Functional and structural analysis of Nip7p, a conserved protein involved in ribosome biogenesis

Author(s)

Patricia Pereira Coltri

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2007-10-12

Examining board members

Nilson Ivo Tonin Zanchin; Jörg Kobarg; Marcelo Menossi; Beatriz Amaral de Castilho; Gustavo Henrique Goldman

• Advisor: Nilson Ivo Tonin Zanchin

Abstract

Ribosome biogenesis is conserved throughout eukaryotes and takes place in the nucleolus, a specialized nuclear compartment where the rRNA precursors are transcribed. More than 170 trans-acting factors coordinately interact to generate the mature rRNAs. Among the proteins identified in the pre-60S particle in Saccharomyces cerevisiae is Nip7p. Highly conserved Nip7p orthologues are found in all eukaryotes and Archaea. The analysis of Nip7p sequence reveals a conserved C-terminal domain named PUA, also found in a number of RNA-interacting proteins. In this work, we performed structural and functional analysis to investigate Nip7p molecular role on rRNA processing and modification. The structure of Pyrococcus abyssi Nip7p ortholog, PaNip7, was solved using X-ray diffraction data to 1,8Å resolution. Structural analysis followed by in vitro assays confirmed the involvement of PUA domain in RNA interaction. S. cerevisiae Nip7p and its archaeal and human counterparts show preference for binding uridine-rich sequences, indicating conserved functional features among the orthologues. The preference for uridine can explain the higher affinity of S. cerevisiae Nip7p for ITS2 sequence, as observed by UV-crosslinking assays. Consistently, functional analysis revealed pre-rRNA processing in the ITS2 region is seriously impaired. Yeast two-hybrid analysis confirmed by pull down assays revealed Nip7p interacts with Nop8p and Nop53p, two nucleolar proteins involved in pre-27S processing and components of pre-60S particle. Although yeast two-hybrid and pull down assays indicated that Nip7p interacts with H/ACA box core proteins, pseudouridylation is not affected under conditions of Nip7p depletion. In addition, yeast two-hybrid analysis confirmed by GST-pull down revealed HsNip7 interaction with FTSJ3 and SUMO-2. Both HsNip7 and FTSJ3 showed nucleolar subcellular localization in HEK293 cells. FTSJ3 is an uncharacterized protein containing the FtsJ domain, initially described in prokaryotic rRNA methyl-transferases. FTSJ3 shows sequence similarity to yeast Spb1p, an rRNA methyl-transferase involved in methylation of Gm2922, indicating that FTSJ3 may be the human orthologue of Spb1p. Sumoylation is a post-transcriptional covalent modification involved in regulation of protein subcellular localization. Putative yeast orthologues of SUMO, such as Smt3p, have been described in the pre-60S ribosomal particle, suggesting that SUMO-2 might play a specific role in 60S subunit biogenesis (AU)