Contribution of protein Gar1 to the RNA-guided and RNA-independent rRNA:Ψ-synthase activities of the archaeal Cbf5 protein

Abstract

Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ₂₆₀₃ in Pyrococcus abyssi 23S rRNA and of Ψ₅₅ in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ₅₅ in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ₂₆₀₇ (Ψ₂₆₀₃ in P. abyssi) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent activity of Cbf5.

Figure 1

Construction of disruption mutants. (A) Schematic diagram of the loci targeted for disruption. The target genes are in gray. The black arrows indicate the primers for RT-PCR analysis (panel C and Table S3). (B) Normalized reads’ coverage as a function of the median coverage observed for all four deleted T. kodakarensis strains. Colors correspond to the coverage observed for ΔTK1509/cbf5 (blue), ΔTK0903/pus10 (red), ΔTK1101/nop10 (yellow) and ΔTK2286/gar1 (green) strains. Identity of outlier points is indicated. TK2276 (pyrF) is affected in all four strains analyzed. (C) Disruption of each targeted ORFs does not impair transcription of their respective downstream contiguous ORF. RT-PCR reactions (gel + RTase) were performed with oligonucleotide primers (Table S3) complementary to the coding sequence of the target gene’s adjacent ORFs (indicated below each lane). The RT-PCR products were fractionated by 0.8% agarose gel electrophoresis. The bands correspond to RNA amplification since no amplified products were obtained in a reaction mixture lacking reverse transcriptase (-RTase). The asterisk indicates non-specific amplification.

Figure 2

Analysis of the effect of gene disruption on cell growth. The various T. kodakarensis wild type (KU216) and mutant strains were grown in ASW-YT medium. Cultures were carried out at the optimal growth temperature of 85 °C, or at 75 °C and 90 °C, and cell growth was monitored by counting the number of cells with Thoma’s cell counter during the course of the culture. The mean values obtained from three independent experiments and the standard deviations are shown with bars. Significance values of *p < 0.05 relative to KU216. square: KU216, diamond: Δcbf5, triangle: Δpus10, circle: Δnop10, and cross: Δgar1.

Figure 3

The effect of gene disruption on Ψ formation in the domain V of T. kodakarensis 23S rRNA. (A) Ψs identified in this work in the T. kodakarensis 23S rRNA, which are conserved in the P. abyssi 23S rRNA are indicated by black dots. The grey dots correspond to other Ψs detected in P. abyssi 23S rRNA in a previous work. The secondary structure of the T. kodakarensis 23S rRNA is adapted from Piekna et al.. The sequence of the two substrates mini–23S–2607 and mini–23S–2589, synthesized by in vitro transcription and used in vitro in Panel C (mini–23S–2607) and in Fig. 5 (mini–23S–2589), is highlighted. (B) Identification of Ψs in the T. kodakarensis 23S rRNA by the CMCT-RT method. As indicated at the top of each lane, total RNAs extracted from the various T. kodakarensis strains were treated in the absence (−) or the presence of CMCT (+), for 2, 10, and 20 min as described in Methods. The CMCT treatment was (+) or was not (−) followed by an alkaline treatment at pH 10.4. The positions of Ψs were identified by primer extension analysis using oligonucleotide O-2941 (represented by the dashed arrow) as described in Methods. Digital image of the autoradiogram was obtained by scanning the x-ray film. The presence of Ψs is revealed by the appearance of alkali-resistant RT stops. Lanes U, G, C, and A correspond to the RNA sequencing ladder. The positions of nucleotides in 23S rRNAs are indicated to the left of the autoradiogram. The two panels correspond to a cropping of two sections of the same autoradiogram. The full-length gel is presented in Supplementary Figure S4. (C) Time course analysis of in vitro modification by recombinant proteins in RNA substrate mini–23S–2607. The RNA substrate was radiolabeled by the incorporation of [α-³²P]CTP during in vitro transcription. The RNA was incubated with different protein combinations: Cbf5 alone (C), Cbf5 and Nop10 (CN), Cbf5, and Gar1 (CG), and a set of the three proteins (CNG). At each time point, an aliquot of the reaction mixture was analyzed by 2D-TLC. The radioactivity was quantified by PhosphorImager analysis. The quantities of Ψ nucleotides formed are expressed in moles per mole of mini–23S–2607.

Figure 4

In vitro activity of P. abyssi Pab91 RNP enzyme for formation of Ψ₂₆₈₅ in a 143-nucleotide-long rRNA substrate. (A) The RNA substrate mimicking the peptidyl transferase center (PTC) region of the 23S rRNA. The secondary structure model of the P. abyssi PTC is represented. The U at position 2685 targeted by the Pab91 RNP is indicated. The region of RNA substrate 23S-143nt is delineated by the black line. The loop CUGA replaced a large portion of 23S rRNA corresponding to domains I, II, III, IV, VI and is used as a linker between the 5′ and 3′ parts of domain V. (B) Single-turnover activity of the P. abyssi Pab91 LCN and LCNG RNP enzymes for modification of radiolabeled 23S–143nt at 65 °C. The ³²P was introduced in the phosphodiester bound preceding U₂₆₈₅ by a splinted ligation (detailed in Material and Methods). (C-D) Multiple-turnover activity of the LCN (C), and LCNG (D) enzymes for modification of radiolabeled 22–U substrate RNA at 65 °C. The unlabeled substrate RNA 23S-143nt was added in a 4-fold (2 μM), 10-fold (5 μM), or 20-fold (10 μM) excess over the RNP (~0.5 µM). A control was performed in absence of the unlabeled RNA (0 µM). (E) Electrophoretic mobility shift analysis (EMSA) of the binding of the radiolabeled substrate 23S–143nt with the Pab91 sRNP assembled with the L7Ae, Cbf5, and Nop10 mix. Incubation was performed at 65 °C during 10 and 60 minutes.

Figure 5

In vitro activity tests suggesting that Cbf5′s RNA dependent activity modifies orphan position U_2589(2585). The mini–23S–2589 RNA fragment was radiolabeled by incorporation of [α-³²P]GTP during in vitro transcription. This substrate was incubated with different combination of the four recombinant proteins from T. kodakarensis L7Ae (L), Cbf5 (C), Nop10 (N), and Gar1 (G) and total RNAs extracted from a S100 fraction from KU216 cells (+RNA). After digestion with RNase T2, the amount of Ψ formation was estimated by two-dimension thin layer chromatography as described in Methods. The control reaction (panel 1) was performed in the presence of LCNG but without the addition of total RNAs. The plates corresponding to each panel were exposed together on a same screen of the PhosphorImager. The full-length image is presented in Supplementary Figure S5.