Published online 28 June 2004
Nucleic Acids Research, Vol. 32 No. 11 © Oxford University Press 2004; all rights reserved
A novel 4-base-recognizing RNA cutter that can remove the single 3' terminal nucleotides from RNA molecules
Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Higashijima 265-1, Niitsu, Niigata 956-8603, Japan
* To whom correspondence should be addressed. Tel: +81 250 25 5119; Fax: +81 250 25 5021; Email: mnashimoto{at}niigatayakudai.jp
Received May 11, 2004; Revised and Accepted June 6, 2004
Mammalian tRNase ZL shows versatility in substrate recognition. This enzyme can not only process pre-tRNAs by cleaving off their 3' trailer sequences, but also recognize and cleave pre-tRNA-like complexes and micro-pre-tRNAs. Here we demonstrate that 24–27 nt hairpin RNAs (hook RNAs) can guide cleavages of separate target RNAs by tRNase ZL through the micro-pre-tRNA-like complexes between the targets and the hook RNAs and that tRNase ZL together with hook RNA works as 4–7-base-recognizing RNA cutters. The cleavage sites were located only after the nucleotide corresponding to the discriminator nucleotide. Cleavage assays for various substrate/hooker complexes showed that the cleavage efficiency changes depending on the maximum number of substrate/hooker recognition base pairings and the stem length of hook RNA and that a 5 nt recognition sequence and a hook RNA containing a 6 or 7 bp stem are the best combination for the optimal target cleavage. We also show that a 4-base RNA cutter can remove the single 3' terminal nucleotides from RNA molecules. These results indicate that this new type of RNA cutter can be utilized to homogenize at their 3' termini RNA transcripts synthesized in vitro with a bacteriophage RNA polymerase.
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