The conserved protein Nip 7 is involved in ribosome biogenesis , being required for proper 27 S pre-rRNA processing and 60 S ribosome subunit assembly in Saccharomyces

The conserved protein Nip7 is involved in ribosome biogenesis, being required for proper 27S pre-rRNA processing and 60S ribosome subunit assembly in Saccharomyces cereVisiae. Yeast Nip7p interacts with nucleolar proteins and with the exosome subunit Rrp43p, but its molecular function remains to be determined. Solution of the Pyrococcus abyssi Nip7 (PaNip7) crystal structure revealed a monomeric protein composed by two alpha-beta domains. The N-terminal domain is formed by a five-stranded antiparallel ! -sheet surrounded by three R-helices and a 310 helix while the C-terminal, a mixed ! -sheet domain composed by strands ! 8 to ! 12, one R-helix, and a 310 helix, corresponds to the conserved PUA domain (after Pseudo-Uridine synthases and Archaeosine-specific transglycosylases). By combining structural analyses and RNA interaction assays, we assessed the ability of both yeast and archaeal Nip7 orthologues to interact with RNA. Structural alignment of the PaNip7 PUA domain with the RNAinteracting surface of the ArcTGT (archaeosine tRNA-guanine transglycosylase) PUA domain indicated that in the archaeal PUA domain positively charged residues (R151, R152, K155, and K158) are involved in RNA interaction. However, equivalent positions are occupied by mostly hydrophobic residues (A/ G160, I161, F164, and A167) in eukaryotic Nip7 orthologues. Both proteins can bind specifically to polyuridine, and RNA interaction requires specific residues of the PUA domain as determined by sitedirected mutagenesis. This work provides experimental verification that the PUA domain mediates Nip7 interaction with RNA and reveals that the preference for interaction with polyuridine sequences is conserved in Archaea and eukaryotic Nip7 proteins. Eukaryotic ribosome biogenesis takes place mainly in the nucleolus where a large pre-rRNA, containing the sequences of the mature 18S, 5.8S, and 25S/28S rRNAs separated by external and internal spacer sequences, is transcribed by RNA polymerase I, and the 5S rRNA is transcribed by RNA polymerase III. The pre-rRNA is assembled with processing factors concomitantly with transcription, undergoing subsequently extensive endoand exonucleolytic cleavage and covalent modification to generate the mature rRNAs (1, 2). Major pre-rRNA processing and ribosome assembly steps have already been described and a large number of factors identified especially in the yeast model system. The large Saccharomyces cereVisiae 35S pre-rRNA is assembled into a 90S particle, which is rapidly converted into the pre-40S and the pre-60S preribosomal particles. While the pre-40S particles are further processed in the cytoplasm, pre-60S prerRNAs undergo several endoand exonucleolytic digestions before export to the cytoplasm, where ribosome assembly terminates (1, 3). Important progress has recently been made regarding the identification of the components of the preribosomal complexes. The 90S preribosome contains the 35S pre-rRNA, the U3 snoRNP, 40S subunit processing factors and a large number of 40S subunit ribosomal proteins, along with a small number of 60S ribosomal proteins (3). Characterization of the U3 snoRNP led to the identification of the small subunit processing complex, termed SSU processome, which contains 28 proteins required for maturation of the 18S rRNA and 40S subunit biosynthesis (4). Absence of 27S pre-rRNA processing factors in the 90S preribosome indicates that the 60S subunit biogenesis factors and structural proteins join the complex later in the process