de la Cruz J, Kressler D, Rojo M, Tollervey D, Linder P
Département de Biochimie Médicale, Centre Médical Universitaire, Université de Genève, Geneva, Switzerland.
RNA. 1998 Oct;4(10):1268-81. doi: 10.1017/s1355838298981158.
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 A0, Al, and A2 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.
Spb4p是一种假定的ATP依赖性RNA解旋酶，是60S核糖体亚基合成所必需的。对基因缺失Spb4p或携带冷敏感spb4 - 1突变的菌株进行多核糖体分析，发现60S核糖体亚基积累不足。通过脉冲追踪标记、Northern杂交和引物延伸对前体rRNA加工进行分析，结果表明这些菌株中25S/5.8S rRNA的合成减少，这是由于27SB前体rRNA的加工受到抑制。在Spb4p缺失的后期或spb4 - 1菌株转移到更严格的温度条件后，A0、A1和A2位点的早期前体rRNA加工步骤也受到抑制。蔗糖梯度分级分离显示，积累的27SB前体rRNA与一种高分子量复合物相关，很可能是66S前核糖体颗粒。带有HA表位标签的Spb4p定位于核仁及相邻的核质区域。在蔗糖梯度上，HA - Spb4p几乎只存在于快速沉降的复合物中，并在含有66S前核糖体的级分中出现峰值。我们认为Spb4p直接参与60S核糖体亚基组装过程中的一个后期关键步骤，推测其作为一种rRNA解旋酶发挥作用。
