The ribosomal translocase homologue Snu114p is involved in unwinding U4/U6 RNA during activation of the spliceosome

Abstract

Snu114p is a yeast U5 snRNP protein homologous to the ribosomal elongation factor EF-2. Snu114p exhibits the same domain structure as EF-2, including the G-domain, but with an additional N-terminal domain. To test whether Snu114p in the spliceosome is involved in rearranging RNA secondary structures (by analogy to EF-2 in the ribosome), we created conditionally lethal mutants. Deletion of this N-terminal domain (snu114 Delta N) leads to a temperature-sensitive phenotype at 37 degrees C and a pre-mRNA splicing defect in vivo. Heat treatment of snu114 Delta N extracts blocked splicing in vitro before the first step. The snu114 Delta N still associates with the tri-snRNP, and the stability of this particle is not significantly impaired by thermal inactivation. Heat treatment of snu114 Delta N extracts resulted in accumulation of arrested spliceosomes in which the U4 RNA was not efficiently released, and we show that U4 is still base paired with the U6 RNA. This suggests that Snu114p is involved, directly or indirectly, in the U4/U6 unwinding, an essential step towards spliceosome activation.

Fig. 1. The N-terminal domain of Snu114p is evolutionarily conserved. (A) Alignment of ∼125 amino acids of the N-terminal domains of potential orthologues of Snu114p from S. cerevisiae and other proteins that display sequence similarity. Lines above the sequences indicate evolutionarily conserved hydrophobic blocks (the numbering follows the Snu114p sequence). Identical residues are highlighted in black and similar residues in grey. (B) Block diagram of the primary structure of Snu114p. G1 to G5 are the conserved motifs of the G-domain. Below the diagram, yeast cells carrying the wild type (wt) and the temperature-sensitive deletion mutant snu114ΔN are shown after growth at 25 and 37°C, respectively.

Fig. 2. Deletion of the N-terminal domain (snu114ΔN) leads to a splicing defect in vivo and in vitro. (A) Total RNA was extracted from snu114ΔN as well as wild-type (wt) cells grown at 25°C (lanes 1 and 7) and after the switch to 37°C for 2 h (lane 2 and 8) and 4 h (lanes 3 and 9). Primer extension was performed to measure the levels of unspliced pre-U3A/U3B transcripts. (B) In vitro splicing reactions were performed at 25°C using snu114ΔN and prp5-1 extracts inactivated at 35°C for the time indicated above each lane. Splicing activity complementation was obtained by mixing equal volumes of the two heat-inactivated extracts prior to the splicing reaction (lanes 6 and 7). The identity of the ³²P-labeled RNA species is indicated (from top to bottom): intron-lariat-exon 2 intermediate, excised lariat-intron, pre-mRNA, mature mRNA and cleaved exon 1 intermediate.

Fig. 3. Tri-snRNP particles are present in snu114ΔN extracts heat inactivated in vitro. Wild-type (wt) and snu114ΔN extracts were treated at 25 or 35°C for 35 min and then sedimented on a 10–30% glycerol gradient. Fractions were analysed for RNA contents by northern blotting.

Fig. 4. Snu114p participates in spliceosome activation. (A) Extracts were heat inactivated for 25 min at 35°C, and then biotinylated pre-mRNA was added and incubated at 25°C under splicing conditions for the time indicated (splice time). Splicing reactions were performed using either wild-type (wt; lanes 1–5) or snu114ΔN (lanes 6–10) extracts. Splicing complexes were isolated using streptavidin beads, and their RNA content was analysed by northern blotting. Lanes 1 and 6 are control reactions using non-biotinylated substrates. The arrow points to the U4 RNA accumulating in the snu114ΔN mutant. (B) Plot of the U4 RNA release in the wild-type (wt) and snu114ΔN spliceosomes. The U4 and U6 RNAs bands were quantified by phosphorimager analysis and the U4/U6 ratio was plotted. The graph was obtained from five independent experiments.

Fig. 5. U4 RNA remains bound to U6 RNA in snu114ΔN heat-inactivated spliceosomes. Wild-type (wt; lanes 4–6) and snu114ΔN (lanes 7–9) spliceosomes were assembled at non-permissive temperature (30°C) using a biotinylated actin pre-mRNA and were affinity purified on streptavidin beads. Spliceosomal RNAs were fractionated on an SDS gel. RNAs were analysed by northern blotting after probing the membrane with U4 (A) and U6 (B) probes. Lane 3 is a control reaction using non-biotinylated substrate. The U4/U6 duplex (lane 1) was obtained after phenol–chloroform extraction at 4°C of the reaction shown in lane 6, before affinity purification. In lane 2, the U4/U6 duplex was dissociated by heating the sample for 5 min at 95°C. (The free U6 RNA is only weakly visible.)