Applications
- Alternative splicing and gene expression studies.
- Intron cDNA production.
- Intronic screening of cDNA libraries.
Ribonuclease R (RNase R) from E. coli is 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.2 Most
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 MgCl2 up
to 1 mM final concentration can be used to compensate for EDTA in the
substrate. Optimal activity is at 37°C. |
Figure 1. Schematic overview showing processing of intron lariats
by RNase R. |
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
- Suzuki, H. et al. (2006) Nucleic Acids Res. 34,
63.
- Vincent, H.A. and Deutscher, M. P. (2006) J. Biol. Chem. 281,
29769.
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