Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae
Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae
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DOI：
10.1017/s1355838298981158
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发表时间：
1998-10-01
期刊：
影响因子：
4.5
通讯作者：
Linder, P
中科院分区：
文献类型：
--
作者：
de la Cruz, J;Kressler, D;Linder, P
Spb4p是一种假定的atp依赖性RNA解旋酶，是合成60S核糖体亚基所必需的。对Spb4p基因缺失或携带冷敏感spb4-1突变菌株的多体分析显示60S核糖体亚基积累不足。通过脉冲追踪标记、northern杂交和引物延伸对pre-rRNA加工进行分析表明，由于抑制了27SB pre-rRNA的加工，这些菌株的25S/5.8S rrna的合成减少。在Spb4p耗尽的后期或将spb4-1菌株转移到更严格的温度后，位点A(0)， A(1)和A(2)的早期pre-rRNA加工步骤也被抑制。蔗糖梯度分离表明，积累的27SB前rnas与一个高分子量复合物有关，最有可能是66S前核糖体颗粒。HA表位标记的Spb4p定位于核仁和邻近的核质区域。在蔗糖梯度上，HA-Spb4p几乎全部存在于快速沉积复合物中，在含有66S前核糖体的组分中表现出峰值。我们认为Spb4p可能作为rRNA解旋酶，直接参与了60S核糖体亚基组装的后期和关键步骤。
Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits. Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an underaccumulation of 60S ribosomal subunits. Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs. At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A(0), A(1), and A(2) were also inhibited. Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area. On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase.