Shining Light on Dim2: Dissecting the Roles of an RNA-Binding Protein in Ribosomal Small Subunit Assembly.

[摘要] Ribosome assembly in eukaryotes requires hundreds of conserved essential proteins not present in the mature particle. Because deletion of individual assembly factors typically disrupts multiple assembly steps and changes the composition of the entire pre-ribosomal particle, determining the essential function of individual assembly factors has become a main focus of ribosomal research. Furthermore, many proteins appear to be present in assembling ribosomes for extended times making it difficult to pinpoint their role to a distinct step. Using combined classical yeast genetics and recombinant protein and RNA biochemistry, this research details the roles of Dim2, and RNA-binding assembly factor, and its interaction with Nob1, the nuclease that generates the 3’-end of 18S rRNA. Analysis of Dim2 mutants, in which the interaction with Nob1 is disrupted in vitro, demonstrates that the interaction between Dim2 and Nob1 is essential. In vivo analysis of Nob1 localization on pre-ribosomal particles containing these Dim2 mutants show that Dim2 is involved in recruiting Nob1 to pre-ribosomes. Additionally, RNA binding experiments show that Dim2 increases Nob1’s RNA affinity. Furthermore, data indicate that Dim2 helps position Nob1 for correct cleavage at the 3’-end of 18S rRNA, as a point mutation that abolishes this interaction in vitro leads to accumulation of pre-ribosomes containing Nob1 and 20S rRNA. Interestingly, the site of interaction with Nob1 is mapped to a KH-like domain in Dim2, suggesting that this domain typically used for RNA binding can additionally interact with proteins.Additionally, mapping of the Dim2 rRNA binding site reveals close proximity to D-site cleavage as well as the Nob1 rRNA binding site.

[发布机构] University of Michigan

[效力级别] Dim2

[关键词] Ribosome Assembly;Dim2;Nob1;Biological Chemistry;Science;Chemical Biology