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A novel RNA binding surface of the TAM domain of TIP5/BAZ2A mediates epigenetic regulation of rRNA genes.

Anosova I, Melnik S, Tripsianes K, Kateb F, Grummt I, Sattler M - Nucleic Acids Res. (2015)

Bottom Line: Here, we show that the NMR structure of the TAM domain of TIP5 resembles the fold of the MBD domain, found in methyl-CpG binding proteins.Mutation of critical amino acids within this surface abolishes RNA binding in vitro and in vivo.Our results explain the distinct binding specificities of TAM and MBD domains to RNA and methylated DNA, respectively, and reveal structural features for the interaction of NoRC with non-coding RNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg D-85764, Germany Biomolecular NMR and Center for Integrated Protein Science Munich (CIPSM), Department Chemie, Technische Universität München, Garching D-85747, Germany.

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pRNA binding of the TAM domain. (A) pRNAmini preserves the structural features of the pRNA stem recognized by TIP5. The secondary structure was calculated by Mfold (55). Nucleotides added for stabilization of the construct are shown in lower-case letters. (B) Confirmation of the secondary structure of pRNAmini by an imino NOESY NMR spectrum. The sequential connectivities between imino protons of neighboring base pairs in the two stem regions are indicated by red and blue lines, respectively, matching the colors indicated for the RNA stem in (a). The characteristic imino signals of the G-U and U-U base pairs have chemical shifts around 11–12 ppm as expected. G-U cross peaks are indicated by magenta dotted lines. The G3-U33 cross peak is broadened possibly due to its terminal position in the pRNAmini stem. (C) Superposition of 1H,15N HSQC NMR spectra of 15N-labeled, 50% random fractional deuterated TIP5/TAM-AT free (black) and in the presence of a 1.2 molar excess of pRNAmini (red). Insets show close-up views for specific residues upon addition of 0.2 (green), 0.5 (blue) and 1.2 (red) molar equivalents of pRNAmini. Arrows indicate the direction of the peak shift. (D) NMR chemical shift perturbations observed for backbone amides in TIP5/TAM-AT at 1.2 molar excess of pRNA. NMR signals that experience line-broadening upon titration of the RNA are shown as orange negative bars. Residues with chemical shift changes larger than one standard deviation from the average (Δδ = 0.078 ppm) are colored yellow.
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Figure 4: pRNA binding of the TAM domain. (A) pRNAmini preserves the structural features of the pRNA stem recognized by TIP5. The secondary structure was calculated by Mfold (55). Nucleotides added for stabilization of the construct are shown in lower-case letters. (B) Confirmation of the secondary structure of pRNAmini by an imino NOESY NMR spectrum. The sequential connectivities between imino protons of neighboring base pairs in the two stem regions are indicated by red and blue lines, respectively, matching the colors indicated for the RNA stem in (a). The characteristic imino signals of the G-U and U-U base pairs have chemical shifts around 11–12 ppm as expected. G-U cross peaks are indicated by magenta dotted lines. The G3-U33 cross peak is broadened possibly due to its terminal position in the pRNAmini stem. (C) Superposition of 1H,15N HSQC NMR spectra of 15N-labeled, 50% random fractional deuterated TIP5/TAM-AT free (black) and in the presence of a 1.2 molar excess of pRNAmini (red). Insets show close-up views for specific residues upon addition of 0.2 (green), 0.5 (blue) and 1.2 (red) molar equivalents of pRNAmini. Arrows indicate the direction of the peak shift. (D) NMR chemical shift perturbations observed for backbone amides in TIP5/TAM-AT at 1.2 molar excess of pRNA. NMR signals that experience line-broadening upon titration of the RNA are shown as orange negative bars. Residues with chemical shift changes larger than one standard deviation from the average (Δδ = 0.078 ppm) are colored yellow.

Mentions: Solution structure of the hTIP5/TAM domain. (A) Cartoon representation of the human TIP5/TAM domain. Secondary structure elements and important loops are annotated. The TAM-specific N-terminal helix and the C-terminal α/β motif are highlighted by black circles. (B) Cartoon representation of the canonical MBD domain of human MBD1 (light blue, PDB ID: 1D9N) in a comparable view as the TAM domain on the left. Secondary structure elements and the loop L1 that mediates DNA binding of the MBD1 domain are annotated. (C) Mapping of the binding surface of TIP5/TAM with pRNAmini based on NMR chemical shift perturbations (Figure 4c) onto a surface representation (left and middle panel) and a ribbon model (right) of the TIP5/TAM domain structure. Left: same view as in (a), middle and right views are rotated by 180°. Colors indicate the RNA binding surface mapped by NMR titrations (Figure 4c and d). Residues with significant chemical shift perturbation are shown in yellow, while residues that exhibit strong intensity reductions upon RNA binding are colored orange as in Figure 4c and d. (D) Electrostatic potential of the TAM domain. Vacuum electrostatics surfaces are shown blue for positive and red for negative charges for the TAM domain in the same orientations as in (c).


