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. 2018 Dec 14;46(22):12126-12138.
doi: 10.1093/nar/gky966.

Multiple RNA-RNA Tertiary Interactions Are Dispensable for Formation of a Functional U2/U6 RNA Catalytic Core in the Spliceosome

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Free PMC article

Multiple RNA-RNA Tertiary Interactions Are Dispensable for Formation of a Functional U2/U6 RNA Catalytic Core in the Spliceosome

Penghui Bao et al. Nucleic Acids Res. .
Free PMC article

Abstract

The active 3D conformation of the spliceosome's catalytic U2/U6 RNA core is stabilised by a network of secondary and tertiary RNA interactions, but also depends on spliceosomal proteins for its formation. To determine the contribution towards splicing of specific RNA secondary and tertiary interactions in the U2/U6 RNA core, we introduced mutations in critical U6 nucleotides and tested their effect on splicing using a yeast in vitro U6 depletion/complementation system. Elimination of selected RNA tertiary interactions involving the U6 catalytic triad, or deletions of the bases of U6-U80 or U6-A59, had moderate to no effect on splicing, showing that the affected secondary and tertiary interactions are not required for splicing catalysis. However, removal of the base of U6-G60 of the catalytic triad completely blocked splicing, without affecting assembly of the activated spliceosome or its subsequent conversion into a B*-like complex. Our data suggest that the catalytic configuration of the RNA core that allows catalytic metal M1 binding can be maintained by Protein-RNA contacts. However, RNA stacking interactions in the U2/U6 RNA core are required for productive coordination of metal M2. The functional conformation of the U2/U6 RNA core is thus highly buffered, with overlapping contributions from RNA-RNA and Protein-RNA interactions.

