E. coli RNase III(E38A) generates discrete-sized products from long dsRNA.
Ribonuclease III (RNase III) represents a highly conserved family of double-strand-specific endoribonucleases that are important for RNA processing and post-transcriptional gene regulation in both prokaryotes and eukaryotes. We constructed a single amino acid substitution (E38A) of RNase III that shows a unique and useful enzymatic activity. It produces a dsRNA product of a discrete size migrating as 23 base pairs (bp) when given a long dsRNA as a substrate in an easy-to-control reaction. We demonstrate that the RNase III(E38A) mutant produces the 23-bp dsRNA product by making a double-strand cleavage of the long dsRNA substrate with the product being protected from further digestion. Using the hairpin RNA R1.1 as a substrate, RNase III(E38A) cleaves at the primary site and remains bound to the RNA, thereby preventing cleavage at the secondary site. The 23-bp dsRNA product is demonstrated to be a pool of dsRNAs representative of the long dsRNA substrate and has RNA interference activity in mammalian tissue culture transfection experiments. The RNA interference activity suggests that the 23-bp dsRNA product has typical 2-nucleotide 3' overhangs and behaves as siRNA thereby making it a useful tool in RNA interference experiments.
Xiao
Jianping
J
New England Biolabs, Ipswich, Massachusetts 01938, USA.
Feehery
Caitlin E
CE
Tzertzinis
George
G
Maina
Claude V
CV
D019943:Amino Acid Substitution; D001483:Base Sequence; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D043244:Ribonuclease III / Q000235:genetics* / Q000378:metabolism*
D029968:Escherichia coli Proteins; D012330:RNA, Double-Stranded; D043244:Ribonuclease III; C468366:ribonuclease III, E coli