A novel RNA binding surface of the TAM domain of TIP5/BAZ2A mediates epigenetic regulation of rRNA genes.

Anosova I, Melnik S, Tripsianes K, Kateb F, Grummt I, Sattler M - Nucleic Acids Res. (2015)

pRNA binding of the TAM domain. (A) pRNAmini preserves the structural features of the pRNA stem recognized by TIP5. The secondary structure was calculated by Mfold (55). Nucleotides added for stabilization of the construct are shown in lower-case letters. (B) Confirmation of the secondary structure of pRNAmini by an imino NOESY NMR spectrum. The sequential connectivities between imino protons of neighboring base pairs in the two stem regions are indicated by red and blue lines, respectively, matching the colors indicated for the RNA stem in (a). The characteristic imino signals of the G-U and U-U base pairs have chemical shifts around 11–12 ppm as expected. G-U cross peaks are indicated by magenta dotted lines. The G3-U33 cross peak is broadened possibly due to its terminal position in the pRNAmini stem. (C) Superposition of 1H,15N HSQC NMR spectra of 15N-labeled, 50% random fractional deuterated TIP5/TAM-AT free (black) and in the presence of a 1.2 molar excess of pRNAmini (red). Insets show close-up views for specific residues upon addition of 0.2 (green), 0.5 (blue) and 1.2 (red) molar equivalents of pRNAmini. Arrows indicate the direction of the peak shift. (D) NMR chemical shift perturbations observed for backbone amides in TIP5/TAM-AT at 1.2 molar excess of pRNA. NMR signals that experience line-broadening upon titration of the RNA are shown as orange negative bars. Residues with chemical shift changes larger than one standard deviation from the average (Δδ = 0.078 ppm) are colored yellow.
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Figure 4: pRNA binding of the TAM domain. (A) pRNAmini preserves the structural features of the pRNA stem recognized by TIP5. The secondary structure was calculated by Mfold (55). Nucleotides added for stabilization of the construct are shown in lower-case letters. (B) Confirmation of the secondary structure of pRNAmini by an imino NOESY NMR spectrum. The sequential connectivities between imino protons of neighboring base pairs in the two stem regions are indicated by red and blue lines, respectively, matching the colors indicated for the RNA stem in (a). The characteristic imino signals of the G-U and U-U base pairs have chemical shifts around 11–12 ppm as expected. G-U cross peaks are indicated by magenta dotted lines. The G3-U33 cross peak is broadened possibly due to its terminal position in the pRNAmini stem. (C) Superposition of 1H,15N HSQC NMR spectra of 15N-labeled, 50% random fractional deuterated TIP5/TAM-AT free (black) and in the presence of a 1.2 molar excess of pRNAmini (red). Insets show close-up views for specific residues upon addition of 0.2 (green), 0.5 (blue) and 1.2 (red) molar equivalents of pRNAmini. Arrows indicate the direction of the peak shift. (D) NMR chemical shift perturbations observed for backbone amides in TIP5/TAM-AT at 1.2 molar excess of pRNA. NMR signals that experience line-broadening upon titration of the RNA are shown as orange negative bars. Residues with chemical shift changes larger than one standard deviation from the average (Δδ = 0.078 ppm) are colored yellow.
Mentions: Solution structure of the hTIP5/TAM domain. (A) Cartoon representation of the human TIP5/TAM domain. Secondary structure elements and important loops are annotated. The TAM-specific N-terminal helix and the C-terminal α/β motif are highlighted by black circles. (B) Cartoon representation of the canonical MBD domain of human MBD1 (light blue, PDB ID: 1D9N) in a comparable view as the TAM domain on the left. Secondary structure elements and the loop L1 that mediates DNA binding of the MBD1 domain are annotated. (C) Mapping of the binding surface of TIP5/TAM with pRNAmini based on NMR chemical shift perturbations (Figure 4c) onto a surface representation (left and middle panel) and a ribbon model (right) of the TIP5/TAM domain structure. Left: same view as in (a), middle and right views are rotated by 180°. Colors indicate the RNA binding surface mapped by NMR titrations (Figure 4c and d). Residues with significant chemical shift perturbation are shown in yellow, while residues that exhibit strong intensity reductions upon RNA binding are colored orange as in Figure 4c and d. (D) Electrostatic potential of the TAM domain. Vacuum electrostatics surfaces are shown blue for positive and red for negative charges for the TAM domain in the same orientations as in (c).

Bottom Line: Here, we show that the NMR structure of the TAM domain of TIP5 resembles the fold of the MBD domain, found in methyl-CpG binding proteins.Mutation of critical amino acids within this surface abolishes RNA binding in vitro and in vivo.Our results explain the distinct binding specificities of TAM and MBD domains to RNA and methylated DNA, respectively, and reveal structural features for the interaction of NoRC with non-coding RNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg D-85764, Germany Biomolecular NMR and Center for Integrated Protein Science Munich (CIPSM), Department Chemie, Technische Universität München, Garching D-85747, Germany.

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