Antisense snRNA

Northern blot analysis of mixed RNAs using multiple snRNA probes Recovered RNA may be analysed by 3 -end labelling with pCp (Section 2.3.3.2) followed by denaturing gel electrophoresis. Breakdown products due to water hydrolysis or contaminating RNases have a 3 -phosphate and will not be 3 -end labelled by pCp (see e.g. Ref. 11). However, if known species of RNA are to be analysed, RNase protection or Northern blotting are the recommended methods. The procedure below describes the quantification of the 5 snRNAs in spliceosomal complexes (Ul, U2, U4, U5, and U6 snRNAs), essentially as described by Grabowsky and Sharp.10 The RNAs are separated in a denaturing polyacrylamide gel, transferred to a membrane and probed with a mixture of five antisense snRNA probes (Fig. 5.4). 

Liu S, Asparuhova M, Brondani V, Ziekau I, Klimkait T, Schumperh D (2004) Inhibition of HlV-1 multiplication by antisense U7 snRNAs and siRNAs targeting cyclophilin A, Nucleic Acids Res 32 3752-3759... 

Of the five snRNAs, U2 and U6 interact with the reaction site (the 5 splice site and the branch point) in the first chemical step. These two snRNAs are known to anneal together to form a stable-based paired structure in the absence of proteins and in the presence of ions as shown in Fig. 13, with U2 acting as an inducer molecule that displaces the U4 (that is an antisense molecule that regulates the catalytic function of U6 RNA) from the initially formed U4-U6 duplex. The secondary (or higher ordered) structure of the U2-U6 complex consists of the active site of the spliceosome. Recent data suggests that these two snRNAs function as the catalytic domain of the spliceosome that catalyzes the first step of the splicing reaction [145]. 

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