Nob1 binds the single-stranded cleavage site D at the 3'-end of 18S rRNA with its PIN domain.
Ribosome assembly is a hierarchical process that involves pre-rRNA folding, modification, and cleavage and assembly of ribosomal proteins. In eukaryotes, this process requires a macromolecular complex comprising over 200 proteins and RNAs. Whereas the rRNA modification machinery is well-characterized, rRNA cleavage to release mature rRNAs is poorly understood, and in yeast, only 2 of 8 endonucleases have been identified. The essential and conserved ribosome assembly factor Nob1 has been suggested to be the endonuclease responsible for generating the mature 3'-end of 18S rRNA by cleaving at site D. Here we provide evidence that recombinant Nob1 forms a tetramer that binds directly to pre-rRNA analogs containing cleavage site D. Analysis of Nob1's affinity to a series of RNA truncations, as well as Nob1-dependent protections of pre-rRNA in vitro and in vivo demonstrate that Nob1's binding site centers around the 3'-end of 18S rRNA, where our data also locate Nob1's suggested active site. Thus, Nob1 is poised for cleavage at the 3'-end of 18S rRNA. Together with prior data, these results strongly implicate Nob1 in cleavage at site D. In addition, our data provide evidence that the cleavage site at the 3'-end of 18S rRNA is single-stranded and not part of a duplex as commonly depicted. Using these results, we have built a model for Nob1's interaction with preribosomes.
Lamanna
Allison C
AC
Departments of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Karbstein
Katrin
K
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D046912:Multiprotein Complexes; C405962:NOB1 protein, S cerevisiae; D009687:Nuclear Proteins; D012322:RNA Precursors; D012337:RNA, Ribosomal, 18S; C096168:RPS14A protein, S cerevisiae; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C030831:ribosomal protein S5
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