Functional and sructural investigation of spliceosomal snRNPs.

The expression of most eukaryotic protein-encoding genes involves precursor messenger RNA (pre-mRNA) processing steps including pre-mRNA splicing. Pre-mRNA splicing is catalyzed by a multi-subunit RNA-protein enzyme, the spliceosome, which emerges from the stepwise recruitment of the U1, U2, U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs) and numerous non-snRNP proteins to conserved sequences of the pre-mRNA substrate. The U2 snRNP is essential for splicing in yeast and humans, participating in the recognition/selection of the so-called branch site (BS) of the pre-mRNA during spliceosome assembly and also during the subsequent catalysis of splicing. 12S U2 snRNPs are composed of U2 snRNA, which is complexed by U2 snRNP specific proteins, U2-A' and U2-B'', and seven Sm proteins. In catalytically active human 17S U2 snRNPs, two heteromeric splicing factors, SF3a and SF3b, are additionally found, which contain three (SF3a120, SF3a66 and SF3a60) and seven (SF3b155, SF3b145, SF3b130, SF3b49, SF3b14a/p14, SF3b14b and SF3b10) proteins, respectively. In the yeast Sacharomyces cerevisiae, SF3b complexes were shown to consist of yeast orthologs Hsh155p, Rse1p, Cus1p, Hsh49p, Rds3p, Rcp10p/Ysf3p, and at least two additional proteins Bud31p and Snu17p/Ist3p. Moreover, Snu17p is found in a complex with proteins Pml1p and Bud13p. Together, these three proteins form the pre-mRNA retention and splicing (RES) complex, which counteracts the escape of unspliced pre-mRNAs from the nucleus and activates splicing of a subset of Mer1p-dependent genes. A homologous complex is present in human pre-catalytic, activated and step 1 spliceosomes. Structural analyses by electron microscopy, nuclear magnetic resonance spectroscopy and X-ray crystallography gave important insight into structural arrangements among 17S U2 snRNP subcomplexes and proteins, but atomic models of 12S U2 snRNPs, SF3b particles and the RES complex remained elusive. To derive atomic models of entire human 12S U2 snRNPs and SF3b particles, I set out to produce these particles for X-ray crystallographic analyses. I was able to optimize purification protocols for both natively isolated 12S U2 snRNPs and SF3b complexes. Since no crystals could be obtained from native 12S U2 snRNPs, presumed unstructured regions of U2 snRNA, Sm proteins Sm B/B' and U2-A' and U2-B'' were successfully removed by a DNA-directed RNaseH-based cleavage and limited proteolysis, respectively. U2 snRNP particles with truncated snRNA or a truncated snRNA and truncated proteins Sm B/B' were subjected to crystallization at a concentration of at least 10 mg/ml. U2 snRNP particles with truncated snRNA, truncated proteins Sm B/B' and truncated U2-A' and U2-B'' were obtained in analytical scale. The established purification protocols would allow preparative production in the future. SF3b particles were purified and screened for crystal growth at concentrations of at least 9 mg/ml. During the preparation of SF3b particles, a yet unknown complex could be isolated and characterized. It comprises at least 4 proteins, including the DEAD-box helicase DDX1 and proteins HSPC117, Family with sequence similarity 98/member B and CGI-99. Its association with SF3b particles is discussed. To expand knowledge about Snu17p and its interaction partners, I set out to characterize yeast RES complexes by biophysical and biochemical methods, with the ultimate goal to elucidate the molecular architecture of the RES complex. GST pull-down experiments and size exclusion chromatography revealed that Snu17p constitutes the central platform of the complex, while Bud13p and Pml1p do not interact with each other. Fluorimetric structure probing showed the entire Bud13p and the N-terminal third of Pml1p to be natively disordered in isolation. Mutational analysis and tryptophan fluorescence confirmed that a conserved tryptophan-containing motif in the C-terminus of Bud13p binds to the core RRM of Snu17p, while a different interaction surface encompassing a C-terminal extension of the Snu17p RRM is required to bind an N-terminal peptide of Pml1p. Isothermal titration calorimetry revealed 1:1 interaction stoichiometries, large negative binding entropies and dissociation constants in the low nanomolar and micromolar ranges for the Snu17p-Bud13p and the Snu17p-Pml1p interactions, respectively. By performing peptide-scanning experiments, the Snu17p interacting regions of Bud13p and Pml1p could be further delineated with single amino acid resolution. Preliminary results from NMR spectroscopy and limited proteolysis indicate that Snu17p persists in a molten globule like structure and folds upon binding of at least one of the interating peptides. Thus, the non-canonical Snu17p RRM concomitantly binds multiple ligand proteins via short, intrinsically unstructured peptide epitopes and thereby acts as a platform that displays functional modules of the ligands, such as a forkhead-associated domain of Pml1p and a conserved poly-lysine motif of Bud13p. The crystal structures of full-length and N-terminally truncated Pml1p were determined and revealed the presence of a forkhead-associated (FHA) domain fold in the C-terminal region. FHA domains are small protein modules, which bind phosphorylated epitopes on proteins. The first 50 residues of Pml1p, encompassing the Snu17p-binding region, are disordered. A non-canonical N-terminal expansion runs across one -sheet and, thereby, critically stabilizes the domain. Structure based alignments identified a similar expansion in the human protein NIPP1, which was previously shown to be associated with spliceosomes. A sulfate ion was found at the putative phosphopeptide-binding loops of full-length Pml1p while the truncated protein lacked a similar phosphopeptide mimic but retained an almost identical structure. A long loop neighboring the phosphopeptide-binding site was disordered in both structures and may confer ligand specificity. It is speculated that Pml1p most likely recognizes the phosphorylated amino acid of ligands by a lock-and-key mechanism, while specificity relies on induced-fit interactions. The results suggest that Snu17p recruits Pml1p as a phosphorylation sensor to the spliceosome. Finally, a model of the molecular architecture of the RES complex is presented.