Barabino S M, Blencowe B J, Ryder U, Sproat B S, Lamond A I
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Cell. 1990 Oct 19;63(2):293-302. doi: 10.1016/0092-8674(90)90162-8.
HeLa cell nuclear splicing extracts have been prepared that are specifically and efficiently depleted of U1, U2, or U4/U6 snRNPs by antisense affinity chromatography using biotinylated 2'-OMe RNA oligonucleotides. Removal of each snRNP particle prevents pre-mRNA splicing but arrests spliceosome formation at different stages of assembly. Mixing extracts depleted for different snRNP particles restores formation of functional splicing complexes. Specific binding of factors to the 3' splice site region is still detected in snRNP-depleted extracts. Depletion of U1 snRNP impairs stable binding of U2 snRNP to the pre-mRNA branch site. This role of U1 snRNP in promoting stable preslicing complex formation is independent of the U1 snRNA-5' splice site interaction.
已制备出HeLa细胞核剪接提取物，通过使用生物素化的2'-O-甲基RNA寡核苷酸进行反义亲和层析，可特异性且高效地去除U1、U2或U4/U6 snRNP。去除每种snRNP颗粒会阻止前体mRNA剪接，但会在组装的不同阶段阻止剪接体形成。将针对不同snRNP颗粒耗尽的提取物混合可恢复功能性剪接复合物的形成。在snRNP耗尽的提取物中仍可检测到因子与3'剪接位点区域的特异性结合。U1 snRNP的耗尽会损害U2 snRNP与前体mRNA分支位点的稳定结合。U1 snRNP在促进稳定的剪接前复合物形成中的这一作用独立于U1 snRNA-5'剪接位点相互作用。
