Limits...
Evidence for a group II intron-like catalytic triplex in the spliceosome.

Fica SM, Mefford MA, Piccirilli JA, Staley JP - Nat. Struct. Mol. Biol. (2014)

Bottom Line: Here we show by genetics, cross-linking and biochemistry in yeast that analogous triples form in U6 and promote catalytic-metal binding and both chemical steps of splicing.Because the triples include an element that defines the 5' splice site, they also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals.Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate Program in Cell and Molecular Biology, University of Chicago, Chicago, Illinois, USA. [2] Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA. [3].

ABSTRACT
To catalyze pre-mRNA splicing, U6 small nuclear RNA positions two metals that interact directly with the scissile phosphates. U6 metal ligands correspond stereospecifically to metal ligands within the catalytic domain V of a group II self-splicing intron. Domain V ligands are organized by base-triple interactions, which also juxtapose the 3' splice site with the catalytic metals. However, in the spliceosome, the mechanism for organizing catalytic metals and recruiting the substrate has remained unclear. Here we show by genetics, cross-linking and biochemistry in yeast that analogous triples form in U6 and promote catalytic-metal binding and both chemical steps of splicing. Because the triples include an element that defines the 5' splice site, they also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals. Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing.

Show MeSH
The U6 triplex forms in vitroa, Stacking interaction between C377 and G288, as observed in the group II intron crystal structure (PDB 4FAQ, ref. 8); left, side view; right, top view; equivalent spliceosome residues are indicated in parentheses. The positions of the 4-thio-uridine (4SU) and of the radioactive label (32p) are indicated in red. b, Denaturing PAGE analysis of U6-4SU80 recovered from in vitro splicing reactions after UV irradiation. Where indicated, unlabeled ACT1 pre-mRNA was present in the reactions. The U6 crosslinks (X1, X2, and X3) are indicated to the right. The efficiency of X1 formation is quantified below the gel; error bars represent s.d. from three technical replicates. c, Predicted products for P1 nuclease and NaOH digestion; note that P1 nuclease is able to cleave 5′ of UV-crosslinked RNA residues46. d, Denaturing PAGE analysis of RNA products following P1 nuclease digestion of un-crosslinked U6 or X1 excised from a gel like that shown in b. A 5′-32pGpG dinucleotide was also digested with P1 as a size marker. Curvy blue arrow signifies a crosslink. For full gels see Supplementary Fig. 8a,b.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4257784&req=5

Figure 4: The U6 triplex forms in vitroa, Stacking interaction between C377 and G288, as observed in the group II intron crystal structure (PDB 4FAQ, ref. 8); left, side view; right, top view; equivalent spliceosome residues are indicated in parentheses. The positions of the 4-thio-uridine (4SU) and of the radioactive label (32p) are indicated in red. b, Denaturing PAGE analysis of U6-4SU80 recovered from in vitro splicing reactions after UV irradiation. Where indicated, unlabeled ACT1 pre-mRNA was present in the reactions. The U6 crosslinks (X1, X2, and X3) are indicated to the right. The efficiency of X1 formation is quantified below the gel; error bars represent s.d. from three technical replicates. c, Predicted products for P1 nuclease and NaOH digestion; note that P1 nuclease is able to cleave 5′ of UV-crosslinked RNA residues46. d, Denaturing PAGE analysis of RNA products following P1 nuclease digestion of un-crosslinked U6 or X1 excised from a gel like that shown in b. A 5′-32pGpG dinucleotide was also digested with P1 as a size marker. Curvy blue arrow signifies a crosslink. For full gels see Supplementary Fig. 8a,b.

Mentions: As a result of triple helix formation in domain V, two of the base-triple partners (C377 and G288) stack through their base rings (Fig. 4a). To investigate whether the corresponding positions in U6 (U80 and G52) form a similar stacking interaction, we designed a crosslinking assay. We reconstituted U6-depleted extract with a synthetic U6 (U6-4SU80) containing 4-thio-uridine at U80 and a single radioactive label at G52 (Fig. 4a; Supplementary Fig. 2a). If these two bases stack, UV irradiation should induce formation of a covalent linkage between U80 and G52 (ref. 30), with the site-specific radiolabel facilitating identification of such a linkage.


