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Investigation of the Structurally Related U4 snRNP, U4atac snRNP and Box C/D s(no)RNPs
von Homa GhaleiSplicing is an essential step in the processing of eukaryotic pre-mRNAs, during which a highly dynamic ribonucleoprotein (RNP), the spliceosome, catalyzes the removal of non-coding sequences from a precursor-messenger RNA (pre-mRNA). Two distinct types of spliceosome have been characterized. The major spliceosome exists in all eukaryotes and is comprised of U1, U2, U5 and U4/U6 snRNPs. The minor spliceosome has been only found in some metazoans and plants and splices a rare class of pre-mRNA introns. The latter spliceosome contains U11/U12 and U4atac/U6atac snRNPs, which are functional analogues of U1, U2 and U4/U6 snRNPs of the major spliceosome. U5 snRNP is common to both spliceosomes. One of the major building blocks of spliceosomes is the 25S tri-snRNP comprising the U4/U6 (U4atac/U6atact) and U5 snRNAs and many distinct proteins. The tri-snRNP joins the pre-spliceosome as a pre-formed complex in which the U4/U6 (U4atac/U6atac) di-snRNPs are connected to U5 snRNP. The interaction of the U5 specific protein, human Prp6 (hPrp6), with the protein human Prp31 (hPrp31) from the U4/U6 (U4atac/U6atac) di-snRNP plays a key role in bringing the tri-snRNP components together. While the relative positions of Prp8, Brr2, Snu114, LSm8, Prp3, Prp6 and Prp31 in the yeast tri-snRNP have been mapped by electron microscopy, there is no atomic model available for the interactions that bridge the U4/U6 and U5 snRNPs. Protein hPrp6 is also considered to constitute an interaction platform for several other U5 specific proteins (i. e. hPrp8, hBrr2 and hSnu114). To find out more about the structural organization of hPrp6 and the bridging interactions within the tri-snRNP, part of the work in this thesis was focused on structural analyses of hPrp6. The expression and purification of recombinant hPrp6 were optimized and its precise domain borders were defined. Using a peptide array of the disordered C-terminal region of hPrp31 and a well-defined fragment of hPrp6, a minimal linear binding epitope of hPrp31 was determined.
Although the sequences of U4atac and U6atac snRNAs differ from those of U4 and U6, the U4atac/U6atac and U4/U6 duplexes bind the same set of proteins. In both cases, the protein 15.5K recognizes a kink-turn (K-turn) region of the 5'-stem loops (5'-SLs) of the snRNAs. Binding of 15.5K to the K-turn sets the stage for subsequent recruitment of hPrp31. The crystal structure of hPrp31-15.5K-U4atac 5'-SL complex revealed that hPrp31 interacts with the central residues of the U4atac 5'-SL in a sequence-independent fashion as previously also seen in the analogous U4-based complex. However, a pentaloop region in U4atac snRNA interacts more intimately with hPrp31 side chains than observed in the U4-based complex. Using fluorescence spectroscopy, we could show that while the primary binding protein 15.5K does not contact the U4atac pentaloop directly, it induces formation of a non-canonical base pair from a distance. The observed folding of the RNA upon protein binding increases the specificity and modulates the stability of the ternary complexes. Using site-directed mutagenesis, it could be confirmed that the locally different interactions between hPrp31 and U4 or U4atac snRNA form the basis for differential stabilities of the U4 and U4atac snRNPs.
