Structural and functional characterization of proteins involved in the biogenesis of spliceosomal U snRNPs

One major hallmark of eucaryotic organisms is the subdivision of their cells into different, membrane enclosed compartments. In their central compartment, the nucleus, eucaryotes store their condensed genetic information. In order to translate this genetic information into the protein sequence the DNA has to be transcribed into RNA. However, transcription does not immediately lead to mature messenger RNA (mRNA) but results in a pre-mature messenger RNA (pre-mRNA) containing coding regions (exons) as well as non-coding regions (introns). Prior to their transport to the cytoplasm the introns of pre-mRNAs are excised by a large ribonucleoprotein complex, the spliceosome. The major components of the spliceosome are the uridyl-rich small nuclear ribonucleoprotein particles (so-called UsnRNPs), whose biogenesis requires a nucleocytoplasmic transport cycle. The major goal of the present work was the structural and functional characterization of two different proteins and a protein complex, which either are required for the biogenesis of UsnRNPs or which bind the m7G-cap. The first protein described is the dimethyltransferase TGS1 (Trimethylguanosine Synthase 1), which catalyzes the dimethylation of the 5 -guanine base of specific RNA caps. The second project concerned the characterization of a nuclear export complex, consisting of the cargo protein snurportin 1, its exportin (exportin 1) as well as the molecular switch RanGTP. The third project comprised the analysis and characterization of the binding mode of the poly(A)-specific ribonuclease (PARN) to the mRNA 5 -cap. The results of the three independent projects are briefly summarized below. The methyltransferase domain of the Trimethylguanosine Synthase 1 (TGS1), which hypermethylates the m7G-cap of spliceosomal UsnRNAs during biogenesis of the corresponding RNP was crystallized and its crystal structure was determined. The active form of the methyltransferase domain comprises the structurally conserved methyltransferase fold as well as a small N-terminal and α-helical domain. Biochemical as well as further crystallographic analyses revealed that this additional N-terminal domain is strictly required for both, substrate binding and catalysis. This functional characterization was enabled by a newly established HPLC-based methyltransferase activity assay. Moreover, a previously postulated structure based reaction mechanism could be verified biochemically by a combination of this assay and site-directed mutagenesis studies. The nuclear export complex comprising exportin 1 (Xpo1, CRM1), its cargo snurportin1 as well as the molecular switch Ran in its GTP bound form was recombinantly expressed, assembled in vitro and crystallized. The crystal structure analysis revealed that CRM1 adopts an overall superhelical, toroid-shaped conformation and that the GTPase Ran is enwrapped by the exportin s inner surface. Unexpectedly, the cargo snurportin 1 binds on the outer surface of CRM1 including three different areas and does not make a single direct contact to the molecular switch RanGTP. However, between the cargo and RanGTP indirect contacts are mediated by the so-called acidic loop of CRM1. This strategy explains on the one hand the extremely broad substrate spectrum of CRM1 and on the other hand the apparent cooperativity of binding between RanGTP and snurportin 1. The RNA recognition motif (RRM) of the poly(A)-specific ribonuclease (PARN) was purified and crystallized in complex with the cap analog m7GTP. The crystal structure as well as further biochemical studies revealed that the positively charged m7G-cap is bound in an unexpected mode, which has not been observed so far. While other structurally defined cap-binding proteins bind the methylated purine base in between two aromatic or hydrophobic residues, PARN stacks the base only on one side by a tryptophan side chain and lacks a protein residue on the opposing side. The binding mode observed in the crystal structure could be verified by means of fluorescence spectroscopy. The change of the emitted tryptophan fluorescence upon cap binding allowed the determination of PARN-cap dissociation constants for the wild type protein as well as for some single amino acid mutants.