Systematic screening of archaeal MazF homologs reveals Tth-MazF1, a versatile, sequence-specific ribonuclease from <i>Thermococcus thioreducens</i>

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-85°C). Tth-MazF1 enabled high sequence coverage of 0.8-8.9 kb messenger RNA (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 ribosomal RNA. 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.