The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence

Abstract

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.

Figure 1.

DM15 binds the 5′TOP sequence of RPS6. (A) Representative EMSAs of WT DM15 with oligonucleotides containing the first 42 nucleotides of the RPS6 5′TOP (RPS6-42mer; top), the RPS6 TOP lacking the 5′ polypyrimidine tract (RPS6 ΔTOP; middle), and the first 20 nucleotides of the RPS6 5′TOP (RPS6-20mer; bottom). (B) EMSAs of point mutants of DM15 at the putative DM15 RNA-binding and non-crystallographic dimerization surfaces. RNA binding of these point mutants was tested with the RPS6-20mer oligonucleotide. (C) Quantification of three independent EMSAs of wild-type DM15 bound to radiolabelled RPS6-20mer RNA oligonucleotide; error bars show standard deviation. In (A) and (B), asterisks denote 1 μM total protein for reference.

Figure 2.

The human DM15 region contains three helix-turn-helix repeats. (A) Ribbon diagram of the structure of LARP1 amino acids 796–946. The eight alpha helices observed are labelled numerically from N- to C-terminus. The two layers of helices are coloured yellow and orange; the loops and the helix (α8) that does not lie parallel to other helices are shown in white. The left panel is related to the right panel by a rotation about the x-axis of ∼95°. (B) The DM15 region contains three helix-turn-helix repeats. DM15A is shown in brown, DM15B in purple, DM15C in pink. (C) Alignment of three structural repeats, coloured as in (B).

Figure 3.

Alignment of the LARP1 DM15 region from plant, protista, fungi and animal species. Alignment of the (A) DM15A, (B) DM15B and (C) DM15C repeats from select plant, protist, fungi and animal species. (D) Alignment of the DM15 A, B and C consensus sequences. Black asterisks, amino acid targeted by mutagenesis (Figure 4). Red asterisks, amino acids that mediate dimer interactions through the co-crystallized sulphate ion (Figure 5). The numbers above each alignment indicate the position in the human LARP1a sequence. The intensity of the sequence colouring is proportional to the level of conservation. The species code is the following: Animal: Dm: Drosophila melanogaster, Dr: Danio rerio, Hs: Homo sapiens, Ta: Trichoplax adhaerens. Fungi: Aspwe: Aspergillus wentii, Batde: Batrachochytrium dendrobatidis, Phybl: Phycomyces blakesleeanus.Plant and algae: At: Arabidopsis thaliana, Cr: Chlamydomonas reinhardii. Protista: Mb: Monosiga brevicollis, Dd: Dictyostelium discoideum. A more complete alignment of the LARP1 DM15 region is shown in Supplementary Figure S4.

Figure 4.

The concave surface of DM15 is conserved and positively charged. (A) Edge-on views of DM15 reveal that it assumes a curved structure. Top, cartoon representation and coloured as in Figure 2A. Amino acids tested for their roles in binding the RPS6 5′TOP oligonucleotide are shown as sticks. Middle, surface electrostatic representation as calculated by PyMOL (blue, electrostatically positive; red, electrostatically negative). Bottom, surface representation coloured by conservation as calculated by Consurf (42) using alignment of DM15 sequences from Supplementary Figure S4 (white, less conserved; dark green, more conserved). (B) View of the concave DM15 surface, coloured as in (A). (C) View of the convex DM15 surface, coloured as in (A). The view in panel (B) is related to that in panel (C) by a 180° rotation about the x-axis.

Figure 5.

Dimerization of DM15 in the crystal. (A) A pseudo-twofold axis of rotation passes through the co-crystallized sulfate ion. The N- and C-termini of each monomer are labelled. (B) A surface representation of the same view as in (A), coloured according to electrostatic potential. A positively charged cleft is generated upon dimerization of DM15. (C) A zoomed view of the area in (A) bordered by the dotted box. The bound sulfate ion interacts directly with K915 and Y911 on both molecules within the non-crystallographic pseudo-dimer.

Figure 6.

Modelling of single-stranded oligopyrimidine RNA with conserved amino acids on the concave surface of the DM15 region. (A) A poly-pyrimidine RNA sequence (CUUUU) was modelled onto the positive patch on the structure of DM15 based on shape and surface complementarity, in addition to hydrogen bonding potential and sulfate ion positioning. (B) A zoomed view of the docked RNA boxed in panel (A). (C) The specific amino acids used to facilitate RNA docking in the structure are labelled. An additional residue, Y911 (shown in Figure 5) was also used to position the 5′ phosphate of the third residue. Predicted hydrogen bonds shown as black dashed lines.