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Structure of the Rna15 RRM-RNA complex reveals the molecular basis of GU specificity in transcriptional 3'-end processing factors.

Pancevac C, Goldstone DC, Ramos A, Taylor IA - Nucleic Acids Res. (2010)

Bottom Line: RNA recognition by CFIA is mediated by an RNA recognition motif (RRM) contained in the Rna15 subunit of the complex.We show here that Rna15 has a strong and unexpected preference for GU containing RNAs and reveal the molecular basis for a base selectivity mechanism that accommodates G or U but discriminates against C and A bases.This mode of base selectivity is rather different to that observed in other RRM-RNA structures and is structurally conserved in CstF64, the mammalian counterpart of Rna15.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

ABSTRACT
Rna15 is a core subunit of cleavage factor IA (CFIA), an essential transcriptional 3'-end processing factor from Saccharomyces cerevisiae. CFIA is required for polyA site selection/cleavage targeting RNA sequences that surround polyadenylation sites in the 3'-UTR of RNA polymerase-II transcripts. RNA recognition by CFIA is mediated by an RNA recognition motif (RRM) contained in the Rna15 subunit of the complex. We show here that Rna15 has a strong and unexpected preference for GU containing RNAs and reveal the molecular basis for a base selectivity mechanism that accommodates G or U but discriminates against C and A bases. This mode of base selectivity is rather different to that observed in other RRM-RNA structures and is structurally conserved in CstF64, the mammalian counterpart of Rna15. Our observations provide evidence for a highly conserved mechanism of base recognition amongst the 3'-end processing complexes that interact with the U-rich or U/G-rich elements at 3'-end cleavage/polyadenylation sites.

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Examination of the binding affinity of U/G-pocket mutants by fluorescence spectroscopy. The panel shows the titration curves [fraction of bound RNA; ΔF/Fmax versus total Rna15(16-111) concentration] derived from the change in fluorescence intensity for Rna15(16-111) (dark blue) and substitution mutants Y21F, Y21A, Y27F, Y27A, R87A and R87K upon binding to the Tet-UGUUGU ribo-oligonucleotide. The Binding constants in the inset table were determined from these plots by fitting of a hyperbolic binding isotherm to the data.
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Figure 5: Examination of the binding affinity of U/G-pocket mutants by fluorescence spectroscopy. The panel shows the titration curves [fraction of bound RNA; ΔF/Fmax versus total Rna15(16-111) concentration] derived from the change in fluorescence intensity for Rna15(16-111) (dark blue) and substitution mutants Y21F, Y21A, Y27F, Y27A, R87A and R87K upon binding to the Tet-UGUUGU ribo-oligonucleotide. The Binding constants in the inset table were determined from these plots by fitting of a hyperbolic binding isotherm to the data.

Mentions: In order test our structural observations and to investigate the contribution that residues in the Site-I and Site-II base-binding pockets make to the affinity and specificity of Rna15-RNA interaction, substitution mutants were prepared. At Site-I, Y27 was replaced by either A or F and R87 by either A or K. At Site-II, Y21 was substituted by A or F. The effects of these mutations on the RNA binding activity of Rna15(16-111) were then examined using fluorescence spectroscopy employing the Tet-UGUUGU ribo-oligonucleotide. Binding isotherms constructed from fluorescence intensity measurements are shown in Figure 5. It is apparent that where substitutions have been made at residues that form the walls of the Site-I base-binding pocket, RNA binding is heavily diminished or even abolished. For instance, Y27A and even the conservative R87K replacement reduce the binding affinity to an immeasurable level in the fluorescence assay. Although, abolition of RNA binding by the Y27A substitution might be anticipated on the basis of the crystal structures, it is somewhat surprising that the lysine mutation also has such strong effect. Presumably, although both K and R maintain a positive charge the absence of delocalization and planarity of lysine side-chain means it is unable to stack against the bound base in the same favourable way as the arginine side-chain does. The conservative substitution Y27F also reduces binding but not to the same degree as the R87 substitutions. Notably, a weak hydrogen bond between the Y27 phenolic hydroxyl and the O4’ of the ribose is observed in the Rna15(P16-S103)-ht complex, so it is possible in Y27F removal of this interaction is responsible for the diminished RNA-binding affinity. At Site-II the Y21F substitution reduces the RNA binding 5-fold and Y21A around 10-fold. However, relative to the larger effects we observe with the Site-I mutations, in Site-II the substitution of a phenolic by an aromatic ring produces only a modest reduction in binding supporting the idea that aromatic ring-base stacking rather than hydrogen bonding is important for the Site-II-nucleobase interaction.Figure 5.


