Section of Molecular Genetics and Microbiology and the Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA.
RNA. 2013 Jun;19(6):828-40. doi: 10.1261/rna.037671.112. Epub 2013 Apr 19.
Bud23 is responsible for the conserved methylation of G1575 of 18S rRNA, in the P-site of the small subunit of the ribosome. bud23Δ mutants have severely reduced small subunit levels and show a general failure in cleavage at site A2 during rRNA processing. Site A2 is the primary cleavage site for separating the precursors of 18S and 25S rRNAs. Here, we have taken a genetic approach to identify the functional environment of BUD23. We found mutations in UTP2 and UTP14, encoding components of the SSU processome, as spontaneous suppressors of a bud23Δ mutant. The suppressors improved growth and subunit balance and restored cleavage at site A2. In a directed screen of 50 ribosomal trans-acting factors, we identified strong positive and negative genetic interactions with components of the SSU processome and strong negative interactions with components of RNase MRP. RNase MRP is responsible for cleavage at site A3 in pre-rRNA, an alternative cleavage site for separating the precursor rRNAs. The strong negative genetic interaction between RNase MRP mutants and bud23Δ is likely due to the combined defects in cleavage at A2 and A3. Our results suggest that Bud23 plays a role at the time of A2 cleavage, earlier than previously thought. The genetic interaction with the SSU processome suggests that Bud23 could be involved in triggering disassembly of the SSU processome, or of particular subcomplexes of the processome.
Bud23 负责核糖体小亚基 P 位上 18S rRNA 的 G1575 的保守甲基化。bud23Δ 突变体的小亚基水平严重降低，并在 rRNA 加工过程中在 A2 位点显示出普遍的切割失败。A2 位点是分离 18S 和 25S rRNA 前体的主要切割位点。在这里，我们采用遗传方法来鉴定 BUD23 的功能环境。我们发现编码 SSU 加工体成分的 UTP2 和 UTP14 基因突变是 bud23Δ 突变体的自发抑制子。这些抑制子改善了生长和亚基平衡，并恢复了 A2 位点的切割。在针对 50 种核糖体反式作用因子的定向筛选中，我们鉴定了与 SSU 加工体成分的强烈正和负遗传相互作用，以及与 RNase MRP 成分的强烈负遗传相互作用。RNase MRP 负责 pre-rRNA 中 A3 位点的切割，这是分离前体 rRNA 的替代切割位点。RNase MRP 突变体与 bud23Δ 之间的强烈负遗传相互作用可能是由于 A2 和 A3 切割的综合缺陷所致。我们的结果表明，Bud23 在 A2 切割时发挥作用，比以前认为的更早。与 SSU 加工体的遗传相互作用表明，Bud23 可能参与触发 SSU 加工体或加工体的特定亚基复合物的解体。
