The ATPase and helicase activities of Prp43p are stimulated by the G-patch protein Pfa1p during yeast ribosome biogenesis - PubMed (original) (raw)

The ATPase and helicase activities of Prp43p are stimulated by the G-patch protein Pfa1p during yeast ribosome biogenesis

Simon Lebaron et al. EMBO J. 2009.

Abstract

Prp43p is a RNA helicase required for pre-mRNA splicing and for the synthesis of large and small ribosomal subunits. The molecular functions and modes of regulation of Prp43p during ribosome biogenesis remain unknown. We demonstrate that the G-patch protein Pfa1p, a component of pre-40S pre-ribosomal particles, directly interacts with Prp43p. We also show that lack of Gno1p, another G-patch protein associated with Prp43p, specifically reduces Pfa1p accumulation, whereas it increases the levels of the pre-40S pre-ribosomal particle component Ltv1p. Moreover, cells lacking Pfa1p and depleted for Ltv1p show strong 20S pre-rRNA accumulation in the cytoplasm and reduced levels of 18S rRNA. Finally, we demonstrate that Pfa1p stimulates the ATPase and helicase activities of Prp43p. Truncated Pfa1p variants unable to fully stimulate the activity of Prp43p fail to complement the 20S pre-rRNA processing defect of Deltapfa1 cells depleted for Ltv1p. Our results strongly suggest that stimulation of ATPase/helicase activities of Prp43p by Pfa1p is required for efficient 20S pre-rRNA-to-18S rRNA conversion.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

Pfa1p interacts directly with Prp43p in vitro. (A) Pfa1p domain structure (top) and Coomassie blue staining (bottom) of purified Prp43p–His, Pfa1p–His, Nob1p–His and truncated versions of Pfa1p–His. (B, C) Pull-down experiments. Purified proteins were mixed in the indicated combinations and incubated with IgG-Sepharose beads coated with polyclonal anti-Prp43p antibodies. Proteins remaining in the supernatant fractions (Sup) were precipitated using TCA. After washing the beads, pulled-down proteins (IP) were re-suspended in SDS–PAGE loading buffer. Proteins were detected by the western blotting procedure using anti-histidine-HRP antibodies.

Figure 2

Figure 2

The pre-40S pre-ribosomal particle composition in Δ_gno1_ cells. (A) Western blot analysis of the steady-state accumulation of the indicated TAP-tagged non-ribosomal pre-40S pre-ribosomal particle components in Δ_gno1_ (Δ_G_) and isogenic wild-type cells (WT). ‘αPAP' refers to the PAP rabbit antibodies (DAKO) used to detect the TAP tag fused to the proteins of interest. (B) Western blot analysis of the interaction between Pfa1p–TAP and Prp43p in yeast extracts. Extracts from Δ_gno1_ cells expressing Pfa1p–TAP (lanes 5 and 6) or from isogenic wild-type cells expressing Pfa1p–TAP (lanes 3 and 4) or wild-type Pfa1p (lanes 1 and 2) were incubated with IgG-Sepharose beads. Total proteins were extracted from the bead pellets (IP) or from 1/30th of the initial input extracts used for immunoprecipitations (Tot) and analysed by western blotting. (C) Interactions between 20S pre-rRNA and non-ribosomal pre-40S pre-ribosomal particle components in wild-type (WT) and Δ_gno1_ (Δ_G_) cells. Extracts from Δ_gno1_ or isogenic wild-type cells expressing the indicated TAP-tagged proteins were incubated with IgG-Sepharose beads. Total RNAs were extracted from the bead pellets (IP) or from 1/30th of the initial input extracts used for immunoprecipitations (Tot). 20S pre-rRNA was detected by northern blotting. Percentages of input 20S pre-rRNA precipitated are indicated below the IP lanes.

Figure 3

Figure 3

The 20S pre-rRNA strongly accumulates in GAL∷ltv1/Δ_pfa1_ cells depleted for Ltv1p. (A) Structure of the primary RNA PolI transcript. Cleavage sites are indicated as well as the hybridization position of probes used in the northern blot analysis. (B) Northern blot analysis of pre-rRNAs extracted from GAL∷ltv1/Δ_pfa1_, Δ_pfa1_ or Δ_ltv1_ cells grown at 25°C in galactose- (Gal) or glucose-containing (Glu) medium, as indicated, is shown. The probes used are indicated above each hybridization panel.

