The U6 small nuclear RNA (snRNA) undergoes major conformational changes during the assembly of the spliceosome and catalysis of splicing. It associates with the specific protein Prp24p, and a set of seven LSm2-8 proteins, to form the U6 small nuclear ribonucleoprotein (snRNP). These proteins have been proposed to act as RNA chaperones that stimulate pairing of U6 with U4 snRNA to form the intermolecular stem I and stem II of the U4/U6 duplex, whose formation is essential for spliceosomal

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... n. However, the mechanism whereby Prp24p and the LSm complex facilitates U4/U6 base-pairing, as well as the exact binding site(s) of Prp24p in the native U6 snRNP, are not well understood. In order to understand the binding site(s) of Prp24 and LSm 2-8 proteins on the U6 snRNA, as well as to shed light on the mechanism whereby Prp24p and the LSm complex facilitate U4/U6 base pairing, purified native U6 snRNPs were thoroughly characterized by chemical structure probing, UV-cross linking and hydroxyl radical footprinting. These three methods demonstrate that the naked U6 snRNA structure is very compact, whereas in the presence of Prp24p and the LSm proteins, the RNA structure in the U6 particle is much more open. This is particularly apparent for the 3´-stem loop and a large internal asymmetrical loop of the U6 snRNA, in which several nucleotides are accessible to chemical modification in the U6 snRNP but are inaccessible to such modification in the naked U6 snRNA. Prp24p binds strongly to the left-hand part of the asymmetrical loop (nucleotides 40-60) and only weakly to the 3´-stem loop in the U6 snRNP. On the contrary, initially a binding of the LSm proteins in the U6 snRNP could not be detected. Interestingly, the 3´-stem loop of the U6 snRNA is strongly contacted by Prp24p when LSm proteins are missing, while, in the presence of both Prp24p and LSm2p-8p the 3´-stem loop assumes a more open conformation. Therefore, we suggest that Prp24p presents the Watson-Crick base pairing positions of the asymmetrical loop. In addition, in cooperation with LSm proteins, Prp24p might be involved in opening up the U6 RNA regions, whereby promoting the formation of stems I and II of the U4/U6 duplex. Interestingly, we find that the open structure of the yeast U6 snRNA in native snRNPs can also be adopted by human U6 and U6atac snRNAs.