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RNA catalyses nuclear pre-mRNA splicing

Abstract

In nuclear pre-messenger RNA splicing, introns are excised by the spliceosome, a dynamic machine composed of both proteins and small nuclear RNAs (snRNAs). Over thirty years ago, after the discovery of self-splicing group II intron RNAs, the snRNAs were proposed to catalyse splicing. However, no definitive evidence for a role of either RNA or protein in catalysis by the spliceosome has been reported so far. By using metal rescue strategies in spliceosomes from budding yeast, here we show that the U6 snRNA catalyses both of the two splicing reactions by positioning divalent metals that stabilize the leaving groups during each reaction. Notably, all of the U6 catalytic metal ligands we identified correspond to the ligands observed to position catalytic, divalent metals in crystal structures of a group II intron RNA. These findings indicate that group II introns and the spliceosome share common catalytic mechanisms and probably common evolutionary origins. Our results demonstrate that RNA mediates catalysis within the spliceosome.

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Figure 1: Chemistry of pre-mRNA splicing and U2/U6 model showing sites sensitive to sulphur substitutions and rescued by thiophilic metal.
Figure 2: U6 snRNA positions metals important for both steps of splicing.
Figure 3: U6 snRNA positions catalytic metals during branching.
Figure 4: U6 snRNA positions a catalytic metal during exon ligation.
Figure 5: Model for catalytic metal interactions during pre-mRNA splicing and comparison to the domain V catalytic core of group II introns.

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Acknowledgements

We thank C. Guthrie for plasmids; S. -C. Cheng for anti-Cwc25p serum; D. Semlow for strains; J. Olvera for reagents and experimental assistance; R. -J. Lin for sharing unpublished data; members of the Staley and Piccirilli laboratories for discussions; and D. Herschlag, A. Macmillan and T. Nilsen for comments on the manuscript. N.T. was supported by an NSF Graduate Research Fellowship and by a CBI Training Grant (5T32GM008720). This work was funded by a grant from the Chicago Biomedical Consortium, with support from The Searle Funds at the Chicago Community Trust, to J.P.S, A. S. Mankin and E. J. Sontheimer, and by a grant from the National Institutes of Health (R01GM088656) to J.P.S. and J.A.P.

Author information

Authors and Affiliations

Authors

Contributions

S.M.F., N.T., T.N., J.P.S. and J.A.P. designed the study; T.N. and P.K. performed initial screening of U6 sulphur substitutions; S.M.F. performed all experiments related to branching; N.T. performed all experiments related to exon ligation; S.M.F. and N.T. together performed Prp8p experiments; J.L., N.-S.L. and Q.D. synthesized RNA oligonucleotides; S.M.F., N.T., J.P.S. and J.A.P. analysed the data and wrote the manuscript.

Corresponding authors

Correspondence to Jonathan P. Staley or Joseph A. Piccirilli.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Affinity purification of spliceosomes with Prp19 requires reconstitution with U6 snRNA and enhances the potential to detect rescue by thiophilic metals.

a, b, Schemes depicting experimental strategy for staging spliceosomes to monitor branching (a) or exon ligation (b) in the absence of ATP. Spliceosomes are depicted as light magenta ovals and Prp19p as a magenta circle. Following affinity purification of spliceosomes by Prp19p (ref. 36), beads were washed to remove ATP and soluble factors and metals ions were added to assay for splicing. c, Prp19p-mediated affinity purification of activated spliceosomes, reflected by immunoprecipitation of pre-mRNA, is specific for properly reconstituted complexes. Note that the affinity purification allows quantification of the branching efficiency for activated complexes, independently of any effects on assembly. RNA from 10% of the reaction (input, I) or from beads after affinity purification (B) was extracted and analysed by denaturing PAGE. Raw data (top); quantification of immunoprecipitation efficiency (bottom); Rec., reconstitution. d, ATP represses the Cd2+ rescue for G78-PS(Sp) spliceosomes. Spliceosomes were assayed as in Fig. 2e; for lane 4, 2 mM ATP-Mg2+ was also present during the incubation. Representative gel (left) and quantification of the extent of ATP repression (right). Values are averages; error bars represent s.d. (n = 3).

