The Archaeal Lsm Protein Binds to Small RNAs
The Archaeal Lsm Protein Binds to Small RNAs
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DOI：
10.1074/jbc.m110.118950
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发表时间：
2010-11-05
影响因子：
4.8
通讯作者：
Marchfelder, Anita
中科院分区：
文献类型：
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作者：
Fischer, Susan;Benz, Juliane;Marchfelder, Anita
Lsm家族的蛋白质，包括真核生物Sm蛋白和细菌Hfq，是RNA代谢的关键参与者。对这些蛋白质的古细菌同源物知之甚少。因此，我们使用体外和体内方法对来自火山盐古菌Haloferax volcanii的Lsm蛋白进行了表征。H. volcanii编码一个单一的Lsm蛋白，属于Lsm 1亚家族。lsm基因与核糖体蛋白L37 e的基因共转录并重叠。北方印迹分析表明lsm基因是差异转录的。Lsm蛋白形成同源七聚体复合物，并且具有4000个分子/细胞的拷贝数。使用电泳迁移率变动分析和超软质谱（激光诱导液珠离子解吸）的体外分析显示重组Lsm蛋白与寡聚（U）-RNA、tRNA和小RNA形成复合物。与FLAG标记的Lsm蛋白在体内产生的免疫共沉淀证实，该蛋白质结合到小RNA。此外，免疫共沉淀揭示了几个蛋白质相互作用的合作伙伴，这表明它参与不同的细胞途径。Lsm基因的缺失是可行的，导致多效性表型，表明盐古菌Lsm参与许多细胞过程，这与蛋白质相互作用伴侣的数量一致。
Proteins of the Lsm family, including eukaryotic Sm proteins and bacterial Hfq, are key players in RNA metabolism. Little is known about the archaeal homologues of these proteins. Therefore, we characterized the Lsm protein from the haloarchaeon Haloferax volcanii using in vitro and in vivo approaches. H. volcanii encodes a single Lsm protein, which belongs to the Lsm1 subfamily. The lsm gene is co-transcribed and overlaps with the gene for the ribosomal protein L37e. Northern blot analysis shows that the lsm gene is differentially transcribed. The Lsm protein forms homoheptameric complexes and has a copy number of 4000 molecules/cell. In vitro analyses using electrophoretic mobility shift assays and ultrasoft mass spectrometry (laser-induced liquid bead ion desorption) showed a complex formation of the recombinant Lsm protein with oligo(U)-RNA, tRNAs, and an small RNA. Co-immunoprecipitation with a FLAG-tagged Lsm protein produced in vivo confirmed that the protein binds to small RNAs. Furthermore, the co-immunoprecipitation revealed several protein interaction partners, suggesting its involvement in different cellular pathways. The deletion of the lsm gene is viable, resulting in a pleiotropic phenotype, indicating that the haloarchaeal Lsm is involved in many cellular processes, which is in congruence with the number of protein interaction partners.