A comprehensive analysis of the La-motif protein superfamily

Abstract

The extremely well-conserved La motif (LAM), in synergy with the immediately following RNA recognition motif (RRM), allows direct binding of the (genuine) La autoantigen to RNA polymerase III primary transcripts. This motif is not only found on La homologs, but also on La-related proteins (LARPs) of unrelated function. LARPs are widely found amongst eukaryotes and, although poorly characterized, appear to be RNA-binding proteins fulfilling crucial cellular functions. We searched the fully sequenced genomes of 83 eukaryotic species scattered along the tree of life for the presence of LAM-containing proteins. We observed that these proteins are absent from archaea and present in all eukaryotes (except protists from the Plasmodium genus), strongly suggesting that the LAM is an ancestral motif that emerged early after the archaea-eukarya radiation. A complete evolutionary and structural analysis of these proteins resulted in their classification into five families: the genuine La homologs and four LARP families. Unexpectedly, in each family a conserved domain representing either a classical RRM or an RRM-like motif immediately follows the LAM of most proteins. An evolutionary analysis of the LAM-RRM/RRM-L regions shows that these motifs co-evolved and should be used as a single entity to define the functional region of interaction of LARPs with their substrates. We also found two extremely well conserved motifs, named LSA and DM15, shared by LARP6 and LARP1 family members, respectively. We suggest that members of the same family are functional homologs and/or share a common molecular mode of action on different RNA baits.

FIGURE 1.

Phylogenetic relationships among LAMs and structural organization of LAM-containing proteins. (A) Phylogenetic relationships among LAMs. The phylogenetic tree was obtained using LAM sequences from the 134 selected proteins as described in the Materials and Methods. Selected informative statistical supports (approximate likelihood-ratio test [aLRT] data) are indicated. (Blue) Proteins from protists, (green) proteins from plants, (brown) proteins from from fungi, (red) proteins from animals. Protein names are given according to the nomenclature proposed in the present paper. For the corresponding accession numbers, see Supplemental Figure 2. Previously utilized names are reported in parentheses when available. Black numbers following protein names correspond to structural organizations presented in B. (B) Schematic representations of the different structural organizations found among the full-length LAM-containing proteins. The color code used is reported in the box at the top of the figure. (Numbers in parentheses) Number of proteins of a given structural organization, (dashed lines) putative significant groups of proteins based on phylogenetic and structural organization criteria. Species names abbreviations: (Am) Apis mellifera, (At) Arabidopsis thaliana, (An) Aspergillus niger, (Ce) Caenorharbditis elegans, (Cs) Capitella sp., (Cr) Chlamydomonas reinhardii, (Ci) Ciona intestinalis, (Dp) Daphnia pullex, (Dd) Dictyostelium discoideum, (Dm) Drosophila melanogaster, (Fr) Fugu rubripes, (Gg) Gallus gallus, (Hs) Homo sapiens, (Lb) Laccaria bicolor, (Lm) Leishmania major, (Lg) Lottia gigantae, (Ng) Naegleria gruberi, (Nv) Nematostella vectensis, (Os) Oriza sativa, (Pc) Phanerochaete chrysosporium, (Pb) Phycomyces blakesleanus, (Php) Physcomitrella patens, (Ps) Phytophthora sojae, (Sc) Saccharomyces cerevisiae, (Sp) Schizosaccharomyces pombe, (Sm) Selaginella moellendorfii, (Stp) Stronggylocentrotus purpuratus, (Tb) Trypanosoma brucei, (Xt) Xenopus tropicalis.

FIGURE 2.

Structural organization of the LAM-RRM1 and LAM-RRM-L domains of genuine La homologs and LARPs. (A) Schematic representation of the predicted structural organizations of the 90 amino acids found immediately downstream from the LAM. (Plain red box) LAM, (colored arrows) β-sheets (β1, β2, β3, and β4), (colored boxes) α-helices (α1 and α2) of the RRM or RRM-L domains. The proposed names of these regions are reported on the right (see Fig. 5). (B) Size of the linker regions (in amino acids) connecting the LAM and RRM1 or RRM-L domains and of the loops connecting the structural features of each domain. Sizes of each of these connecting regions were determined for every protein, and the average sizes are reported together with standard deviations.

FIGURE 3.

Analysis of the DM15/LARP1 region. (A) Complete sequence alignment of every detected DM15 box. The multiple sequence alignment was edited by Boxshade at the Mobyle portal (http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form=boxshade). (Black) Identical residues, (gray) similar residues. Protein names are reported on the left. These names are according to the nomenclature used in Figure 1A. The bold letter added to the protein name (A, B, or C) corresponds to the type of DM15 box. Amino acids identical in 90%–100% of the sequences are reported on top of the alignment. (Black dots) Residues present in 100% of the sequences, (gray dots) amino acids present in 90%–100% of the sequences. (B) Phylogenetic relationships among the DM15 boxes and structural organization of the full-length proteins. A phylogenetic tree was reconstructed (see Materials and Methods) from the multiple sequence alignment presented in A. Protein names from which the DM15 box originates are reported at the tip of each branch. Selected informative statistical supports are reported. (Black numbers) Structural organization of the full-length proteins as pictured on the right-hand side. (Plain red box) The LAM, (blue box) the RRM-L5 motif, (brown boxes) type A DM15 repeats (pink boxes) type B DM15 repeats, (purple boxes) type D DM15 repeats. The number of proteins presenting a given type of organization is reported in parentheses. (C) Multiple sequence alignments performed between DM15 boxes of the same type: (brown bar) type A, (pink bar) type B, (purple bar) type C. Protein names reported on the left and alignment editing are as in A.

FIGURE 4.

Identification of a novel conserved region, the LSA motif. Multiple sequence alignment of the C-terminal region of proteins with a LAM/RRM-L3a or LAM/RRM-L3b organization. (Black) Identical residues, (gray) similar residues. The names of the proteins are reported on the left-hand side as in Figure 1A. The consensus sequence is reported below the alignment. (X) Any amino acid. Plant protein consensus sequences contain an additional three amino acids (PRM) between positions six and seven and display two instead of four undefined amino acids between positions 13 and 14.

FIGURE 5.

Phylogenetic relationships among the LAM-RRM1(RRM-L) domains. (A) Phylogenetic tree obtained using sequences of LAM-RRM1 or LAM-RRM-L regions (see Supplemental Figs. 3–6 for the sequences used) from the 113 LAM proteins and the ciliate p43 and p65 proteins. Selected statistical supports are reported and relevant clusters, defining the different LAM-containing protein families, are labeled with pink numbers (1–5). Phylogenetic groups of proteins previously identified in Figure 1A are also indicated (orange uppercase letters between parentheses). Protein names, species color codes, and species abbreviation names are as in Figure 1, plus: (Ea) Euplotes aediculatus, (Tth) Tetrahymena thermophila. Black numbers following protein names correspond to the structural organization of the protein as represented in B. (B) Schematic representations of the different structural organizations found among the full-length LAM-RRM(RRM-L)-containing proteins. The color code used is reported in the box at the top of the figure. (Dashed lines) Boundaries of each significant cluster.

FIGURE 6.

Nomenclature and definition of the five families of LAM-containing proteins, summarizing the proposed families definition of LAM-containing proteins. Names are defined according to these rules: Xy: species abbreviation name; La: genuine La or LARP: La-related Protein; 1, 4, 6, or 7; family number, z: lower case letter (a, b, c, …) added in cases when there are several members of the same family in a given species. The structural organization of each family member is shown. Color code is identical to that of Figure 5B.