Extended Data Figure 2 Broad rescue specificity of A59-PS(Sp) and U80-PS(Sp) spliceosomes.

a, Pre-incubation with EDTA reveals a branching defect for A59-PS(Sp). Extracts were pre-incubated at 4 °C in the presence or absence of 2 mM EDTA and then incubated with the 3′O substrate. After affinity-purification, branching efficiency was quantified without further incubation (bottom). A representative gel is shown (top). EDTA pre-incubation caused an eightfold reduction in branching efficiency, indicating that splicing extracts may contain a thiophilic metal that supports branching by A59-PS(Sp) spliceosomes; note that pre-mRNA still immunoprecipitated efficiently, indicating catalytic activation of the spliceosome. In contrast EDTA pre-incubation has no effect on U6 wild-type spliceosomes (data not shown). b, The branching defects for A59-PS(Sp) and U80-PS(Sp) spliceosomes are rescued by either Mn2+ or Cd2+. Assays were as in Fig. 2c. A representative gel (top) and quantification (bottom) are shown; no inc, no incubation. Both Mn2+ and Cd2+ strongly rescue A59-PS(Sp) and U80-PS(Sp) spliceosomes (lanes 3, 4, 7 and 8), suggesting that even the weaker Mn2+-S interaction38 at these positions can support branching. This broad specificity for branching may also explain why A59-PS(Sp) and U80-PS(Sp) spliceosomes also catalysed exon ligation in the presence of thiophilic metals, whereas G60-PS(Rp), G78-PS(Sp), and U80-PS(Rp) spliceosomes, for which branching was only rescued in the presence of Cd2+, stalled after branching. Values are averages; error bars, s.d. (n = 3).

Extended Data Figure 3 A divalent metal binds the 5′ splice site pro-Rp oxygen during branching.

a, UBC4 pre-mRNAs bearing the indicated modifications at the 5′ splice site (top panel) were assayed as in Fig. 3a. The band marked * results from 5′ to 3′ exonucleolytic degradation that is blocked by the sulphur; ** denotes statistical significance of Cd2+ rescue compared to Mg2+ splicing (P = 0.004, paired, one-tailed t-test, n = 3). The data from Fig. 3a are reproduced here to aid comparison. Values are averages; error bars, s.d. (n = 3); no inc, no incubation. b, Mapping of the site of branching. Purified, 32P-labelled, intact lariat intermediates (LI 0.2 fmol) resulting from branching of the UBC4 3′O-PO or 3′S-PS(Rp) substrates were used as templates for reverse transcriptase (RT) using primer IP3 (10 fmol), which binds at nucleotides +5 to +24 of the intron (see lower diagram). Lanes 6 and 7 show that the major RT stop occurs at the same position when either the 3′O-PO or 3′S-PS(Rp) lariats are used as template. This stop migrates at the expected position, which is the position of the ddC stop resulting from extension of primer IP3 with pre-mRNA as template and therefore corresponds to position +1 of the intron, the expected branch site. Lower diagram shows mapping of the primer and expected RT stop onto the UBC4 pre-mRNA sequence; bold exon; italics intron. Note that the band marked *** is present in the 32P pre-mRNA lane and thus is probably a nonspecific band resulting from contamination with pre-mRNA degradation products that can anneal to the primer and serve as template.

Extended Data Figure 4 The UBC4 3′S substrate can be branched efficiently only in the presence of thiophilic metals.

a, A sulphur at the 5′ splice site leaving group alone blocked splicing in extract. Following affinity-purification branching efficiency was quantified (bottom) without further incubation. Note that immunoprecipitation of pre-mRNA indicated that the sulphur did not block the catalytic activation of the spliceosome. b, Thiophilic metals rescued the branching defect of the 3′S substrate. Spliceosomes were assayed in buffer PK (pH 7.0) in the presence of 2 mM total metal (1 mM MgCl2 plus 1 mM MnCl2 or 1 mM CdCl2) for 1.5 h. The bar graph quantifies the relative stimulation by specific metals (normalized to Mg2+). c, d, Thiophilic metals specifically stimulate branching of the 3′S, but not the 3′O substrate. Affinity-purified spliceosomes from extracts depleted of Cwc25p, to stall spliceosomes independent of the sulphur substitution, were incubated as in Fig. 3a except that washes and incubation were done in buffer DK without EDTA and with 1 mM MgCl2; rCwc25 as well as an HP extract fraction were also added to complement (Supplementary Note 4; Methods). Quantification of the thiophilic metal stimulation relative to Mg2+ is shown both for reaction endpoints (c) and for the rate of branching (d). Values are averages; error bars, s.d. (n = 3). The band marked * results from 5′ to 3′ exonucleolytic degradation that is blocked by the sulphur.

