Structure-function relationships of archaeal Cbf5 during in vivo RNA-guided pseudouridylation

Abstract

In Eukarya and Archaea, in addition to protein-only pseudouridine (Ψ) synthases, complexes containing one guide RNA and four proteins can also produce Ψ. Cbf5 protein is the Ψ synthase in the complex. Previously, we showed that Ψ's at positions 1940, 1942, and 2605 of Haloferax volcanii 23S rRNA are absent in a cbf5-deleted strain, and a plasmid-borne copy of cbf5 can rescue the synthesis of these Ψ's. Based on published reports of the structure of archaeal Cbf5 complexed with other proteins and RNAs, we identified several potential residues and structures in H. volcanii Cbf5, which were expected to play important roles in pseudouridylation. We mutated these structures and determined their effects on Ψ production at the three rRNA positions under in vivo conditions. Mutations of several residues in the catalytic domain and certain residues in the thumb loop either abolished Ψ's or produced partial modification; the latter indicates a slower rate of Ψ formation. The universal catalytic aspartate of Ψ synthases could be replaced by glutamate in Cbf5. A conserved histidine, which is common to Cbf5 and TruB is not needed, but another conserved histidine of Cbf5 is required for the in vivo RNA-guided Ψ formation. We also identified a previously unreported novelty in the pseudouridylation activity of Cbf5 where a single stem-loop of a guide H/ACA RNA is used to produce two closely placed Ψ's and mutations of certain residues of Cbf5 abolished one of these two Ψ's. In summary, this first in vivo study identifies several structures of an archaeal Cbf5 protein that are important for its RNA-guided pseudouridylation activity.

Keywords: H/ACA RNA; Haloferax volcanii; RNA modification; mutagenesis; protein structure; ribonucleoprotein.

FIGURE 1.

Multiple sequence alignment of archaeal and eukaryal Cbf5 proteins. Sequences represented here are from four Archaea: Haloferax volcanii (H. volcanii), Pyrococcus furiosus (P. furiosus), Methanocaldococcus jannaschii (M. jannaschii), and Sulfolobus solfataricus (S. solfataricus); and five Eukarya: Saccharomyces cerevisiae (S. cerevisiae), Caenorhabditis elegans (C. elegans), Drosophila melanogaster (D. melanogaster), Arabidopsis thaliana (A. thaliana), and Homo sapiens (H. sapiens). Secondary structural elements are marked above the sequences, with α-helices depicted as cylinders and β-strands as arrows. Secondary structure of P. furiosus protein is based on the crystal structure (PDB 2EY4) (Rashid et al. 2006). Secondary structure of H. volcanii protein is the resulting modeled structure from I-TASSER. One new secondary structural element for HvCbf5 was predicted by I-TASSER, which is not shown in the cited reference (Rashid et al. 2006). The PUA domain regions comprising the structures at the two termini are marked by green lines above the sequence. Thumb loop sequences are boxed in orange. The conserved catalytic aspartate (D) residue is enclosed within a red box. Certain residues that differ between Archaea and Eukarya are enclosed in blue boxes. Residues used for mutagenesis in this study are indicated with red asterisks above the sequence. Highly conserved residues (>80%) among all proteins are shaded in dark blue, and at least 60% conserved residues are shown in medium blue. Light blue represents at least 40% conservation. Numbers after each sequence denote ending residues of each block. Numbering of P. furiosus Cbf5 has been adjusted to match a previous report (Rashid et al. 2006), which is commonly used in the literature. Numbers in parentheses indicate the length of the sequence not shown here.

FIGURE 2.

