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

Abstract

The chromatin remodeling complex NoRC, comprising the subunits SNF2h and TIP5/BAZ2A, mediates heterochromatin formation at major clusters of repetitive elements, including rRNA genes, centromeres and telomeres. Association with chromatin requires the interaction of the TAM (TIP5/ARBP/MBD) domain of TIP5 with noncoding RNA, which targets NoRC to specific genomic loci. 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. However, the TAM domain exhibits an extended MBD fold with unique C-terminal extensions that constitute a novel surface for RNA binding. 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.

Figure 1.

Domain organization of TIP5 and its TAM domain. (A) Top: domain organization of human TIP5. Bottom: the role of pRNA in NoRC-mediated silencing of rRNA genes (1). Intergenic transcripts (red dashed line) originating from the rDNA intergenic spacer are processed into 150–250 nt transcripts, termed pRNA (red waved lines). The interaction of pRNA with TIP5 guides NoRC to the rDNA promoter, an essential step in NoRC-dependent silencing of rRNA genes. (B) Multiple sequence alignment of the TAM domain of hTIP5 (BAZ2A) with phylogenetically related BAZ2A homologs (14) and canonical human MBDs. Sequences were aligned using MUSCLE algorithm (47), color coding according to CLUSTAL-X (48). Secondary structure observed in human TAM (Figure 3) is indicated above the sequence. Structural features of the TAM domain that are not present in MBD folds are indicated by black boxes. Black stars mark residues that are strongly affected in NMR titrations of TIP5/TAM-AT with pRNA^mini. Red and black filled circles indicate residues that were mutated to probe the TAM/pRNA interaction interface showing strong or no effect on RNA binding, respectively. Black triangles indicate key residues of the MBD1 that are crucial for its interaction with methylated CpG (52). Many of these residues are not conserved in TAM domains, i.e. TIP5 TAM R538, Q547, Q562. Residue numbering refers to human TIP5.

Figure 2.

NMR and biochemical analysis of RNA binding by the TIP5/TAM domain. (A) NMR analysis of the TIP5/TAM-AT domain. ¹³C secondary chemical shifts Δδ(¹³C_α) − Δδ(¹³C_β), ¹⁵N R₁, R₂ relaxation rates and {¹H}-¹⁵N heteronuclear NOE values are plotted versus TIP5 residue numbers. Secondary structure elements of the TAM domain are indicated on top. (B) The TAM-AT domain represents the minimal RNA binding region of TIP5. Electrophoretic mobility shift assays (EMSA) showing binding of TIP5, TIP5/TAM and TIP5/TAM-AT to RNA. 0, 10, 20 and 30 nM of the respective proteins were incubated with 1 nM of [³²P]-MCS-RNA probe. Positions of free RNA probe and RNA:TIP5 complexes are marked with arrows. Super-shifted binding products are marked with asterisks. (C) TIP5/TAM-AT has almost the same RNA binding efficiency as TIP5. Amounts of bound RNA in the EMSAs shown in panel (B) were quantitated using the ImageGauge software and are presented as percentage of input probe. (D) Scatchard plot analysis of in vitro binding of TIP5/TAM-AT to pRNA. Binding kinetics to pRNA were monitored by filter binding assay. The dissociation constant (K_D) was calculated using Scatchard plot analysis from the ratio of bound to bound/free probes. The resulting fit (R² = 0.6) yields an RNA binding affinity constant for the TIP5/TAM-AT/pRNA interaction of K_D = 5.5 nM.

Figure 3.

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 pRNA^mini 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).

Figure 4.

pRNA binding of the TAM domain. (A) pRNA^mini 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 pRNA^mini 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 pRNA^mini 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 pRNA^mini (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 pRNA^mini. 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.

Figure 5.

Mutational analysis of TAM/pRNA interaction. (A) Side chains of residues probed by mutational analysis are shown in magenta on the structure of the TIP5/TAM domain. Residues which are involved in RNA binding are annotated with larger font. Side chains shown in pink are controls, not expected to be involved in RNA binding. (B) RNA binding of wild-type and mutant TIP5/TAM-AT. The triangle on top indicates increasing protein concentrations (0, 5, 10, 20, 40 and 80 nM) of wild-type TIP5/TAM-AT (WT) or the indicated TIP5/TAM-AT mutants, which were incubated with 10 nM of [³²P]-pRNA. Protein–RNA complexes were retained on nitrocellulose filters and visualized by phosphorimaging. Mutants selected for further analyses (R545E and W546A) are marked with black stars. (C) EMSA experiments monitoring binding of wild-type TIP5/TAM-AT and mutants R545E and W546A to RNA. 12.5, 25, 50 nM of the respective proteins were incubated with 2.5 nM of [³²P]-labeled RNA. Positions of free RNA and RNA:TIP5 complexes are indicated by arrows. Supershifts indicating higher order complexes of TIP5 binding with several RNA molecules are marked with asterisks. (D) RNA immunoprecipitation experiments monitoring binding of wild-type and mutant TIP5 to pRNA in vivo. HEK293T cells were transfected with expression vectors encoding FLAG-tagged wild-type TIP5, TIP5/R545E or TIP5/W546A. TIP5 was immunoprecipitated with anti-FLAG antibodies and associated pRNA was monitored by RT-qPCR.

Figure 6.

Comparison of TAM and MBD nucleic acid binding domains. (A) The pRNA binding surface of the TIP5/TAM domain. Side chains of residues important for RNA binding are shown on a cartoon representation of the TIP5/TAM domain structure. (B) Cartoon representation of the canonical MBD domain from MBD1 (light blue) in complex with methylated DNA shown as orange cartoon (PDB ID: 1IG4) (52).