Extended Data Figure 5 The 3′S-PS(Rp) substrate specifically improves rescue for spliceosomes containing U6 sulphur substitutions that compromise catalytic metal binding.

a, b, Spliceosomes were assayed as in Fig. 3b. Note that the 3′S-PS(Sp) substrate does not significantly improve Cd2+ rescue when compared to the 3′O-PO substrate (a), despite having similar reactivity to the 3′S-PS(Rp) substrate with wild-type U6 (b). c, The U6 double mutation U80g/C61g permitted both spliceosome assembly and activation, as reflected by the stable association of Prp19p with the splicing substrate. 10% of the RNA in the input (I) for the immunoprecipitation or 100% of the RNA associated with affinity-purified spliceosomes (B) were analysed by denaturing PAGE (top). Total immunoprecipitation efficiency was quantitated for all splicing species combined (bottom). d, The 3′S-PS(Rp) substrate did not significantly improve splicing for U80g and U80g/C61g spliceosomes. Assays were as in Fig. 3b. e, f, Representative raw data for Fig. 3. Assays were as in Fig. 3. In e, for wild-type U6 lanes 1–3 and 4–6 were taken from two different gels, for G78-PS(Sp) lanes 7–12 and 13–15 were taken from two different gels. In all other cases the lanes for different substrates assembled with spliceosomes bearing the same U6 modification were taken from the same gel. Values are averages; error bars, s.d. (n = 3 for a, b, d; n = 2 for c); no inc., no incubation. g, Summary of combinations of sulphur substitutions in U6 and the substrate tested for rescue of branching. The + sign indicates that branching was observed in the presence of thiophilic metal.

Extended Data Figure 6 U6 snRNA positions catalytic metals during branching: controls for the U80-PS(Sp) induced shift in the Cd2+ transition midpoint for rescue of the 3′S-PS(Rp) substrate.

a, The shift induced by U80-PS(Sp) in the Cd2+ midpoint for rescue was also observed when reaction rates, instead of amplitudes, were compared. Initial rates are plotted versus CdCl2 concentration. Assays were as in Fig. 3d. Values are averages; error bars represent s.d. (n = 2). Initial rates, rather than apparent overall rates, were used here because the branching efficiency did not level off by 120 min at Cd2+ concentrations below 0.025 mM (assuming an endpoint of 0.4). In support of this approach, at saturating Cd2+ both wild-type and U80-PS(Sp) spliceosomes branched similar fractions of the 3′S-PS(Rp) substrate (see c), indicating that addition of a sulphur in U6 within the catalytic core did not necessarily alter the population of complexes that are competent for catalysis. b, Transcribed (txt.) and ligated (lig.) wild-type U6 behaved similarly relative to U80-PS(Sp) for branching of the 3′S-PS(Rp) substrate at a limiting Cd2+ concentration. Initial rates are shown for branching of the 3′S-PS(Rp) substrate in the presence of 0.01 mM Cd2+. c, A sulphur at U80 pro-Sp shifts the Cd2+ titration midpoint for rescue of the 3′S-PS(Rp) substrate relative to the 3′S-PS(Sp) substrate. Assays were as in Fig. 3d; the 3′S-PS(Rp) data from Fig. 3d are shown again here for comparison. Curves represent Hill fits to the data. Values are averages; error bars, s.d. (n = 3). Although the Cd2+ titration for rescue of U80-PS(Sp) spliceosomes assembled on the 3′S-PS(Sp) substrate did not plateau under our experimental conditions (panel c), the data nevertheless set a lower limit for the apparent transition midpoint; further, this midpoint is equal to or greater than that observed for U6 wild-type spliceosomes, indicating that the shift by U80-PS(Sp) of the Cd2+ transition midpoint for rescue was specific for the 3′S-PS(Rp) substrate. d, With the 3′S-PS(Rp) substrate, U80-PS(Sp) decreased the Cd2+ titration midpoint for rescue of branching by sixfold. The apparent midpoint for G78-PS(Rp) spliceosomes is shown as an additional specificity control for the shift observed for U80-PS(Sp) (actual titration data not shown). The apparent Cd2+ rescue midpoints were obtained by fitting titration curves to the general Hill equation (see Methods). Error bars represent error of the Hill fit. e, Representative raw data for Fig. 3d. Assays were in the presence of 0.01 mM Cd2+ when indicated.