Box C/D s(no)RNPs are small (nucleolar) ribonucleoprotein particles that guide the sitespecific 2'-O-methylation of nucleotides in target rRNAs and are therefore essential for the biogenesis and the maturation of ribosomes. The box C/D sRNAs contain evolutionarily conserved sequence elements, i. e. the box C/D and the box C'/D' motifs, which adopt K-turns. Protein 15.5K also recognizes the K-turn region of the box C/D snoRNAs. Binding of 15.5K to the box C/D RNA is the prerequisite for association of Nop56/Nop58 proteins (Nop5 in archaea). Despite relaxed RNA sequence requirements for binding of hPrp31 to the 15.5KsnRNPs, hPrp31 does not bind to the 15.5K-box C/D snoRNPs. Nop56/58 proteins share a highly conserved RNA binding module with hPrp31, the so-called NOP domain. An additional N-terminal domain in these proteins is responsible for interaction with the methyl-transferase, fibrillarin. The conserved box C/D and C'/D' elements in box C/D s(n)RNAs are intervened by guide regions, which base pair to complementary sequences in the substrate RNA, thereby specifying and positioning nucleotides for modification by fibrillarin. To elucidate the molecular mechanisms underlying the specific RNP assemblies and to reveal how the components of snoRNPs bring about functional enzymatic machinery, we aimed at structural characterization of an archaeal box C/D sRNP. Recombinant expression and in vitro reconstitution of a Pyrococcus furiosus (P. fur.) box C/D sRNP were established. Using analytical gel filtration and electron microscopy (EM), it was shown that P. fur.-sRNP components assemble into di-sRNPs. Using cryo-EM, the dynamic network of interactions within box C/D elements and the structural rearrangements of the complex were revealed. After complete structural refinement, which is still ongoing, the EM structural data should shed light on the mechanism of catalytic activation of the box C/D sRNPs. RNA annealing assays suggested an additional role for fibrillarin in substrate recognition and binding, besides its role in the methylation process. Analysis of UVinduced protein-RNA crosslinks in the in vitro assembled P. fur.-box C/D sRNPs revealed a novel interaction between the ALFR motif, in the NOP domain of Nop5, and the guide regions of the sRNA. Mutational analyses showed that the ALFR motif and the guide sequence adjacent to box C or C' are important for box C/D sRNP assembly in vitro. The presented cross-link data therefore reveal new RNA-protein contacts in the box C/D sRNP and suggest a role for Nop5 in substrate binding and/or release.
In vivo and in vitro studies have shown that eukaryotic box C/D snoRNPs require complex assembly machineries. Archaeal box C/D sRNPs are simplified homologs of the eukaryotic snoRNPs. However, owing to their extreme growth conditions and efficient in vitro assembly, archaeal box C/D sRNPs have so far not been investigated with regards to their possible in vivo assembly factors. One candidate assembly factor that is selectively present in certain hyperthermophilic archaeal species is Rbp18. In this thesis, possible functions of Rbp18 were investigated by structural analyses, RNA binding and RNA annealing assays. Two crystal structures of Rbp18 from Pyrococcus furiosus and Aeropyrum pernix were determined. The structures revealed a conserved novel fold and a surprising architectural diversity among the members of Rbp18 family. The N-terminal domain of the protein showed structural homology to the conserved N-terminal domain of ribosomal protein L11. Biochemical studies revealed that Rbp18 can bind to RNA and enhance annealing of two complementary RNA strands. These data therefore suggest a role for Rbp18 in RNA protection and chaperoning at elevated temperatures.
Although the sequences of U4atac and U6atac snRNAs differ from those of U4 and U6, the U4atac/U6atac and U4/U6 duplexes bind the same set of proteins. In both cases, the protein 15.5K recognizes a kink-turn (K-turn) region of the 5'-stem loops (5'-SLs) of the snRNAs. Binding of 15.5K to the K-turn sets the stage for subsequent recruitment of hPrp31. The crystal structure of hPrp31-15.5K-U4atac 5'-SL complex revealed that hPrp31 interacts with the central residues of the U4atac 5'-SL in a sequence-independent fashion as previously also seen in the analogous U4-based complex. However, a pentaloop region in U4atac snRNA interacts more intimately with hPrp31 side chains than observed in the U4-based complex. Using fluorescence spectroscopy, we could show that while the primary binding protein 15.5K does not contact the U4atac pentaloop directly, it induces formation of a non-canonical base pair from a distance. The observed folding of the RNA upon protein binding increases the specificity and modulates the stability of the ternary complexes. Using site-directed mutagenesis, it could be confirmed that the locally different interactions between hPrp31 and U4 or U4atac snRNA form the basis for differential stabilities of the U4 and U4atac snRNPs.