Structure of the Rna15 RRM-RNA complex reveals the molecular basis of GU specificity in transcriptional 3'-end processing factors.

Pancevac C, Goldstone DC, Ramos A, Taylor IA - Nucleic Acids Res. (2010)

Examination of the binding affinity of U/G-pocket mutants by fluorescence spectroscopy. The panel shows the titration curves [fraction of bound RNA; ΔF/Fmax versus total Rna15(16-111) concentration] derived from the change in fluorescence intensity for Rna15(16-111) (dark blue) and substitution mutants Y21F, Y21A, Y27F, Y27A, R87A and R87K upon binding to the Tet-UGUUGU ribo-oligonucleotide. The Binding constants in the inset table were determined from these plots by fitting of a hyperbolic binding isotherm to the data.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875009&req=5

Figure 5: Examination of the binding affinity of U/G-pocket mutants by fluorescence spectroscopy. The panel shows the titration curves [fraction of bound RNA; ΔF/Fmax versus total Rna15(16-111) concentration] derived from the change in fluorescence intensity for Rna15(16-111) (dark blue) and substitution mutants Y21F, Y21A, Y27F, Y27A, R87A and R87K upon binding to the Tet-UGUUGU ribo-oligonucleotide. The Binding constants in the inset table were determined from these plots by fitting of a hyperbolic binding isotherm to the data.
Mentions: In order test our structural observations and to investigate the contribution that residues in the Site-I and Site-II base-binding pockets make to the affinity and specificity of Rna15-RNA interaction, substitution mutants were prepared. At Site-I, Y27 was replaced by either A or F and R87 by either A or K. At Site-II, Y21 was substituted by A or F. The effects of these mutations on the RNA binding activity of Rna15(16-111) were then examined using fluorescence spectroscopy employing the Tet-UGUUGU ribo-oligonucleotide. Binding isotherms constructed from fluorescence intensity measurements are shown in Figure 5. It is apparent that where substitutions have been made at residues that form the walls of the Site-I base-binding pocket, RNA binding is heavily diminished or even abolished. For instance, Y27A and even the conservative R87K replacement reduce the binding affinity to an immeasurable level in the fluorescence assay. Although, abolition of RNA binding by the Y27A substitution might be anticipated on the basis of the crystal structures, it is somewhat surprising that the lysine mutation also has such strong effect. Presumably, although both K and R maintain a positive charge the absence of delocalization and planarity of lysine side-chain means it is unable to stack against the bound base in the same favourable way as the arginine side-chain does. The conservative substitution Y27F also reduces binding but not to the same degree as the R87 substitutions. Notably, a weak hydrogen bond between the Y27 phenolic hydroxyl and the O4’ of the ribose is observed in the Rna15(P16-S103)-ht complex, so it is possible in Y27F removal of this interaction is responsible for the diminished RNA-binding affinity. At Site-II the Y21F substitution reduces the RNA binding 5-fold and Y21A around 10-fold. However, relative to the larger effects we observe with the Site-I mutations, in Site-II the substitution of a phenolic by an aromatic ring produces only a modest reduction in binding supporting the idea that aromatic ring-base stacking rather than hydrogen bonding is important for the Site-II-nucleobase interaction.Figure 5.

Bottom Line: RNA recognition by CFIA is mediated by an RNA recognition motif (RRM) contained in the Rna15 subunit of the complex.We show here that Rna15 has a strong and unexpected preference for GU containing RNAs and reveal the molecular basis for a base selectivity mechanism that accommodates G or U but discriminates against C and A bases.This mode of base selectivity is rather different to that observed in other RRM-RNA structures and is structurally conserved in CstF64, the mammalian counterpart of Rna15.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

ABSTRACT
Rna15 is a core subunit of cleavage factor IA (CFIA), an essential transcriptional 3'-end processing factor from Saccharomyces cerevisiae. CFIA is required for polyA site selection/cleavage targeting RNA sequences that surround polyadenylation sites in the 3'-UTR of RNA polymerase-II transcripts. RNA recognition by CFIA is mediated by an RNA recognition motif (RRM) contained in the Rna15 subunit of the complex. We show here that Rna15 has a strong and unexpected preference for GU containing RNAs and reveal the molecular basis for a base selectivity mechanism that accommodates G or U but discriminates against C and A bases. This mode of base selectivity is rather different to that observed in other RRM-RNA structures and is structurally conserved in CstF64, the mammalian counterpart of Rna15. Our observations provide evidence for a highly conserved mechanism of base recognition amongst the 3'-end processing complexes that interact with the U-rich or U/G-rich elements at 3'-end cleavage/polyadenylation sites.

Show MeSH