Evidence for a group II intron-like catalytic triplex in the spliceosome.

Fica SM, Mefford MA, Piccirilli JA, Staley JP - Nat. Struct. Mol. Biol. (2014)

The U6 triplex forms in vitroa, Stacking interaction between C377 and G288, as observed in the group II intron crystal structure (PDB 4FAQ, ref. 8); left, side view; right, top view; equivalent spliceosome residues are indicated in parentheses. The positions of the 4-thio-uridine (4SU) and of the radioactive label (32p) are indicated in red. b, Denaturing PAGE analysis of U6-4SU80 recovered from in vitro splicing reactions after UV irradiation. Where indicated, unlabeled ACT1 pre-mRNA was present in the reactions. The U6 crosslinks (X1, X2, and X3) are indicated to the right. The efficiency of X1 formation is quantified below the gel; error bars represent s.d. from three technical replicates. c, Predicted products for P1 nuclease and NaOH digestion; note that P1 nuclease is able to cleave 5′ of UV-crosslinked RNA residues46. d, Denaturing PAGE analysis of RNA products following P1 nuclease digestion of un-crosslinked U6 or X1 excised from a gel like that shown in b. A 5′-32pGpG dinucleotide was also digested with P1 as a size marker. Curvy blue arrow signifies a crosslink. For full gels see Supplementary Fig. 8a,b.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257784&req=5

Figure 4: The U6 triplex forms in vitroa, Stacking interaction between C377 and G288, as observed in the group II intron crystal structure (PDB 4FAQ, ref. 8); left, side view; right, top view; equivalent spliceosome residues are indicated in parentheses. The positions of the 4-thio-uridine (4SU) and of the radioactive label (32p) are indicated in red. b, Denaturing PAGE analysis of U6-4SU80 recovered from in vitro splicing reactions after UV irradiation. Where indicated, unlabeled ACT1 pre-mRNA was present in the reactions. The U6 crosslinks (X1, X2, and X3) are indicated to the right. The efficiency of X1 formation is quantified below the gel; error bars represent s.d. from three technical replicates. c, Predicted products for P1 nuclease and NaOH digestion; note that P1 nuclease is able to cleave 5′ of UV-crosslinked RNA residues46. d, Denaturing PAGE analysis of RNA products following P1 nuclease digestion of un-crosslinked U6 or X1 excised from a gel like that shown in b. A 5′-32pGpG dinucleotide was also digested with P1 as a size marker. Curvy blue arrow signifies a crosslink. For full gels see Supplementary Fig. 8a,b.
Mentions: As a result of triple helix formation in domain V, two of the base-triple partners (C377 and G288) stack through their base rings (Fig. 4a). To investigate whether the corresponding positions in U6 (U80 and G52) form a similar stacking interaction, we designed a crosslinking assay. We reconstituted U6-depleted extract with a synthetic U6 (U6-4SU80) containing 4-thio-uridine at U80 and a single radioactive label at G52 (Fig. 4a; Supplementary Fig. 2a). If these two bases stack, UV irradiation should induce formation of a covalent linkage between U80 and G52 (ref. 30), with the site-specific radiolabel facilitating identification of such a linkage.

Bottom Line: Here we show by genetics, cross-linking and biochemistry in yeast that analogous triples form in U6 and promote catalytic-metal binding and both chemical steps of splicing.Because the triples include an element that defines the 5' splice site, they also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals.Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate Program in Cell and Molecular Biology, University of Chicago, Chicago, Illinois, USA. [2] Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA. [3].

ABSTRACT
To catalyze pre-mRNA splicing, U6 small nuclear RNA positions two metals that interact directly with the scissile phosphates. U6 metal ligands correspond stereospecifically to metal ligands within the catalytic domain V of a group II self-splicing intron. Domain V ligands are organized by base-triple interactions, which also juxtapose the 3' splice site with the catalytic metals. However, in the spliceosome, the mechanism for organizing catalytic metals and recruiting the substrate has remained unclear. Here we show by genetics, cross-linking and biochemistry in yeast that analogous triples form in U6 and promote catalytic-metal binding and both chemical steps of splicing. Because the triples include an element that defines the 5' splice site, they also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals. Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing.

Show MeSH