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The La and related RNA‐binding proteins (LARPs): structures, functions, and evolving perspectives

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La was first identified as a polypeptide component of ribonucleic protein complexes targeted by antibodies in autoimmune patients and is now known to be a eukaryote cell‐ubiquitous protein. Structure and function studies have shown that La binds to a common terminal motif, UUU‐3′‐OH, of nascent RNA polymerase III (RNAP III) transcripts and protects them from exonucleolytic decay. For precursor‐tRNAs, the most diverse and abundant of these transcripts, La also functions as an RNA chaperone that helps to prevent their misfolding. Related to this, we review evidence that suggests that La and its link to RNAP III were significant in the great expansions of the tRNAomes that occurred in eukaryotes. Four families of La‐related proteins (LARPs) emerged during eukaryotic evolution with specialized functions. We provide an overview of the high‐resolution structural biology of La and LARPs. LARP7 family members most closely resemble La but function with a single RNAP III nuclear transcript, 7SK, or telomerase RNA. A cytoplasmic isoform of La protein as well as LARPs 6, 4, and 1 function in mRNA metabolism and translation in distinct but similar ways, sometimes with the poly(A)‐binding protein, and in some cases by direct binding to poly(A)‐RNA. New structures of LARP domains, some complexed with RNA, provide novel insights into the functional versatility of these proteins. We also consider LARPs in relation to ancestral La protein and potential retention of links to specific RNA‐related pathways. One such link may be tRNA surveillance and codon usage by LARP‐associated mRNAs. WIREs RNA 2017, 8:e1430. doi: 10.1002/wrna.1430 This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications
Schematic representation of architectures of La proteins and selected La‐related proteins (LARPs). The La motif (LaM) and RNA recognition motif (RRM) comprise the La module. The PDBs used to depict the three‐dimensional structures for the La module and hLa RRM2α are 2VOD and 1OWX, respectively. Other symbols refer to the following: N, nuclear localization (import) sequence; E, nuclear export sequence; R, nuclear retention element; S, Serine‐366; SBM, short basic motif important for recognition of 5′ pppG of nascent RNA and nucleolar localization; DM15, important for direct binding to 7mGpppC‐cap‐5′TOP motif. The RRM2α, xRRM, and DM15 RNA‐interaction motifs/domains are reviewed in a separate section. PAM2, poly(A)‐binding protein interaction motif‐2; PAM2*, LARP1‐associated PAM2 candidate with atypical features (see text); LSA, LaM and S1‐associated motif; PBM, poly(A)‐binding protein interaction protein motif; RIR, RACK1‐interaction region. EI4L in LARP1 refers to eIF4‐like region. The different species’ La proteins depicted are referred to in the text; for simplicity, only the human versions of the LARPs (hLARP) are shown.
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Working models of translation and La‐related protein‐poly(A)‐binding protein (LARP‐PABP) factors. (a) Schematic showing factors involved in an example network of protein–protein and RNA–protein interactions that serve to bridge the 5′ and 3′ end regions of the mRNA according to a closed‐loop model of translation. A translation initiation (start) codon AUG, and a termination (stop) codon, UAA are to orient polarity. Individual factors are referred to in the text. Multiple copies of PABP can bind to the poly(A) tail via its four RNA recognition motif (RRM) domains (depicted in highly schematized form), some of which also serve as separate docking sites for other factors including eIF4G and Paip1. The MLLE domain of PABP, depicted as red circles, is used to interact with different PAM2‐consensus sequence‐containing proteins involved in translation initiation (Paip1), termination (eRF3), or recruitment of deadenylase‐containing complexes (CAF1‐CCR4 and PAN3‐PAN2). The