Box C/D s(no)RNPs are small (nucleolar) ribonucleoprotein particles that guide the sitespecific 2'-O-methylation of nucleotides in target rRNAs and are therefore essential for the biogenesis and the maturation of ribosomes. The box C/D sRNAs contain evolutionarily conserved sequence elements, i. e. the box C/D and the box C'/D' motifs, which adopt K-turns. Protein 15.5K also recognizes the K-turn region of the box C/D snoRNAs. Binding of 15.5K to the box C/D RNA is the prerequisite for association of Nop56/Nop58 proteins (Nop5 in archaea). Despite relaxed RNA sequence requirements for binding of hPrp31 to the 15.5KsnRNPs, hPrp31 does not bind to the 15.5K-box C/D snoRNPs. Nop56/58 proteins share a highly conserved RNA binding module with hPrp31, the so-called NOP domain. An additional N-terminal domain in these proteins is responsible for interaction with the methyl-transferase, fibrillarin. The conserved box C/D and C'/D' elements in box C/D s(n)RNAs are intervened by guide regions, which base pair to complementary sequences in the substrate RNA, thereby specifying and positioning nucleotides for modification by fibrillarin. To elucidate the molecular mechanisms underlying the specific RNP assemblies and to reveal how the components of snoRNPs bring about functional enzymatic machinery, we aimed at structural characterization of an archaeal box C/D sRNP. Recombinant expression and in vitro reconstitution of a Pyrococcus furiosus (P. fur.) box C/D sRNP were established. Using analytical gel filtration and electron microscopy (EM), it was shown that P. fur.-sRNP components assemble into di-sRNPs. Using cryo-EM, the dynamic network of interactions within box C/D elements and the structural rearrangements of the complex were revealed. After complete structural refinement, which is still ongoing, the EM structural data should shed light on the mechanism of catalytic activation of the box C/D sRNPs. RNA annealing assays suggested an additional role for fibrillarin in substrate recognition and binding, besides its role in the methylation process. Analysis of UVinduced protein-RNA crosslinks in the in vitro assembled P. fur.-box C/D sRNPs revealed a novel interaction between the ALFR motif, in the NOP domain of Nop5, and the guide regions of the sRNA. Mutational analyses showed that the ALFR motif and the guide sequence adjacent to box C or C' are important for box C/D sRNP assembly in vitro. The presented cross-link data therefore reveal new RNA-protein contacts in the box C/D sRNP and suggest a role for Nop5 in substrate binding and/or release.
In vivo and in vitro studies have shown that eukaryotic box C/D snoRNPs require complex assembly machineries. Archaeal box C/D sRNPs are simplified homologs of the eukaryotic snoRNPs. However, owing to their extreme growth conditions and efficient in vitro assembly, archaeal box C/D sRNPs have so far not been investigated with regards to their possible in vivo assembly factors. One candidate assembly factor that is selectively present in certain hyperthermophilic archaeal species is Rbp18. In this thesis, possible functions of Rbp18 were investigated by structural analyses, RNA binding and RNA annealing assays. Two crystal structures of Rbp18 from Pyrococcus furiosus and Aeropyrum pernix were determined. The structures revealed a conserved novel fold and a surprising architectural diversity among the members of Rbp18 family. The N-terminal domain of the protein showed structural homology to the conserved N-terminal domain of ribosomal protein L11. Biochemical studies revealed that Rbp18 can bind to RNA and enhance annealing of two complementary RNA strands. These data therefore suggest a role for Rbp18 in RNA protection and chaperoning at elevated temperatures.