Mung Bean nuclease mapping of RNAs 3' end
© Bellavia et al; licensee BioMed Central Ltd. 2009
Received: 27 February 2009
Accepted: 21 May 2009
Published: 21 May 2009
A method is described that allows an accurate mapping of 3' ends of RNAs. In this method a labeled DNA probe, containing the presumed 3' end of the RNA under analysis is allowed to anneals to the RNA itself. Mung-bean nuclease is then used to digest single strands of both RNA and DNA. Electrophoretic fractionation of "protected" undigested, labeled DNA is than performed using a sequence reaction of a known DNA as length marker. This procedure was applied to the analysis of both a polyA RNA (Interleukin 10 mRNA) and non polyA RNAs (sea urchin 18S and 26S rRNAs). This method might be potentially relevant for the evaluation of the role of posttrascriptional control of IL-10 in the pathogenesis of the immune and inflammatory mediated diseases associated to ageing. This might allow to develop new strategies to approach to the diagnosis and therapy of age related diseases.
S1 mapping and primer extension are methods used to map the 5' end of an RNA . Although the mapping of 3' ends of RNAs is often as important as the mapping of correspondent 5' ends (i.e. for the presence in the so-called 3' untranslated regions of sequence motifs linked to mRNA stability and/or to map 3'-ends of non polyA RNAs) there is, however, no standard procedure for mapping the 3' end of RNAs.
We developed a simple, reliable method to map the 3' ends of both poly-A and non poly-A RNAs.
After denaturation, the undigested single strand DNA complementary to the 3' end of RNA is electrophoretically fractionated side by side with a sequence reaction of a known DNA used as a marker, which allows the measurement of the length of the undigested, labelled DNA probe. The procedure is reassumed in Figure 1.
We have recently demonstrated (manuscript submitted) in human white blood cells cultured in the presence of LPS, the existence of two interleukin-10 (IL-10) mRNAs, which differ in the length of the 5' UTR regions. To verify if the 3' ends of these mRNA also differ in their respective lengths we used the procedure reported above.
Moreover, to demonstrate that our procedure works well also with non poly-A RNAs, we mapped the 3' ends of the sea urchin Paracentrotus lividus 18S and 26s mature ribosomal RNAs (rRNAs).
For this purpose we used labelled DNA clones of known sequence [EMBL AM981272, EMBL NC000001]  as it is certain that they contain the 3' ends of the aforementioned RNAs, obtained by specific PCR  on correspondent genomic DNAs in the presence of a labelled dNTP.
In lanes 2, 4 and 6, bands of 127 nucleotides (nt), 120 nt and 110 nt, corresponding to the IL-10, 18S and 26S RNA digestions, respectively, indicates the length of the "protected" regions, permitting an accurate mapping of respective 3' ends by comparison with the sequence reaction. In lanes 1, 3 and 5, IL-10, 18S and 26S undigested probes, respectively, were fractionated.
The accuracy and sensibility of our system is also demonstrated by the presence of a faint band in lane 4, which represents the 3' end of a 21S rRNA, precursor of the mature 18S rRNA. The presence of low amounts of this precursor was previously demonstrated  in P. lividus unfertilized eggs. If a "long run" of the electrophoresis shown in Figure 2 is performed, it would be easy to map also the 3' end of this pre-rRNA.
Taking into account the well known role of IL-10 in longevity and in age-related diseases [5–7], we have described a method that might be potentially relevant for the evaluation of the role of posttrascriptional control of IL-10 in the pathogenesis of the immune and inflammatory mediated diseases associated to ageing. This might allow to develop new strategies to approach to the diagnosis and therapy of age related diseases.
This work was supported by funds of Italian MIUR (ex 60%).
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