Nob1 binds the single-stranded cleavage site D at the 3'-end of 18S rRNA with its PIN domain

摘要

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 NobVs affinity to a series of RNA truncations, as well as Nob 1-dependent protections of pre-rRNA in vitro and in vivo demonstrate that NobVs binding site centers around the 3-end of 18S rRNA, where our data also locate NobVs 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 NobVs interaction with preribosomes.