Reconstitution of recombinant human LSm complexes for biochemical, biophysical, and cell biological studies

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.