Kos Martin, Tollervey David
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.
Mol Cell. 2005 Oct 7;20(1):53-64. doi: 10.1016/j.molcel.2005.08.022.
Around 70 yeast snoRNAs guide rRNA modification, frequently forming base-paired interactions predicted to be very stable at physiological temperatures. Eighteen putative RNA helicases are required for ribosome synthesis, but their actual substrates were not known. We report that depletion of the DEAD box helicase Dbp4p dramatically increased cosedimentation of the snoRNAs U14 and snR41 with preribosomes. Cosedimentation was maintained after deproteinization by proteinase K, indicating that the snoRNAs remained base paired to the pre-rRNA. Affinity purification showed that U14 was strongly accumulated in early 90S preribosomes and depleted from later pre-40S complexes. U14 is required for pre-rRNA processing, and depletion of Dbp4p caused a very similar pre-rRNA processing defect, perhaps due to the reduced pool of free U14. Point mutations in helicase motifs I and III of Dbp4p blocked release of U14 from preribosomes. We conclude that the helicase activity of Dbp4p is required to unwind U14 and snR41 from the pre-rRNA.
约70种酵母小核仁RNA（snoRNA）指导核糖体RNA（rRNA）修饰，它们常形成碱基配对相互作用，预计在生理温度下非常稳定。核糖体合成需要18种假定的RNA解旋酶，但其实际底物尚不清楚。我们报告称，DEAD盒解旋酶Dbp4p的缺失显著增加了小核仁RNA U14和snR41与前核糖体的共沉降。用蛋白酶K进行脱蛋白处理后，共沉降仍得以维持，这表明小核仁RNA仍与前体rRNA碱基配对。亲和纯化显示，U14在早期90S前核糖体中强烈积累，而在后期40S前体复合物中减少。U14是前体rRNA加工所必需的，Dbp4p的缺失导致了非常相似的前体rRNA加工缺陷，这可能是由于游离U14池减少所致。Dbp4p解旋酶基序I和III中的点突变阻止了U14从前核糖体中释放。我们得出结论，Dbp4p的解旋酶活性是将U14和snR41从前体rRNA上解开所必需的。
