Mutational Analysis of the Nsa2 N-Terminus Reveals Its Essential Role in Ribosomal 60S Subunit Assembly

• Published in: International journal of molecular sciences 2020-11, Vol.21 (23), p.9108
• Main Authors: Paternoga, Helge, Früh, Alexander, Kunze, Ruth, Bradatsch, Bettina, Baßler, Jochen, Hurt, Ed
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biosynthesis; Catalytic Domain; DNA Mutational Analysis; Domains; Evolution; Genetic analysis; Genotype & phenotype; GTP-Binding Proteins - metabolism; Helices; Meanders; Models, Molecular; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Mutation; Mutation - genetics; N-Terminus; Nog1; Nsa2; Nuclear Proteins - metabolism; Phenotype; Proteins; Recruitment; Ribosomal Proteins - chemistry; Ribosomal Proteins - genetics; Ribosomal Proteins - metabolism; ribosome assembly; ribosome biogenesis; Ribosome Subunits, Large, Eukaryotic - metabolism; rRNA; rRNA 25S; Rsa4; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; TINP1; Yeast
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21239108

The ribosome assembly factor Nsa2 is part of the Rea1-Rsa4-Nsa2 interconnected relay on nuclear pre-60S particles that is essential for 60S ribosome biogenesis. Cryo-EM structures depict Nsa2 docked via its C-terminal β-barrel domain to nuclear pre-60S particles, whereas the extended N-terminus, consisting of three α-helical segments, meanders between various 25S rRNA helices with the extreme N-terminus in close vicinity to the Nog1 GTPase center. Here, we tested whether this unappreciated proximity between Nsa2 and Nog1 is of functional importance. Our findings demonstrate that a conservative mutation, Nsa2 Q3N, abolished cell growth and impaired 60S biogenesis. Subsequent genetic and biochemical analyses verified that the Nsa2 N-terminus is required to target Nsa2 to early pre-60S particles. However, overexpression of the Nsa2 N-terminus abolished cytoplasmic recycling of the Nog1 GTPase, and both Nog1 and the Nsa2-N (1-58) construct, but not the respective Nsa2-N (1-58) Q3N mutant, were found arrested on late cytoplasmic pre-60S particles. These findings point to specific roles of the different Nsa2 domains for 60S ribosome biogenesis.