Structure And Mechanism Of Box C/D RNA Protein Complexes

Box C/D RNAs are a large and ancient family of noncoding RNAs present in archaea and eukaryotes.They associate with three proteins Nop5,L7Ae and fibrillarin(Fib)in archaea and four proteins Nop56,Nop58,Snu13 and Fib in eukaryotes to form ribonucleoproteins(RNPs).Majority of C/D RNPs catalyze 2'-O-methylation of rRNAs and snRNAs with the substrate specificity determined by C/D guide RNAs.A few special eukaryotic C/D snoRNAs are involved in rRNA processing and ribosome assembly.Box C/D RNAs have a bipartite structure and contain the terminal boxes C and D and the related internal boxes C' and D'.Each pair of boxes C and D or C' and D' combine into a kink-turn(K-turn)or K-loop structure.The spacer regions between boxes C to D' and boxes C' to D contain the guide sequence that can form a stretch of base pairs with substrate RNAs,thereby selecting the fifth nucleotide from box D or D' for modification.A crystal structure of substrate-bound C/D RNP from the archaeon Su.folobus so.fataricus(Ss)has been previously determined in the active conformation.The structure illustrates the organization of a monomeric C/D RNP(mono-RNP),in which a single bipartite C/D RNA is anchored by its two K-turn motifs onto the Nop5 dimer platform.A 10-bp guide-substrate duplex is placed on the dimerized coiled-coil domains of Nop5 and capped at two ends by the C-terminal domain of Nop5.In vitro reconstitution of archaeal C/D RNPs also yielded a dimeric RNP(di-RNP).The structural organization and biological relevance of di-RNP is still a matter of debate at present.The C/D RNA was proposed to be swapped between two different protein complexes in the di-RNP,rather than associating with the same Nop5 dimer as shown in the mono-RNP structure.In this thesis,we study the structure and mechanism of C/D RNP by biochemical and crystallographic approaches.The guide region of C/D RNAs is variable in length,particularly in eukaryotes,and by prediction,it can form 10-21 base pairs with substrates.In the previously determined structure,a 12-nt long spacer forms a 10-bp duplex with the substrate.It remains unknown how spacers and guide-substrate duplexes of other lengths are accommodated.We analyze how the lengths of spacers and guide-substrate duplexes affect the modification activity of Ss C/D RNP.We find that elongated guide-substrate duplexes inhibit the modification activity at low reaction temperature.We also determine a crystal structure of C/D RNP with 13-nt spacers.The spacer can potentially form an 11-bp duplex with the substrate by design,but only forms a 10-bp duplex in the structure.This indicates that the protein channel is rather rigid and restraints the size of the duplex.Our data shows that the guide can only form a duplex of maximal 10 base pairs with the substrate during modification.Slightly shorter duplexes are tolerated,but longer duplexes must be unwound to fit into a capped protein channel for modification.Spacers with<12 nucleotides are defective mainly because they cannot load the substrate in the active conformation.For spacers with>12 nucleotides,the excessive unpaired sequences near the box C/C' side are looped out.Our results provide novel insight into the substrate recognition mechanism of C/D RNA and refute the RNA-swapped model for dimeric C/D RNP.We have reconstituted in vitro for the first time an active eukaryotic C/D RNPs with recombinant proteins from the thermophilic yeast Chaetomium thermcphilum(Ct).The Ct C/D RNP is capable of site-specific modification and the activity requires the presence of both Nop56 and Nop58.Extensive guide-substrate duplexes inhibit the activity,indicating that eukaryotic C/D RNPs also recognize the substrate with the 10-bp rule.We show that the C/D and C'/D' motifs are functionally independent from each other in both archaeal and eukaryotic RNPs.Mutation or deletion of boxes C/D or C'/D' affects only the D or D'spacer,respectively.Halfmer C/D RNAs with one K-turn motif and one guide are fully functional.Search of experimentally identified C/D RNAs in Drosophila and humans suggest that halfmer C/D RNAs likely exist in vivo and represent a new type of box C/D RNAs.Finally,we show that both K-turns of C/D RNA can be replaced by simple RNA duplexes in the Ss C/D RNP.Such RNAs without K-turn likely represent primordial methylation guide RNAs,providing insight into the evolution of C/D RNA.