Figure 4

Figure 4

Phenotypes of GAL∷ltv1/Δ_pfa1_ cells expressing truncated variants of Pfa1p. GAL∷ltv1/Δ_pfa1_ cells were transformed with centromeric plasmids directing expression of wild-type ZZ-tagged Pfa1p (Pfa1p-ZZ), the ZZ tag alone, or truncated variants of Pfa1p, Pfa1p(1–565)–ZZ, Pfa1p(574–767)–ZZ, Pfa1p(1–202)–ZZ, Pfa1p(201–565)–ZZ and Pfa1p(201–767)–ZZ. (A) Growth of the transformed cells was assessed by performing serial dilutions of the cultures, aliquots of which were spotted on glucose-containing minimal medium plates incubated at 25°C for 4 days. (B) Western blot analysis of the expression of ZZ-tagged Pfa1p or truncated derivatives in GAL∷ltv1/Δ_pfa1_ cells grown in galactose- or glucose-containing medium. (C) Northern blot analysis of pre-rRNA and rRNA steady-state levels in GAL∷ltv1/Δ_pfa1_ cells expressing ZZ-tagged Pfa1p or truncated derivatives and grown in galactose- or glucose-containing medium at 25°C.

Figure 5

Figure 5

Stimulation of the ATPase activity of Prp43p by Pfa1p. (A) Rate of ATP hydrolysis by Prp43p–His (100 nM) as a function of ATP concentration in the absence of RNA and in the absence (solid circles) or in the presence (open circles) of Pfa1p–His at 500 nM (two independent experiments are shown). Experimental points were fitted with the Michaelis–Menten equation. Deviations are from the fit. (B) Same as in (A) except that total yeast RNA (150 μM) was added. (C) Pfa1p–His does not increase the affinity of Prp43p–His for ATP. UV cross-linking of Prp43p–His (1 μM) with [γ-32P]-ATP was performed in the absence (top panel) or presence (bottom panel) of total yeast RNA at 150 μM (as nucleotides). Pfa1p–His was added to the reactions loaded in lanes 3–5 at the following concentrations: lane 3: 0.5 μM; lane 4: 0.75 μM; lane 5: 1 μM. After irradiation, cross-linked Prp43p–His was subjected to SDS–polyacrylamide gel electrophoresis and detected by autoradiography. (D) Stimulation of ATP hydrolysis requires the G-patch-containing domain of Pfa1p. Where indicated, ATP hydrolysis was performed with Prp43p–His (100 nM) and either full-length Pfa1p–His or various truncated Pfa1p–His variants (500 nM). ATP and ADP were separated by TLC and the percentage of ADP produced quantified by phosphorimager.

Figure 6

Figure 6

Pfa1p–His stimulates the helicase activity of Prp43p–His. (A) Schematics of the RNA–DNA hybrid substrates used for the electrophoretic mobility shift assays (EMSAs) and helicase assays. The 113 nucleotide-long RNA probe corresponds to the 5′ stem loop and H box of snR5 snoRNA. It was annealed to either one of two 21 nucleotide-long radiolabelled DNA oligonucleotides. Annealing produced either a 3′ single-stranded overhang (3′–5′ helicase substrate) or a 5′ single-stranded overhang (5′–3′ substrate). (B) EMSAs performed with the 3′–5′ (left panel) or the 5′–3′ (right panel) substrates, Prp43p–His at a concentration of 10 nM and increasing concentrations of Pfa1p–His (from 0.5 to 100 nM). Positions of the free probe, the Prp43p–His-bound probe and the Prp43p–His/Pfa1p–His/probe ternary complex (‘supershift') are indicated. (C) Helicase assays performed with the 3′–5′ substrate (left panel) or the 5′–3′ substrate (right panel). Positions of the RNA/labelled DNA substrate and of the unwound oligonucleotide are indicated. Where indicated, Prp43p–His and/or Pfa1p–His were added to the unwinding reactions at 100 nM and 500 nM, respectively. (D) Duplex unwinding is ATP dependent. The unwinding reaction was performed with Prp43p–His at 100 nM, Pfa1p–His at 500 nM, and different nucleotides at 1 mM. (E) Pfa1p domains required for the stimulation of Prp43p–His helicase activity. Helicase assays were performed with the 3′–5′ substrate as in (C), Prp43p–His (100 nM) and either full-length Pfa1p–His or various truncated Pfa1p–His variants (500 nM), as indicated.

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