Extended Data Figure 7 Further evidence for two distinct catalytic metal sites during branching.

a, Branching requires two catalytic divalent metals. bd, The 5′ splice site pro-Rp oxygen and the U80 pro-Sp oxygen interact with a metal distinct from the metal that interacts with the 5′ splice site pro-Rp oxygen and the U80 pro-Rp and G78 pro-Sp oxygens. See Supplementary Note 7 for discussion. Spliceosomes were assayed for branching as in Fig. 3e; where indicated, MgCl2 or MnCl2 were present at 1 mM. Curves represent Hill fits to the data. The data in a are reproduced here from Fig. 3e to aid comparison. In panels a–d, values are averages; error bars, s.d. (n = 3). Cd2+ was limiting in b to sensitize the assay to binding of a second metal, and Cd2+ was saturating in c to show that G78-PS(Sp) and U80-PS(Sp) have the potential to be rescued at levels comparable to wild-type U6. Panel d shows that U80-PS(Sp) eliminates the affect of Mn2+ on the titration curves. e–j, Metal binding during branching for different combinations of sulphur substitutions in the substrate and U6 in the presence of the indicated metals, as reflected by the data in panels a–d. Panels e and f reflect data in a, b and c; panels g and h reflect data in b and c; panels i and j reflect data in b, c and d. Relevant U6 ligands are coloured red in each panel; the nucleophile is coloured orange. Metals are coloured magenta (Cd2+) and blue (Mn2+), and their interactions with specific U6 ligands are depicted as dashed lines, with differential shading intensity meant to illustrate differences in the expected strength of interaction with oxygen compared with sulphur, as inferred from studies with model compounds38,61. Shading of metals bound at M1 and M2 is further adjusted to reflect experimental observations. Panels for G78-PS(Sp) would look the same as those for U80-PS(Rp) (g and h).

Extended Data Figure 8 A sulphur at the 3′ splice site but not the 5′ splice site leaving group alters the metal specificity for rescue of U80-PS(Sp) spliceosomes: further evidence that U80 interacts with a catalytic metal during exon ligation.

a, A sulphur at the 3′ splice site leaving group during exon ligation alters the metal specificity for rescue of U80-PS(Sp) spliceosomes. Assays were as in Fig. 2j. The bar graph depicts quantification of exon ligation efficiency. b, A sulphur at the 5′ splice site leaving group during branching does not alter the metal specificity for rescue of U80-PS(Sp) spliceosomes. Assays were as in Fig. 3a, except with the 3′S-PO substrate. Values are averages; error bars, s.d (n = 3). c–f, Inferred metal binding during exon ligation (c, d) and branching (e, f) for the indicated combinations of sulphur substitutions in the substrate and U6 in the presence of the indicated metals. Relevant U6 ligands are coloured red in each panel; the nucleophile is coloured orange. Metals and ligand interactions are coloured as in Extended Data Fig. 7.

Extended Data Figure 9 Thiophilic metals rescue exon ligation for substrates containing a sulphur at the 3′ splice site leaving group in both a mutated and wild-type 3′ splice site context.

a, Cd2+ specifically stimulates the rate of exon ligation for the mutated, UAc-3′S substrate. Assays were as in Fig. 4a. b, Exon ligation occurs at the correct site for the 3′S substrate. RNA from affinity-purified spliceosomes assembled on ACT1-3′O, ACT1-3′S, and ACT1-UAc-3′O and chased as in Fig. 4a was subjected to RT–PCR using the primers depicted in blue arrows. c, Diagram of the photocaged linkage at the 3′ splice site. d–f, Cd2+ specifically stimulates the rate of exon ligation for the wild-type, UAG-3′S substrate. Assays were as in Fig. 4a, except that before addition of divalent metals, samples were irradiated with 308 nm light on ice for 5 min to remove the photocage. Shown are a representative gel (d), quantification of the reaction end points (e) and quantification of reaction rates (f). g, Representative raw data for Fig. 4b; bands within each set came from non-adjacent lanes on the same gel. Values are averages; error bars, s.d. (n = 3). h, Summary of combinations of sulphur substitutions in U6 and the substrate tested for rescue of exon ligation. The + sign indicates that exon ligation was observed in the presence of thiophilic metal.

Extended Data Figure 10 Residue D1853 of the RNaseH-like domain of Prp8 does not have a direct role in metal-mediated catalysis of exon ligation.

a, b, Spliceosomes assembled on the indicated ACT1 UAG (a) or UAc (b) 3′O or 3′S substrates were assayed as in Fig. 2j, in the absence of ATP. Splicing extracts were prepared from either a wild-type PRP8 strain or a mutant strain having the prp8-D1853C mutation. See Supplementary Note 15 for details and a discussion. Values are averages; error bars, s.d. (n = 2); no inc, no incubation.

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Fica, S., Tuttle, N., Novak, T. et al. RNA catalyses nuclear pre-mRNA splicing. Nature 503, 229–234 (2013). https://doi.org/10.1038/nature12734

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