Zaric Bozidarka L, Kambach Christian
Institut Curie, UMR 7147, Equipe: Recombinaison et Instabilité Génétique, Paris, France.
Methods Enzymol. 2008;448:57-74. doi: 10.1016/S0076-6879(08)02604-9.
Sm and Sm-like (LSm) proteins are an ancient family of proteins present in all branches of life. Having originally arisen as RNA chaperones and stabilizers, the family has diversified greatly and fulfills a number of central tasks in various RNA processing events, ranging from pre-mRNA splicing to histone mRNA processing to mRNA degradation. Defects in Sm/LSm protein-containing ribonucleoprotein assembly and function lead to severe medical disorders like spinal muscular atrophy. Sm and LSm proteins always assemble into and function in the form of ringlike hexameric or heptameric complexes whose composition and architecture determine their intracellular location and RNA and effector protein binding specificity and function Sm/LSm complexes that have been assembled in vitro from recombinant components provide a flexible and invaluable tool for detailed cell biological, biochemical, and biophysical studies on these biologically and medically important proteins. We describe here protocols for the construction of bacterial LSm coexpression vectors, expression and purification of LSm proteins and subcomplexes, and the in vitro reconstitution of fully functional human LSm1-7 and LSm2-8 heptameric complexes.
Sm蛋白和Sm样（LSm）蛋白是一类古老的蛋白质家族，存在于生命的各个分支中。该家族最初作为RNA伴侣和稳定剂出现，后来发生了很大的分化，并在各种RNA加工事件中承担了许多核心任务，从mRNA前体剪接、组蛋白mRNA加工到mRNA降解。含Sm/LSm蛋白的核糖核蛋白组装和功能缺陷会导致严重的医学疾病，如脊髓性肌萎缩症。Sm蛋白和LSm蛋白总是以环状六聚体或七聚体复合物的形式组装并发挥功能，其组成和结构决定了它们在细胞内的定位以及与RNA和效应蛋白的结合特异性和功能。由重组成分体外组装的Sm/LSm复合物为深入研究这些在生物学和医学上具有重要意义的蛋白质提供了一个灵活且极具价值的工具。我们在此描述了构建细菌LSm共表达载体、表达和纯化LSm蛋白及亚复合物，以及体外重建全功能人LSm1-7和LSm2-8七聚体复合物的方法。
