Brown J D, Beggs J D
Institute of Cell and Molecular Biology, University of Edinburgh, UK.
EMBO J. 1992 Oct;11(10):3721-9. doi: 10.1002/j.1460-2075.1992.tb05457.x.
Three different approaches have been used to investigate the roles of the yeast U5 snRNP protein PRP8 in spliceosome assembly: genetic depletion of PRP8 protein in vivo, heat inactivation of temperature-sensitive prp8 protein in protoplasts and inhibition of PRP8 function with antibodies in vitro. In each case, U5 and U4/U6 snRNPs failed to assemble into the forming spliceosomes. In addition, extract prepared from PRP8-depleted cells and extract containing inactivated PRP8 protein had substantially reduced amounts of U4/U6.U5 triple snRNP complexes. Thus, functional PRP8 protein is required for the stable formation of U4/U6.U5 complexes without which spliceosomes fail to form. As spliceosome formation was also blocked by anti-PRP8 antibodies that apparently do not disrupt triple snRNPs, PRP8 protein may play a separate role in the assembly of triple snRNPs into spliceosomes. As a consequence of PRP8 depletion the levels of the U4, U5 and U6 snRNAs declined dramatically. We discuss this in the context of the known genetic interactions between PRP8 and putative RNA helicase (DEAD box protein) genes and propose that PRP8 protein plays a role in regulating dynamic RNA-RNA interactions in spliceosome assembly, possibly ensuring the correct directionality of these events.
人们采用了三种不同的方法来研究酵母U5 snRNP蛋白PRP8在剪接体组装中的作用：在体内对PRP8蛋白进行基因敲减，对原生质体中温度敏感的prp8蛋白进行热灭活，以及在体外使用抗体抑制PRP8的功能。在每种情况下，U5和U4/U6 snRNP都无法组装到正在形成的剪接体中。此外，从PRP8敲减细胞中制备的提取物以及含有失活PRP8蛋白的提取物中，U4/U6.U5三聚体snRNP复合物的量大幅减少。因此，功能性PRP8蛋白是U4/U6.U5复合物稳定形成所必需的，没有它剪接体就无法形成。由于明显不会破坏三聚体snRNP的抗PRP8抗体也会阻断剪接体的形成，所以PRP8蛋白可能在将三聚体snRNP组装到剪接体的过程中发挥独立作用。由于PRP8敲减，U4、U5和U6 snRNA的水平急剧下降。我们将在PRP8与假定的RNA解旋酶（DEAD盒蛋白）基因之间已知的遗传相互作用的背景下讨论这一现象，并提出PRP8蛋白在剪接体组装过程中调节动态RNA-RNA相互作用，可能确保这些事件的正确方向性。
