Discovery of nucleotide and sequence-specific endoribonucleases
Ribonucleases catalyze the degradation of RNA molecules into smaller fragments. They are not only involved in degrading cellular RNA, but also play a key role in maturation of coding and non-coding RNA. RNases are commonly used in molecular biology as a tool. Among those, nucleotide and sequence-specific RNases are particularly useful for mass spectrometry applications for the detection and mapping of covalent RNA modifications. To discover novel tools for molecular biology and biotechnology applications, we are screening for RNases that are sensitive to or specific to covalent RNA modifications. To accomplish this, we take two different approaches. First, we choose candidate enzymes based on protein homology search. Second, we design genetic screens to search metagenomic libraries to find specific activities. Finally, candidate enzymes are produced recombinantly to high degree of purity, and biochemically characterized.
In E. coli cells, RNase I is found in a complex with a calcium ion, and it exhibits both single-stranded and double-stranded specific activities. Interestingly, removal of the calcium ion from RNase I has no effect on its activity on single-stranded RNA, whereas it reduces its activity on double-stranded RNA nearly 100-fold.