Homology model of HvCbf5. (A) The structure of PfuCbf5 (red) has been extracted from the Cbf5-Nop10-Gar1 crystal structure (PDB 2RFK) (Liang et al. 2007). The homology model of HvCbf5 (tan) based on I-TASSER predicted structure is overlaid. (B) The HvCbf5 model structure from A is shown with thumb loop in blue and purple and the PUA domain in yellow. The purple part of the thumb loop denotes the C loop (Ala115, Val116, and Ser117). The catalytic Asp53 (red) and Cbf5 residues (Pro25, Pro28, and Pro54 in cyan) of the “proline spine” of the RNP complex are shown as van der Waal's spheres. (C) Model of HvCbf5 showing positions of the residues that are individually substituted in this study. The α-carbons of these residues are shown as colored spheres. The colors of these spheres are by residue type as defined in VMD. Effects of Ala substitution of residues are indicated by colors of the labels: absence of (red) or partial (yellow) modification at the three positions (1940, 1942, and 2605) of 23S rRNA; partial modification at positions 1940 and 2605, but no modification at position 1942 (cyan); partial modification at position 1940, and normal modification at positions 1942 and 2605 (blue); and no effect (white), i.e., normal modification at all three positions. (See Table 1 for the effects of mutations of single and multiple residues of HvCbf5.) (D) Structural details near the active site of HvCbf5. Most residues that affected activity of HvCbf5 after mutation are shown. Colors of residues and labels are as in C.

FIGURE 3.

Ala substitutions of HvCbf5 residues conserved across all Ψ synthases abolish or reduce Cbf5-mediated Ψ formation at the three positions of 23S rRNA. (A) Interactions between the two H/ACA motifs of the sRNA and their 23S rRNA target sequences are shown. The two lines above the sRNA indicate the sequences that pair to form the lower stem of the H/ACA RNA. H and ACA sequences are boxed. Regions of sRNAs above the Ψ pocket are illustrated as stem–loops. Positions of the primers (arrows) HVLSUR1 and HVLSUR2 relative to rRNA sequences are shown. Positions of Ψ’s (1940, 1942, and 2605) in the rRNA sequence are indicated. (B) U-specific analyses to determine the modification status of U1940 and U1942 of 23S rRNA were done using primer HVLSUR1 (see panel A) and total RNA of Δcbf5 strain transformed with different mutant pHCbf5 plasmids (marked above each panel). Lanes 1 and 2: Primer extensions on untreated RNA and on RNA following U-specific reactions, respectively. A dark band at a position in lane 2, but not in lane 1 indicates an unmodified U. Positions of certain U's in 23S rRNA are indicated on the side. In WT cells, U1940 and U1942 are converted to Ψ, and U1936 (used as indicator for the positions) remains unmodified. (C) The primer and total RNA used in B are also used for CMCT-primer extension analyses. Total RNAs were either untreated (−) or treated with CMCT (+) for the indicated time (in minutes), followed by alkali (OH⁻) treatment (+) or no treatment (−). Positions of Cbf5-mediated modifications are indicated on the side. A dark band in CMCT followed by alkali, in 10 min and 20 min lanes, indicates Ψ. (D,E) Analyses similar to those in B and C, respectively, using primer HVLSUR2 (see panel A), were done to determine the modification status of U2605 of 23S rRNA. Unmodified U2604 and U2612 served as indicators for positions in D.

FIGURE 4.

Of the two conserved histidines of HvCbf5, H31 is required but H48 is not for rRNA Ψ formation in vivo. Effects of Ala substitution of conserved histidines (marked above each panel) of HvCbf5 for Ψ formation at positions 1940, 1942, and 2605 were determined by primer extensions following U-specific (A,C) and CMCT reactions (B,D). The reactions were done as in Figure 3.

FIGURE 5.

Proper structure of the thumb loop is important for the activity of Cbf5. Effects of the mutations (marked above each panel) in the thumb loop of HvCbf5 on Ψ formation at positions 1940, 1942, and 2605 were determined by primer extension following U-specific (A,C) and CMCT (B,D) reactions. The reactions were done as in Figure 3.

FIGURE 6.

Certain residues of the thumb loop are important for the guide RNA-dependent activity of Cbf5. Effects of alanine substitution of some conserved residues (marked above each panel) of the thumb loop of HvCbf5 for Ψ formation at positions 1940, 1942, and 2605 were determined by primer extensions following U-specific (A,C) and CMCT reactions (B,D). The reactions were done as in Figure 3.

FIGURE 7.

Relevance of PUA domain and certain catalytic core residues of Cbf5 in Ψ formation in vivo. Effects of deletion of PUA domain and Ala substitution of certain conserved residues (marked above each panel) of HvCbf5 for Ψ formation at positions 1940, 1942, and 2605 were determined by primer extensions following U-specific (A,C) and CMCT reactions (B,D). The reactions were done as in Figure 3.