Nucleolar protein Nop12p participates in synthesis of 25S rRNA in Saccharomyces cerevisiae

A genetic screen for mutations synthetically lethal with temperature sensitive alleles of nop2 led to the identification of the nucleolar proteins Nop12p and Nop13p in Saccharomyces cerevisiae. NOP12 was identified by complementation of a synthetic lethal growth phenotype in strain YKW35, which contains a single nonsense mutation at codon 359 in an allele termed nop12-1. Database mining revealed that Nop12p was similar to a related protein, Nop13p. Nop12p and Nop13p are not essential for growth and each contains a single canonical RNA recognition motif (RRM). Both share sequence similarity with Nsr1p, a previously identified, non-essential, RRM-containing nucleolar protein. Likely orthologs of Nop12p were identified in Drosophila and Schizosaccharomyces pombe. Deletion of NOP12 resulted in a cold sensitive (cs) growth phenotype at 15°C and slow growth at 20 and 25°C. Growth of a nop12Δ strain at 15 and 20°C resulted in impaired synthesis of 25S rRNA, but not 18S rRNA. A nop13 null strain did not produce an observable growth phenotype under the laboratory conditions examined. Epitope-tagged Nop12p, which complements the cs growth phenotype and restores normal 25S rRNA levels, was localized to the nucleolus by immunofluorescence microscopy. Epitope-tagged Nop13p was distributed primarily in the nucleolus, with a lesser portion localizing to the nucleoplasm. Thus, Nop12p is a novel nucleolar protein required for pre-25S rRNA processing and normal rates of cell growth at low temperatures.

以与nop2温度敏感等位基因存在合成致死作用的突变为靶标开展遗传筛选，本研究在酿酒酵母（Saccharomyces cerevisiae）中成功鉴定得到核仁蛋白Nop12p与Nop13p。研究人员通过互补实验验证菌株YKW35的合成致死生长表型，从而克隆得到NOP12基因；该菌株携带等位基因nop12-1，其359号密码子处存在单个无义突变。数据库挖掘结果显示，Nop12p与另一相关蛋白Nop13p具有序列相似性。Nop12p与Nop13p并非酵母生长所必需的蛋白，二者均包含单个典型的RNA识别基序（RNA recognition motif, RRM）。二者均与此前已鉴定的、非必需的含RRM核仁蛋白Nsr1p存在序列相似性。本研究在果蝇（Drosophila）与裂殖酵母（Schizosaccharomyces pombe）中均鉴定得到了Nop12p的潜在直系同源蛋白。敲除NOP12基因会导致酵母在15℃下出现冷敏感（cold sensitive, cs）生长表型，并在20℃与25℃下生长缓慢。在15℃与20℃条件下培养nop12Δ缺失菌株时，其25S核糖体RNA（rRNA）的合成过程受到损伤，但18S rRNA的合成未受明显影响。nop13基因的敲除菌株在本研究检测的实验室培养条件下未表现出可观测的生长表型。经表位标签标记的Nop12p可互补冷敏感生长表型并恢复正常的25S rRNA水平，免疫荧光显微镜观察显示其定位于核仁中。经表位标签标记的Nop13p主要分布于核仁，仅有少量定位于核质。综上，Nop12p是一种新型核仁蛋白，其参与pre-25S rRNA的加工过程，并对低温下细胞的正常生长速率至关重要。