project description:Nucleotide- and sequence-specific ribonucleases are essential tools for dissecting RNA structure, modification, and composition, yet the enzymatic repertoire available for high-resolution RNA analysis remains limited. Here, we systematically screened 186 MazF homologs, including 164 from archaeal organisms, and identified 132 active endoribonucleases with diverse sequence preferences. Using a semi-high-throughput expression and purification workflow combined with an Illumina-based RNA-seq assay, we defined recognition motifs for 90 enzymes, uncovering a striking diversity of UG-rich cleavage specificities across the archaeal domain. Among these, Tth-MazF1, a thermostable endoribonuclease from the hyperthermophilic archaeon Thermococcus thioreducens, displayed robust and specific cleavage at U↓GN motifs across a broad range of temperatures (4 ˚C to 85 ˚C). Tth-MazF1 enabled high sequence coverage of 0.8-8.9 kb mRNA transcripts by LC-MS/MS and direct detection of key structural features, including the 5′ cap, poly(A) tail, and mass-altering epitranscriptomic modifications in rRNA. Tolerance of Tth-MazF1 to high temperatures, salt, and chemical modifications highlights it as a powerful enzymatic tool for analyzing chemically modified and structured RNAs. These findings position archaeal MazF proteins as a rich source of customizable RNases for biotechnology applications, such as mass spectrometry-based analysis of RNA-based vaccines and therapeutics as well as cellular RNAs.
Project description:Thermococcus sp. 2319x1E is able to grow on different mono- or polysaccharides as carbon source, including xylan. To investigate xylan degradation in Thermococcus sp. 2319x1E, cells were grown on 4 different carbon sources for 12 h (mid-exponential phase) and harvested by centrifugation. Using a label-free quantification approach the whole proteomes were analyzed. In another experiment a glycosidase specific activity-based probe (JJB384) was used to isolate specific Thermococcus glycosidases activated during growth on xylan or xylose.
