The box C/D sRNP dimeric architecture is conserved across domain Archaea
The box C/D sRNP dimeric architecture is conserved across domain Archaea
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DOI：
10.1261/rna.033134.112
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发表时间：
2012-08-01
期刊：
影响因子：
4.5
通讯作者：
Baserga, Susan J.
中科院分区：
文献类型：
--
作者：
Bower-Phipps, Kathleen R.;Taylor, David W.;Baserga, Susan J.
盒C/D小(核仁)核糖核蛋白[S(NO)RNPs]催化核糖核酸引导的2‘-O-核糖甲基化。最近的结构研究导致了关于box C/D rRNPs在功能上组装为单体大分子还是二聚体大分子的争论。通过甘油梯度沉淀、凝胶过滤层析、天然凝胶分析和单粒子电子显微镜(EM)分析表明，来自詹纳斯氏甲烷球菌(Mj)的古菌box C/D sRNP采用di-sRNP结构，每个box C/D核心蛋白包含4个拷贝，Mj SR8 sRNA包含2个拷贝。随后，研究人员使用双链人工引导sRNA CD45，将来自Sulfolobus solfararicus的盒C/D sRNP与短RNA甲基化底物组装在一起，产生了单-sRNP的晶体结构。为了更仔细地研究Box C/D sRNP结构，我们研究了无所不在的sRNA环作为sRNP组装的结构决定因素的作用。我们通过SRNA突变、天然凝胶电泳和单粒子EM表明，当用含有内环的天然或人工sRNAs重组Box C/D sRNP时，disRNP是获得的近乎排他性的结构。我们的结果跨越了三个远亲的古菌物种--Sulfolobus solfararicus、Pyroins abyssi和Archaeoglobus fulgidus--表明di-sRNP结构在整个古菌领域广泛保守。
Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze RNA-guided 2'-O-ribose methylation in two of the three domains of life. Recent structural studies have led to a controversy over whether box C/D sRNPs functionally assemble as monomeric or dimeric macromolecules. The archaeal box C/D sRNP from Methanococcus jannaschii (Mj) has been shown by glycerol gradient sedimentation, gel filtration chromatography, native gel analysis, and single-particle electron microscopy (EM) to adopt a di-sRNP architecture, containing four copies of each box C/D core protein and two copies of the Mj sR8 sRNA. Subsequently, investigators used a two-stranded artificial guide sRNA, CD45, to assemble a box C/D sRNP from Sulfolobus solfataricus with a short RNA methylation substrate, yielding a crystal structure of a mono-sRNP. To more closely examine box C/D sRNP architecture, we investigate the role of the omnipresent sRNA loop as a structural determinant of sRNP assembly. We show through sRNA mutagenesis, native gel electrophoresis, and single-particle EM that a di-sRNP is the near exclusive architecture obtained when reconstituting box C/D sRNPs with natural or artificial sRNAs containing an internal loop. Our results span three distantly related archaeal species-Sulfolobus solfataricus, Pyrococcus abyssi, and Archaeoglobus fulgidus-indicating that the di-sRNP architecture is broadly conserved across the entire archaeal domain.