RNase R

• Alternative splicing and gene expression studies.
• Intron cDNA production.
• Intronic screening of cDNA libraries.

Ribonuclease R (RNase R) fromE. coliis a magnesium-dependent 3´→5´ exoribonuclease that digests essentially all linear RNAs but does not digest lariat or circular RNA structures1,2, or doublestranded RNA with 3´ overhangs shorter than seven nucleotides.2Most cellular RNAs will be digested completely by RNase R, with the exception of tRNAs, 5S RNA, and intron lariats (Fig. 1). The 3´ tails of lariats will be trimmed by RNase R to the branch point nucleotide, where there is a 2´,5´-phosphodiester linkage. Lariats are produced during pre-mRNA splicing of intron regions and can be isolated from a mixture of total RNA by digestion with RNase R. The ArrayPure™ Kit, and MasterPure™ RNA and Yeast RNA Purification Kits are ideal for such total RNA preparations.

RNA isolated using this method can be used as a template to produce labeled cDNA as a target for microarrays containing potential intron sequences, or for tiling arrays containing overlapping regions of complete chromosomes or genomes. The cDNA produced will not be a linear representation of the intron, but the sequences contained in it will be intron-derived.

Unit Definition:One unit of RNase R converts 1 µg of poly(A) into acid-soluble nucleotides in 10 minutes at 37°C under standard assay conditions.

Storage Buffer:RNase R is supplied in a 50% glycerol solution containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton® X-100.

RNase R 10X Reaction Buffer:0.2 M Tris-HCl (pH 8.0), 1 M KCl, and 1 mM MgCl2.

Note: RNase R requires low (0.1-1.0 mM) magnesium concentrations for activity. Low EDTA concentrations in substrate RNA solutions can negatively affect RNase R activity. Additional MgClup to 1 mM final concentration can be used to compensate for EDTA in the substrate. Optimal activity is at 37°C

Quality Control:RNase R is function-tested in a reaction containing a mixture of linear and circularized RNA oligonucleotides. Only the linear RNA is digested.

References

1. Suzuki, H.et al.(2006)Nucleic Acids Res.34, 63.
2. Vincent, H.A. and Deutscher, M. P. (2006)J. Biol. Chem.281, 29769.