Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome

Abstract

Introns are removed from nuclear messenger RNA precursors through two sequential phospho-transesterification reactions in a dynamic RNA-protein complex called the spliceosome. But whether splicing is catalysed by small nuclear RNAs in the spliceosome is unresolved. As the spliceosome is a metalloenzyme, it is important to determine whether snRNAs coordinate catalytic metals. Here we show that yeast U6 snRNA coordinates a metal ion that is required for the catalytic activity of the spliceosome. With Mg2+, U6 snRNA with a sulphur substitution for the pro-Rp or pro-Sp non-bridging phosphoryl oxygen of nucleotide U80 reconstitutes a fully assembled yet catalytically inactive spliceosome. Adding a thiophilic ion such as Mn2+ allows the first transesterification reaction to occur in the U6/sU80(Sp)- but not the U6/sU80(Rp)-reconstituted spliceosome. Mg2+ competitively inhibits the Mn2+-rescued reaction, indicating that the metal-binding site at U6/U80 exists in the wild-type spliceosome and that the site changes its metal requirement for activity in the Sp spliceosome. Thus, U6 snRNA contributes to pre-messenger RNA splicing through metal-ion coordination, which is consistent with RNA catalysis by the spliceosome.

Figure 1

U6 snRNA with a phosphorothioate substitution at U₈₀ fails to reconstitute splicing. U6-depleted prp2 mutant extracts were reconstituted with T7 polymerase-transcribed, unmodified U6 (lanes 1 and 3) or ligated, synthetic U6 (lanes 4–8) in the absence (lane 1) or presence (lanes 2–8) of wild-type PRP2 protein. No U6 was added in lane 2 (−). Both input pre-mRNA and U6 RNA were labelled with ³²P and were analysed by denaturing gels and autoradiography. Synthetic U6 used: O, no phosphorothioate substitution (lane 4); M, unpurified R_P/S_P phosphorothioate mixture with some unsulphurized RNA (lane 5); and the R_P diastereomer (lane 6), S_P (lane 7) and the unsulphurized RNA (lane 8) purified from the mixture. The intron–exon2 lariat (IVS–E2*) and exon 1 (E1) are products from the first transesterification reaction, and the intron lariat (IVS*) and ligated exon 1/exon 2 (E1–E2) are from the second reaction.

Figure 2

Spliceosome maturation is not affected by phosphorothioate substitution at U₈₀ of U6 but splicing only occurs in the S_P diastereomer with a thiophilic metal ion. a, U6/sU₈₀ forms mature spliceosome. Extracts were reconstituted with unmodified U6 (lanes 1 and 2) or U6/sU₈₀ (R_P, lanes 3–4; S_P, lanes 5–6), in the absence (lanes 1, 3 and 5) or presence (lanes 2, 4 and 6) of PRP2. The reaction mixture was sedimented through a glycerol gradient and the fraction containing the spliceosome was analysed on a native gel. The pre-catalytic and post-PRP2 spliceosomal markers (M; lanes 7 and 8) were generated as described. b, The first transesterification reaction occurs in U6/sU₈₀(S_P)-containing post-PRP2 spliceosome in the presence of Mn²⁺. Spliceosomes isolated from glycerol gradients were incubated in buffer containing combinations of Mg²⁺, Mn²⁺, ATP, PRP2 and HP (a protein factor that facilitates splicing in purified spliceosomes). c, Other thiophilic divalent metal ions can also rescue the transesterification reaction. The post-PRP2 U6/sU₈₀(S_P) spliceosome was incubated with metal ions without the addition of ATP, PRP2 or HP. Mg²⁺ was added as the sole divalent metal ion in lanes 1 (2.5 mM) and 2 (5 mM). The concentrations (mM) of the thiophilic metal (Mn²⁺, Ca²⁺, Cd²⁺ or Co²⁺) in each set were 0.02, 0.1, 0.5 and 2.5. Mg²⁺ was added to lanes 3–18 to make the total concentration of divalent metal ions 5 mM.

Figure 3

With U6/sU₈₀(S_P) RNA, only the first of the two transesterification reactions occurs in the presence of Mn²⁺, which is competitively inhibited by Mg²⁺. a, Mn²⁺ rescues the first but not the second step of splicing in U6/sU₈₀(S_P)-reconstituted reaction. Extract was reconstituted with U6 (lanes 1–6), U6/sU₈₀(R_P) (lanes 7–12), or U6/sU₈₀(S^P) (lanes 13–18) and PRP2 in the presence of Mn²⁺ and Mg²⁺. In each set of six reactions, the concentrations (mM) of Mn²⁺/Mg²⁺ were 0/2.5, 2.5/2.5, 1.25/1.25, 1.7/0.8; 2.0/0.5, 2.5/0. b, Mg²⁺ competitively inhibits the Mn²⁺-rescued reaction. Extract was reconstituted in the absence of PRP2 with U6 (lanes 1–9) or U6/sU₈₀(S_P) (lanes 10–18) and with Mg²⁺ (2.5 mM) as the sole divalent metal ion. Splicing was then triggered by the addition of PRP2 with 1mM Mn²⁺ (lanes 2–9 and 11–18) and an increasing concentration (mM) of Mg²⁺ (2.5, 5.0, 7.5, 10, 15, 20, 25, 30). Reactions in lanes 1 and 2 contained 2.5mM of Mg²⁺ and no Mn²⁺ (asterisk). c, The ratio of splicing efficiency between the U6/sU₈₀(S_P)- and U6-reconstituted reactions (S_P/O). Splicing efficiency in each reaction (Fig. 3b) was calculated as the ratio between the products (IVS–E2*, IVS* and E1–E2) and the total (products plus pre-mRNA).

Figure 4

A representation of RNAs and metal ions in the yeast spliceosome before the first catalytic step. Intermolecular helices between U6 and U2 (Ia and Ib), U2 and pre-mRNA at the branch site (U2/BS), U6 and pre-mRNA at the 5′ splice site (U6/5′SS), as well as the 3′ intramolecular stem-loop (3′ISL) in U6 are depicted^-,. The branchpoint adenine is highlighted with a star. The U₈₀ of U6 is shaded. Three Mg²⁺ ions are shown as circles: one stabilizes the leaving oxygen at the 5′ scissile phosphate (lined); one activates the attacking oxygen (dotted); and one binds to the pro-S_P oxygen of U₈₀ (filled). The tertiary interaction between G₅₂ of U6 and A₂₅ of U2 (refs 26, 27), a putative interaction between 3′ISL of U6 and the U6/5′SS helix based on the λ-λ′ interaction in a group II intron, and the potential relationship between the metals are linked with a line.