Institut für Biochemie, Justus-Liebig-Universität Gießen, Gießen, Germany.
RNA Biol. 2011 Jan-Feb;8(1):90-100. doi: 10.4161/rna.8.1.13985. Epub 2011 Jan 1.
Pre-mRNA splicing in trypanosomes requires the SMN-mediated assembly of small nuclear ribonucleoproteins (snRNPs). In contrast to higher eukaryotes, the cellular localization of snRNP biogenesis and the involvement of nuclear-cytoplasmic trafficking in trypanosomes are controversial. By using RNAi knockdown of SMN in T. brucei to investigate its functional role in snRNP assembly, we found dramatic changes in the steady-state levels of snRNAs and snRNPs: The SL RNA accumulates, whereas U1, U4, and U5 snRNA levels decrease, and Sm core assembly in particular of the SL RNA is strongly reduced. In addition, SMN depletion blocks U4/U6 di-snRNP formation; the variant Sm core of the U2 snRNP, however, still forms efficiently after SMN knockdown. Concerning the longstanding question, whether nuclear-cytoplasmic trafficking is involved in trypanosomal snRNP biogenesis, fluorescence in situ hybridization (FISH) and immunofluorescence assays revealed that the SL RNA genes and transcripts colocalize with SMN. Remarkably, SMN silencing leads to a nucleoplasmic accumulation of both SL RNA and the Sm proteins. In sum, our data demonstrate an essential and snRNA-selective role of SMN in snRNP biogenesis in vivo and strongly argue for a nucleoplasmic Sm core assembly of the SL RNP.
在原生动物中，前体 mRNA 的剪接需要 SMN 介导的小核核糖核蛋白 (snRNP) 的组装。与高等真核生物不同，snRNP 生物发生的细胞定位以及核质运输在原生动物中的参与存在争议。通过使用 RNAi 敲低 T. brucei 中的 SMN 来研究其在 snRNP 组装中的功能作用，我们发现 snRNA 和 snRNP 的稳态水平发生了明显变化：SL RNA 积累，而 U1、U4 和 U5 snRNA 水平下降，特别是 SL RNA 的 Sm 核心组装强烈减少。此外，SMN 耗竭会阻止 U4/U6 二 snRNP 的形成；然而，在 SMN 敲低后，U2 snRNP 的变体 Sm 核心仍能有效地形成。关于核质运输是否参与原生动物 snRNP 生物发生的长期问题，荧光原位杂交 (FISH) 和免疫荧光检测显示 SL RNA 基因和转录物与 SMN 共定位。值得注意的是，SMN 沉默导致 SL RNA 和 Sm 蛋白在核质中的积累。总之，我们的数据表明 SMN 在体内 snRNP 生物发生中具有重要的 snRNA 选择性作用，并强烈支持 SL RNP 的核质 Sm 核心组装。
