Noncanonical RNA binding of human La-related protein 6

Figures

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  Figure 1.. Canonical RNA binding of LARPs. Close-up views of the canonical RNA binding pocket in structural models of La domains from HsLARP1 binding an AAA-3′OH RNA (top left), HsLARP3 (top right), and HsLARP7 (bottom left) binding UUU-3′OH RNA are shown alongside the protein structure of the A2M5-bound state of the La domain of HsLARP6 presented here (bottom right). Side chains of residues relevant for RNA binding are shown and labeled. Structural representations of HsLARP1, HsLARP3, HsLARP6, and HsLARP7 were generated using PDB entries 7SOR, 2VOD, 9NGX, and 4WKR, respectively [28, 30, 53].

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  Figure 2.. Secondary structure of 5′SL RNA. Sequences and secondary structure predictions of wild type A1 and A2 RNA as well as the 35-mer A2M5 model RNA. Nucleotides shared between the wild type A2 RNA and A2M5 model sequences are indicated by a gray, dashed box. Sequence and secondary structure shared with the 14-mer model RNA are indicated by a red, dashed box.

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  Figure 3.. Imino protons resonance assignment of unbound and La domain-bound A2M5. (A) [1H, 15N]-HSQC (top) and [1H, 1H]-NOESY (bottom) spectra of the imino proton region of unbound [13C,15N]-A2M5 recorded at 279 K. Sequential assignment of this region in unbound A2M5 required a 180 ms NOE mixing time and reduced temperature due to otherwise excessive exchange with bulk water limiting detection of diagonal and cross peaks. (B) [1H, 15N]-HSQC (top) and [1H, 1H]-NOESY (bottom) spectra of the imino proton region of La domain-bound [13C, 15N]-A2M5 recorded at 298 K with a 250 ms NOE mixing time. 1D 1H spectra of the imino region of each species are shown on top. Residues of stem 1, stem 2, and the internal loop regions of A2M5 (shown on the right) are labeled with red, blue, and green, respectively. Dashed lines indicate select NOEs showing a sequential walk of base-paired residues. Residues U12 and U25 cannot be distinguished from each other and are labeled accordingly. Imino proton resonances from the internal loop which appear in the La domain-bound state are labeled based on their nucleobase identity URA and GUA (gray boxes).

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  Figure 4.. Structure and topology of the A2M5-bound La domain of HsLARP6. (A) Secondary structure topology of the La domain of HsLARP6. (B) The 20 structures of the A2M5-bound La domain as determined by the solution NMR spectroscopy (PDB: 9NGX). The structures are displayed in cartoon representation with α-helices, β-strands, and loops are shown in red, blue, and gray, respectively. (C) The surface charge of the La domain as determined by APBS shows a continuous basic patch (blue) [104]. The structure is oriented identically to the structure shown in panel (B). (D–F) Sections of interest show structural details surrounding residues W85/W145 (D), the RNK motif (E), and the hydrophobic cluster involving the C-terminus (F). Side chains of select residues are shown and labeled.

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  Figure 5.. Comparison of the A2M5-bound and unbound structures of the La domain of HsLARP6. The structures of (A) the A2M5-bound La domain (PDB: 9NGX) and (B) the unbound La domain (PDB: 2MTF) are shown in cartoon representations. Regions with RMSD values above 4.0 Å are shown in red and side chains are shown for select residues. (C) Average residue-specific RMSDs between the ensembles of the A2M5-bound and unbound structures. RMSD values above 4.0 Å within the well-defined core structure excluding the more flexible N- and C-terminal tails are shown in red.

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  Figure 6.. Mapping the binding surface for 5′SL RNA on the La domain of HsLARP6. (A) CSPs between the A2M5-bound and unbound La domain are shown on a representative model of the A2M5-bound state. Residues with CSPs below 0.1 ppm are colored gray, residues with CSPs between 0.1 and 0.15 ppm, between 0.15 and 0.2 ppm, and above 0.2 ppm are colored light blue, blue, and dark blue, respectively. Side chains are shown and are labeled for residues with a CSP > 0.15 ppm. (B) Solvent accessibility is shown on a representative model of the A2M5-bound state as reported by the ratio of NMR signal intensity in the presence (I) or absence (I0) of co-solvent gadodiamide. Highly (I/I0 > 0.8) and moderately (0.8 > I/I0 > 0.6) inaccessible residues are colored in dark and light red, respectively. Side chains are shown and are labeled for residues with an I/I0 > 0.8. (C) 2D [1H, 1H]-NOESY spectrum showing intermolecular NOE contacts between A2M5 RNA and the La domain. Parts of the imino region (left) and methyl region (right) are shown. Dashed lines link diagonal and cross peaks observed in both spectra while labels indicate the identity of the observed residues from each molecule. A larger portion of the…

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  Figure 7.. Mutagenesis of key residues in the La domain of HsLARP6. (A) Locations of single-point alanine mutants throughout the La domain are shown on a representative structure of the A2M5-bound structure. (B) KD and ΔΔG values determined for each measurable mutation (Supplementary Table S8). Mutated residues are color-coded based on the relative effect their mutation had on the dissociation constant (KD) of A2M5 binding. ΔΔG values above 2.0 kcal/mol are shown in dark red, ΔΔG values between 1.0 and 2.0 kcal/mol are shown in red, ΔΔG values between 0.5 and 1.0 kcal/mol are shown in light red, and ΔΔG values below 0.5 kcal/mol are shown in gray.