Structural and functional analysis of the archaeal endonuclease Nob1

Abstract

Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site.

atBRX1-1 and atBRX1-2 are involved in an alternative rRNA processing pathway in Arabidopsis thaliana

“…These fragments are comparable to the aberrant 23S, 22S, and 21S pre-rRNAs in yeast and 26S and 21S pre-rRNAs in mammalian cells (Henras et al 2014). The 18S-A 3 pre-rRNA is then cleaved at site D by the homolog of the endonuclease Nob1 (Fatica et al 2003;Veith et al 2012;Missbach et al 2013). It remains unclear whether the 18S-A 3 pre-rRNA is first processed to the 20S pre-rRNA before D-site cleavage or if atNOB1 can directly cleave the 18S-A 3 pre-rRNA.…”

Essential ribosome assembly factor Fap7 regulates a hierarchy of RNA–protein interactions during small ribosomal subunit biogenesis

“…4A). Mapping of these chemical shift changes on the structure of PhNob1 (24) showed that significant alterations were observed for residues in the zinc ribbon RNA-binding domain and in the linker connecting this domain to the catalytic PilT N-terminus (PIN) like domain (Fig. 4B).…”

“…PhFap7 and PhNob1 were produced as described previously (24,32). PhS11 was overexpressed from a pET11a-derived vector and purified as described for PhNob1.…”

“…Archaeal homologs of eukaryotic ribosome assembly factors have served as valuable models for structural, functional, and biophysical studies (19)(20)(21)(22)(23)(24). Here we use the homologs of Fap7, Rps14, and Nob1 from the hyperthermophilic archaeon Pyrococcus horikoshii (PhFap7, PhS11, and PhNob1, respectively) to delineate the functional consequences of their interactions in vitro.…”

WBSCR22/Merm1 is required for late nuclear pre-ribosomal RNA processing and mediates N7-methylation of G1639 in human 18S rRNA

“…Interestingly, and in contrast to what is known from yeast, the 21S pre-rRNA then undergoes exonucleolytic processing generating 18SE pre-rRNAs (Preti et al 2013;Sloan et al 2013). Final 40S subunit maturation steps occur in the cytoplasm, including formation of the mature 3 ′ end of the 18S rRNA, which has been shown to be performed by the endonuclease Nob1 in yeast, plants, and archaea (Pertschy et al 2009;Veith et al 2012;Missbach et al 2013). The rRNA modifications cluster in highly conserved areas of the ribosome, such as the peptidyl-transferase center, sites of A-and P-tRNA binding, the peptide exit tunnel and the intersubunit bridge (Piekna-Przybylska et al 2008).…”