Purification and crystallization of spliceosomal snRNPs

Abstract

In eukaryotes, the non-coding sequences (introns) of precursor messenger RNA are excised by a large ribonucleoprotein complex, the spliceosome. The core components of the spliceosome are small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5 and U6. Besides the U1 snRNA, U1 snRNP comprises three particle-specific proteins, U1-70k, U1-A and U1-C, and a set of seven proteins, also present in other snRNPs, termed Sm proteins. The Sm proteins form a ring-like structure, the Sm core. U2 snRNP is associated with two multi-protein factors, SF3a and SF3b, forming a 17S particle. SF3a comprises three protein factors, SF3a 60, 66 and 120. U4, U5 and U6 snRNPs constitute the 25S U4/U6 U5 tri-snRNP, with 36 proteins and three RNAs. Electron microscopic reconstructions of HeLa U1 snRNP, SF3a and tri-snRNP are available and several substructures solved by NMR and X-ray crystallography are known. Atomic structures of SF3a, tri-snRNP and U1 snRNP would yield fundamental insight into splicing. The objective of this work was to obtain U1 snRNP, tri-snRNP and SF3a preparations suitable for X-ray crystallography.In the work presented here, SF3a and tri-snRNP were purified from HeLa cells, concentrated and subjected to crystallization. HeLa tri-snRNP was sensitive to ultrafiltration or pelleting. Concentration was achieved by ammonium sulfate precipitation followed by dialysis. Highly concentrated and stable tri-snRNP fractions were obtained and subjected to crystallization. Previously, HeLa U1 snRNP crystals were obtained, but not reproducible, and diffracted X-rays to 20 Å. The deliberate inclusion of proteases to the crystallization setup (in situ proteolysis) dramatically increased size and reproducibility of U1 snRNP crystals. An analysis of the contents of the crystals demonstrated that a truncation of several U1 snRNP proteins was a prerequisite for crystal formation. The purification protocol of U1 snRNP was improved by entirely separating U1 and U2 snRNPs. This allowed a combination of a high throughput screening approach with in situ proteolysis in the presence of various U1 snRNP ligands. The combined strategies led to the reproducible production of U1 snRNP crystals, diffracting to 4 Å resolution. Entire datasets to a resolution of 4.5 Å were collected of U1 snRNP crystals, complexed with a short DNA oligonucleotide, mimicking a 5 splice site. Initial phase information for these crystals was obtained. Three heavy atom derivatives were characterized at low resolution and a potential molecular replacement solution, encompassing U1-A and a model of the Sm core, was found. The different phasing strategies were confirmed by cross-validation. These results represent a fundamental step towards the structure at atomic resolution of a major part of U1 snRNP.