Identification of determinants in the protein partners aCBF5 and aNOP10 necessary for the tRNA: 55-synthase and RNA-guided RNA: -synthase activities

Abstract

Protein aNOP10 has an essential scaffolding function in H/ACA sRNPs and its interaction with the pseudouridine(Ψ)-synthase aCBF5 is required for the RNA-guided RNA:Ψ-synthase activity. Recently, aCBF5 was shown to catalyze the isomerization of U55 in tRNAs without the help of a guide sRNA. Here we show that the stable anchoring of aCBF5 to tRNAs relies on its PUA domain and the tRNA CCA sequence. Nonetheless, interaction of aNOP10 with aCBF5 can counterbalance the absence of the PUA domain or the CCA sequence and more generally helps the aCBF5 tRNA:Ψ55-synthase activity. Whereas substitution of the aNOP10 residue Y14 by an alanine disturbs this activity, it only impairs mildly the RNA-guided activity. The opposite effect was observed for the aNOP10 variant H31A. Substitution K53A or R202A in aCBF5 impairs both the tRNA:Ψ55-synthase and the RNA-guided RNA:Ψ-synthase activities. Remarkably, the presence of aNOP10 compensates for the negative effect of these substitutions on the tRNA: Ψ55-synthase activity. Substitution of the aCBF5 conserved residue H77 that is expected to extrude the targeted U residue in tRNA strongly affects the efficiency of U55 modification but has no major effect on the RNA-guided activity. This negative effect can also be compensated by the presence of aNOP10.

Long-distance placement of substrate RNA by H/ACA proteins

“…Addition of Gar1 accelerated this process and led to a rapid reduction of the fluorescence intensity. This interpretation is consistent with the known catalytic properties of the CNL and the full CNLG complex (Muller et al 2007). …”

“…Similarly, these results support previous data on Nop10's effect on substrate placement. Nop10 is positioned along the linear track of the guide RNA and has essential roles in anchoring it (Li and Ye 2006;Muller et al 2007). Like L7Ae, it places the substrate RNA indirectly via positioning the guide RNA upper stem.…”

“…2C). Previously, Branlant's laboratory showed that Nop10 is required for substrate binding (Charpentier et al 2005), and that mutation of a residue near Arg34 (His31) in Nop10 impaired Cbf5's ability to modify a substrate RNA (Muller et al 2007). Our result using CNmG corroborates this result and, more importantly, links the specific Nop10-guide RNA interaction directly to the behavior of the substrate's 2-AP nucleotide and potentially also to substrate docking.…”

Archaeal Pus10 proteins can produce both pseudouridine 54 and 55 in tRNA

“…Archaeal and eukaryal Cbf5 are homologous and belong to the TruB family. Archaeal Cbf5 has also been shown to function without guide RNA (Roovers et al 2006;Gurha et al 2007;Muller et al 2007Muller et al , 2008. However, this protein-alone activity of archaeal Cbf5 is mostly observed in association with Gar1 and/or Nop10.…”

“…Archaeal tRNAs also contain C55. Two different enzymes in Archaea, Cbf5 and Pus10 (PsuX), are able to catalyze the in vitro formation of C55 in tRNA in a protein-only manner (Roovers et al 2006;Gurha et al 2007;Muller et al 2007). Since C55 synthesis by these two enzymes appears to be redundant in Archaea, the question remains whether these proteins also serve some other purpose.…”