Figures

Figure 1.
Figure 1.
Structure of the core RNA–RNA network in group IIC introns and catalytically-active yeast spliceosomes. (A) Schematic diagram of the RNA core of a group IIC intron (6) highlighting domain V (DV) and the triple interations of the bulged C and the J2/3 linker. Catalytic triad nucleotides are boxed and nucleotides forming the triple helix are indicated by gray shading. Nucleotides coordinating the catalytic metals are shown in white, with metal M1 coordination by C377 and U375, and M2 by C358 and G359. The dashed line with an open rectangle indicates a stacking interaction. The dotted lines indicate the highly folded intron domains III and IV. (B) RNA–RNA network in the catalytically-active yeast spliceosome. The U6 and U2 snRNAs are colored black and blue, respectively, and intron nucleotides near the 5′ ss (G+1 is circled) are grey. Hoogsteen interactions are indicated by purple lines and stacking interactions in the catalytic triad AGC (boxed) are indicated by open brown bricks. Nucleotides forming the triple helix are indicated by gray shading. Dashed line indicates the base stacking interaction between U80 and G52. Metal binding nucleotides are white with purple borders. (C) Schematic diagram of metal M1 and M2 coordination by the phosphate backbone of nucleotides of the U6 ISL and catalytic triad.
Figure 2.
Figure 2.
RNP architecture of the catalytic core of the spliceosome. (A) Schematic of the domain organization of PRP8 in S. cerevisiae. Regions binding the U6 ISL and U2/U6 helix I are indicated below. NTD, N-terminal domain; NTDL, NTD linker; HB, helical bundle; RT, reverse transcriptase-like; En, endonuclease-like; RH, RNase H-like; Jab1, Jab1/MPN-like. (B) Protein–RNA contacts in the U2/U6 RNA core of the S. cerevisiae Bact (PDB ID: 5GM6 (13)) or and C complex (PDB ID: 5GMK (16)) (see also Supplementary Table S3). Large dots indicate highly favourable geometry for hydrogen bonding (3 stars in Supplementary Table S3) and small dots indicate less favourable geometry for hydrogen bonding (1 star in Supplementary Table S3). (C) Space filling model of proteins or PRP8 domains forming the U2/U6 RNA core binding pocket (with the RNA excluded) in the S. cerevisiae C complex (PDB ID: 5GMK (16)). For orientation, the position of catalytic metal M1 is shown as a brown dot (also in panels D–F). Prp8 domains are coloured as in (A). Other proteins are coloured as in (B) and (C). (D) Space filling model with surface potential of the proteins and protein domains shown in panel (C). The electrostatic potential was calculated using the pyMOL vacuum electrostatics program. Charges are shown as a heat map, with blue for positive and red for negative potential (±5 kT). (E) Space filling model of the U2/U6 RNA network, including the U2/BS helix, in the yeast C complex. Intron nucleotides, light yellow; U2 snRNA (aside from those comprising U2/U6 helix 1b which is black), light green; U6 snRNA, dark green except for the ISL (dark yellow) or nucleotides in helix Ib (black). (F) Fit of the U2/U6 RNA network into the protein space filling model from panel C, where the top layer of protein has been cut away. The cutaway plane is in grey and the regions of the U2/U6 RNA network (compare to panel (E)) which are below the plane are darkened.
Figure 3.
Figure 3.
The Hoogsteen interactions of U6 nucleotides A59 and G60 are not essential for splicing. (AE) Kinetics of splicing of actin pre-mRNA in U6-depleted yeast extract supplemented with different synthetic U6 snRNAs. Splicing performed with (A) wild-type U6 (wt), (B) N7-deaza-A59 U6 (7cA59), (C) N7-deaza-G60 U6 (7cG60) or (D) N7-deaza-A59/N7-deaza-G60 U6 (7cA59/7cG60). The positions of the pre-mRNA, and of the splicing intermediates and products are indicated on the right. (E) Quantification of spliced mRNA production (left panel) and intron lariat-3′ exon (denoted as intermediate) production (right panel). The average amount of mRNA or intermediate present (plus the standard error) at each time point was calculated from three independent experiments. The production of mRNA at all time points was normalized to the amount of mRNA produced at the 60 min time point with wt U6. Similarly, lariat-3′ exon production (intermediate) was normalized to the amount of splicing intermediate produced at the 60 min time point with wt U6.
Figure 4.
Figure 4.
Limited effects on splicing of atomic or abasic mutations of U6 nucleotides in the triple helix structure. (AD) Quantification of the kinetics of mRNA and intermediate production after performing splicing with indicated synthetic U6 snRNA mutants. The corresponding splicing assays are shown in Supplementary Figure S2 and quantification was as in Figure 3. The mutants in panels A-D were all analysed in one experimental series to ensure that the quantification relative to the wt U6 was reliable. The average amount of mRNA or intron lariat-3′ exon (denoted as intermediate) present (plus the standard error) at each time point was calculated from three independent experiments, with the exception of panel D which was derived from two independent experiments.
Figure 5.
Figure 5.
Base stacking interactions within the spliceosomal U2/U6 RNA core. Close-up of the base stacking interactions of the AGC triad nucleotides and U6-U80 in the U2/U6 RNA core of the yeast C complex (PDB ID: 5GMK). Stacks are shown as towering disks between the involved centres of the nucleobases. Base stacking geometries are classified according to (60,61), with fully stacked bases in blue, and staggered stacked bases in yellow. The stacking interactions, in particular the staggered stacks, correspond to real densities in the C complex EM density map (EMD-9525; (16)).
Figure 6.
Figure 6.
Stacking of the base of U6-G60 is required for splicing catalysis, but not for Bact and Prp2-mediated B* complex formation. (A) Quantification of the splicing kinetics of mRNA and intermediate production for different abasic mutants of the catalytic triad. Splicing assays are shown in Supplementary Figure S2 and quantification was as in Figure 3. (B and C) The U6-G60 abasic (G60ab) mutant allows for assembly of Bact complexes and their conversion into a B*-like complex. Bact complexes containing the U6-G60ab mutant were assembled in prp2-1 yeast extract and purified Bact complexes were analysed on a gradient directly (B) or (C) after incubation with Prp2 and Spp2 proteins to form B* (41). Gradient fractions (indicated above) of Bact (B) and B* (C) complex preparations were probed simultaneously with anti-Prp19 and anti-Cwc24 antibodies. (D) Magnesium dependence of splicing with select U6 mutants. Production of mRNA after 20 min was quantified as in Figure 3 at the different magnesium concentrations indicted. In panels A and D, the average amount of mRNA or intron lariat-3′ exon (denoted as intermediate) present (plus the standard error) at each time point was calculated from three independent experiments.

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