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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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The Rna15 RRM-RNA interaction. (A) Superimposition of three 15N-1H correlation NMR experiments recorded during titrations of Rna15(1-94)-ht with either the UGGCG or UAUUU ribo-oligonucleotides. The spectrum free Rna15(1-94)-ht (magenta), together with 1: 3 molar ratios of Rna15(1-94)-ht:UAUUU (blue) and Rna15(1-94)-ht:UGGCG (gold) are shown. Arrows connect the free and bound positions of two representative resonances. The resonances of the same residues are perturbed by interaction with the two RNAs, but larger shifts occur when the UGCGG sequence is added. (B) RNA binding affinity for different sequences measured by changes in fluorescence intensity of 5′-Tetrachloro-fluorescein labelled RNAs. Titration curves for UGUUGU, UUUUUU, UGUUUG, UCUUCU, UAUUAU, AGAAGA and ACAACA are shown. The fraction of bound RNA (ΔF/Fmax) is plotted against total Rna15(16–111) concentration. Equilibrium association constants determined by fitting a hyperbolic binding isotherm to the titration data are shown below.
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Figure 1: The Rna15 RRM-RNA interaction. (A) Superimposition of three 15N-1H correlation NMR experiments recorded during titrations of Rna15(1-94)-ht with either the UGGCG or UAUUU ribo-oligonucleotides. The spectrum free Rna15(1-94)-ht (magenta), together with 1: 3 molar ratios of Rna15(1-94)-ht:UAUUU (blue) and Rna15(1-94)-ht:UGGCG (gold) are shown. Arrows connect the free and bound positions of two representative resonances. The resonances of the same residues are perturbed by interaction with the two RNAs, but larger shifts occur when the UGCGG sequence is added. (B) RNA binding affinity for different sequences measured by changes in fluorescence intensity of 5′-Tetrachloro-fluorescein labelled RNAs. Titration curves for UGUUGU, UUUUUU, UGUUUG, UCUUCU, UAUUAU, AGAAGA and ACAACA are shown. The fraction of bound RNA (ΔF/Fmax) is plotted against total Rna15(16–111) concentration. Equilibrium association constants determined by fitting a hyperbolic binding isotherm to the titration data are shown below.

Mentions: Previously, it has been proposed that, in vitro, in the absence of other protein factors Rna15 binds RNA sequences only weakly and/or non-specifically (16,17). This observation, combined with the lack of a strong Rna15 consensus-binding site in S. cerevisiae 3′-UTRs suggests it is unlikely that the domain recognizes a long specific RNA sequence within the mRNA 3′-end. However, one possibility is that Rna15 displays weak sequence preference for the multiple low complexity sequences flanking the cleavage/polyadenylation sites. To test this notion, we examined the interaction of the core RRM domain, Rna15(1-94)-ht, with two dissimilar pentameric ribo-oligonucleotides UAUUU and UGGCG using NMR spectroscopy, Figure 1A. These data show that the same residues mediate the interaction with both sequences and that the complexes are in a moderately fast regime of exchange. However, because of the low complexity of these RNA sequences it is likely that multiple binding frames are present where small chemical shift differences between resonances of proteins bound in different frames are averaged by the fast regime of exchange. Accordingly, the titrations likely represent the interaction of Rna15(1-94)-ht with a base-averaged sequence rather than a unique RNA–protein interaction. Indeed, considering the low complexity of target sequences in the 3′-UTR multiple binding frames may be important to enhance the RNA binding affinity of Rna15 in vivo. Nevertheless, regardless of the dynamics, quite unexpectedly, the induced chemical shift perturbations are significantly larger upon interaction with UGGCG than they are with the UAUUU sequence, indicating that Rna15(1-94)-ht binds to the G-rich sequence with a higher affinity than the AU sequence.Figure 1.


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)

The Rna15 RRM-RNA interaction. (A) Superimposition of three 15N-1H correlation NMR experiments recorded during titrations of Rna15(1-94)-ht with either the UGGCG or UAUUU ribo-oligonucleotides. The spectrum free Rna15(1-94)-ht (magenta), together with 1: 3 molar ratios of Rna15(1-94)-ht:UAUUU (blue) and Rna15(1-94)-ht:UGGCG (gold) are shown. Arrows connect the free and bound positions of two representative resonances. The resonances of the same residues are perturbed by interaction with the two RNAs, but larger shifts occur when the UGCGG sequence is added. (B) RNA binding affinity for different sequences measured by changes in fluorescence intensity of 5′-Tetrachloro-fluorescein labelled RNAs. Titration curves for UGUUGU, UUUUUU, UGUUUG, UCUUCU, UAUUAU, AGAAGA and ACAACA are shown. The fraction of bound RNA (ΔF/Fmax) is plotted against total Rna15(16–111) concentration. Equilibrium association constants determined by fitting a hyperbolic binding isotherm to the titration data are shown below.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: The Rna15 RRM-RNA interaction. (A) Superimposition of three 15N-1H correlation NMR experiments recorded during titrations of Rna15(1-94)-ht with either the UGGCG or UAUUU ribo-oligonucleotides. The spectrum free Rna15(1-94)-ht (magenta), together with 1: 3 molar ratios of Rna15(1-94)-ht:UAUUU (blue) and Rna15(1-94)-ht:UGGCG (gold) are shown. Arrows connect the free and bound positions of two representative resonances. The resonances of the same residues are perturbed by interaction with the two RNAs, but larger shifts occur when the UGCGG sequence is added. (B) RNA binding affinity for different sequences measured by changes in fluorescence intensity of 5′-Tetrachloro-fluorescein labelled RNAs. Titration curves for UGUUGU, UUUUUU, UGUUUG, UCUUCU, UAUUAU, AGAAGA and ACAACA are shown. The fraction of bound RNA (ΔF/Fmax) is plotted against total Rna15(16–111) concentration. Equilibrium association constants determined by fitting a hyperbolic binding isotherm to the titration data are shown below.
Mentions: Previously, it has been proposed that, in vitro, in the absence of other protein factors Rna15 binds RNA sequences only weakly and/or non-specifically (16,17). This observation, combined with the lack of a strong Rna15 consensus-binding site in S. cerevisiae 3′-UTRs suggests it is unlikely that the domain recognizes a long specific RNA sequence within the mRNA 3′-end. However, one possibility is that Rna15 displays weak sequence preference for the multiple low complexity sequences flanking the cleavage/polyadenylation sites. To test this notion, we examined the interaction of the core RRM domain, Rna15(1-94)-ht, with two dissimilar pentameric ribo-oligonucleotides UAUUU and UGGCG using NMR spectroscopy, Figure 1A. These data show that the same residues mediate the interaction with both sequences and that the complexes are in a moderately fast regime of exchange. However, because of the low complexity of these RNA sequences it is likely that multiple binding frames are present where small chemical shift differences between resonances of proteins bound in different frames are averaged by the fast regime of exchange. Accordingly, the titrations likely represent the interaction of Rna15(1-94)-ht with a base-averaged sequence rather than a unique RNA–protein interaction. Indeed, considering the low complexity of target sequences in the 3′-UTR multiple binding frames may be important to enhance the RNA binding affinity of Rna15 in vivo. Nevertheless, regardless of the dynamics, quite unexpectedly, the induced chemical shift perturbations are significantly larger upon interaction with UGGCG than they are with the UAUUU sequence, indicating that Rna15(1-94)-ht binds to the G-rich sequence with a higher affinity than the AU sequence.Figure 1.

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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