Multiple functions for the invariant AGC triad of U6 snRNA

Abstract

The invariant AGC triad of U6 snRNA plays an essential, unknown role in splicing. The triad has been implicated in base-pairing with residues in U2, U4, and U6. Through a genetic analysis in S. cerevisiae, we found that most AGC mutants are suppressed both by restoring pairing with U2, supporting the significance of U2/U6 helix Ib, and by destabilizing U2 stem I, indicating that this stem regulates helix Ib formation. Intriguingly, one of the helix Ib base pairs is required specifically for exon ligation, raising the possibility that the entirety of helix Ib is required only for exon ligation. We also found that U4 mutations that reduce complementarity in U4 stem I enhance U2-mediated suppression of an AGC mutant, suggesting that U4 stem I competes with the AGC-containing U4/U6 stem I. Implicating an additional, essential function for the triad, three triad mutants are refractory to suppression--even by simultaneous restoration of pairing with U2, U4, and U6. An absolute requirement for a purine at the central position of the triad parallels an equivalent requirement in a catalytically important AGC triad in group II introns, consistent with a role for the AGC triad of U6 in catalysis.

FIGURE 1.

The AGC triad is complementary to sequences within U2, U4, and U6. (A) U2/U6 helix Ib, in which the AGC triad is complementary to U2 (Madhani and Guthrie 1992). Helix Ib is mutually exclusive with U4/U6 stem I and U2 stem I. Helix II is also mutually exclusive with U2 stem I. (B) U6 3′ stem extension, in which the AGC triad is complementary to downstream residues in U6. The extension could form in base-paired U2/U6, as suggested by Sun and Manley (1995), or in free U6. (C) U4/U6 stem I, in which the AGC triad is complementary to U4 (Brow and Guthrie 1988). U4/U6 stem I is mutually exclusive with U4 stem I (Fig. 3C ▶). (D) U2 stem I. Throughout, structures containing the AGC triad are boxed.

FIGURE 2.

Compensatory mutations in U2 but not in downstream residues of U6 can suppress the growth and splicing defects of AGC mutations. (A) Base-pairing of the AGC triad with U2 to form helix Ib, as proposed by Madhani and Guthrie (1992). (B–D) Compensatory analysis of helix Ib base pairs (B) between U6-A59 and U2-U23, (C) between U6-G60 and U2-C22, and (D) between U6-C61 and U2-G21. (E) Alternative base-pairing of the AGC triad with U6 to form the U6 3′ stem extension, as proposed by Sun and Manley (1995). (F–H) Compensatory analysis of U6 3′ stem extension base pairs (F) between U6-A59 and U6-U88, (G) between U6-G60 and U6-U87, and (H) between U6-C61 and U6-G86 in the presence of wild-type U2ΔFD (F) or wild-type, full-length U2 (G,H). The matrices show the growth phenotypes of single and double base pair mutations at 15°C (B,F) or 25°C (C,D,G,H) in strain yHM118. Wild-type residues are capitalized. White boxes highlight Watson–Crick combinations. The growth defects of U6-A59C and -A59G are suppressed by U2 compensatory mutations at all tested temperatures from 15°C to 37°C (data not shown; Madhani and Guthrie 1992). The growth defects of U6-G60A and -C61G are suppressed by U2 compensatory mutations at all tested temperatures except 34°C and 37°C (data not shown). (I) Compensatory analysis, by primer extension of splicing in vivo at 37°C, of the helix Ib base pair between U6-A59 and U2ΔFD-U23. The identities of the bases are shown below the graph. (Top) The RP51A lariat intermediate; the two RP51A mRNAs, which differ in their transcription start sites; and U14, which serves as an internal control, are highlighted. (Bottom) The mRNA:lariat intermediate ratio (mRNA/L.I.) is shown; the standard deviation is derived from the variance of two samples. ¹Compensatory mutation U2-U12A maintains the U2 stem I bulge. ²Compensatory mutations at U2-G13 (C) or U2-C14 (D) preserve Watson–Crick base-pairing in U2 stem I. ³Compensatory mutations at U6-C92 preserve Watson–Crick base-pairing in U2/U6 helix II.

FIGURE 3.

Compensatory mutations in U2, U4, and U6 fail to suppress three lethal AGC mutants. (A,B) Compensatory analysis, by growth, of the U4/U6 stem I base pair (A) between U6-A59 and U4-U60 and (B) between U6-G60 and U4-C59. Helix Ib and the U6 3′ stem, including the wobble between U6-G60 and U6-U87, are maintained in each experiment. The matrices are labeled as in Figure 2 ▶ and show growth phenotypes at 37°C in yJPS628. (C) Proposed structure of U4 stem I (Myslinski and Branlant 1991), which is mutually exclusive with the interaction between the AGC triad and U4. (D) Summary of AGC mutations that are viable alone or in the presence of compensatory U2 mutations. ¹Compensatory mutations at U6-A93 (A) or at U6-C92 (B) maintain Watson–Crick base pairing in U2/U6 helix II. ²Mutation U2-U12A maintains the U2 stem I bulge. ³Compensatory mutations at U2-G13 maintain Watson–Crick base-pairing in U2 stem I.

FIGURE 4.

Mutations that destabilize U2 stem I suppress AGC mutants. (A) Suppression of U6-A59C, -G60A, and -C61A by mutations that destabilize U2 stem I. (B) Suppression of U6-C61A by additional mutations that destabilize U2 stem I. The matrices are labeled as in Figure 2 ▶ and show the growth phenotypes at 34°C in yHM118. Arrows mark the position of nucleotide changes in U2 stem I. ¹Compensatory mutation U6-C92U maintains U2/U6 helix II in the presence of the U2-G13A mutation.