eIF4E cap‐binding positive initiation factor and its association with eIF4G are depicted (for more comprehensive reviews see Refs ). An inset reveals the eukaryotic mRNA two different cap structures; 5′‐TOP mRNAs contain a cap‐C (m7GpppC), and other mRNAs contain a cap‐G/A (m7Gppp‐G/A). (b) Under repressive conditions such as nutritional or other stress, 4E‐BPs (eIF4E‐binding proteins), sequester the cap‐binding protein eIF4E. A potential working model for LARP1 involvement in mRNA metabolism in relation to translation initiation and stabilization. It can bind via the DM15 domain to the 7mGpppC‐TOP motif of TOP mRNAs and to PABP, and possibly to poly(A) (or other regions) of the mRNA via its La module (see text), although whether it would do so simultaneously as depicted here is unknown. LARP1 may either stimulate or inhibit translation and stabilize poly(A) under different conditions (see text). (c) Proposed working model for LARP4/4B involvement in translation and mRNA stability. LARP4/4B interacts with RACK1 and with PABP, although whether either of them would do so simultaneously is unknown, three of the combinatorial possibilities are depicted. LARP4B is proposed to bind to mRNA 3′ UTR sequences (see text). LARP4 has been shown to bind PABP via the PAM2 motif, and through a second PABP‐interaction motif (PBM). It is unknown to which part of PABP the PBM binds. LARP4 can also bind poly(A) RNA. Through competition with the PAM2 motifs of the deadenylases for the MLLE domain of PABP, LARP4/4B may protect mRNA 3′ ends from deadenylation, leading to poly(A) length modulation. 4E‐BPs, 4E‐binding proteins, repressors of translation; Paip1, poly(A)‐binding protein interacting protein 1, a stimulator of translation; eIF4E, eukaryotic initiation factor 4E, a.k.a., cytoplasmic cap‐binding protein; eIF4G, eukaryotic initiation factor 4G; eIF3, eukaryotic initiation factor 3; eIF4A, eukaryotic initiation factor 4A; RACK1, receptor for activated kinase C, a 40S ribosome subunit; eRF1 and eRF3 are eukaryotic translation termination/release factors. Tob2, transducer of ERBB2; CAF1‐CCR4, chromatin assembly factor 1 and CCR4‐NOT transcription complex subunit 6, two proteins with poly(A) deadenylase activity; PAN2, PAB‐dependent poly(A)‐specific ribonuclease subunit PAN2, catalytic subunit of the poly(A)‐nuclease (PAN) deadenylation complex; PAN3, PAB‐dependent poly(A)‐specific ribonuclease subunit PAN3, regulatory subunit of the poly(A)‐nuclease (PAN) deadenylation complex.
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Structure of the DM15 domain of hLARP1 in complex with m7GTP and 5′TOP RNA. The DM15 domain is composed of three helix–turn–helix repeats denoted by DM15 boxes A, B, and C. The figure shows a superimposition of two crystallographic structures: (1) DM15 bound to the RNA sequence 5′‐CUUUUCCG‐3′ (PDB 5V7C) and (2) the complex DM15‐m7GTP (PDB 5V4R). For clarity, the bases beyond U4 in the 5′TOP mRNA have been omitted. Selected side chains involved in protein–RNA contacts are shown as sticks and dashed lines indicate hydrogen bonds.
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Proposed arrangements of the La module for RNA recognition by La‐related proteins (LARPs). (a) Domain arrangement of hLa and hLARP7 in complex with RNA (RNA removed for clarity). The LaM and RRM1 adopt a V‐shaped conformation to create the binding pocket to accommodate the RNA ligand. (b) Cartoon representation of possible domain–domain orientation of the La modules of hLARP6 and hLARP4. Current knowledge on wing 2 conformation and interdomain linker for these proteins suggests that LaM and RRM1 will adopt a more elongated arrangement to interact with RNA (see text).
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Comparison of RRM1 and RRM2 domains in La‐related proteins (LARPs). The RRM1 domains of LARPs are structurally diverse: (a) hLa RRM1 (PDB 1S79); (b) hLARP7 RRM1 (PDB 4WKR); (c) hLARP6 RRM1 (PDB 2MTG). hLARP7 RRM1 lacks strand β4; hLARP6 RRM1 contains additional helices α0′ and α1′. The RRMs2 of (d) hLa (PDB 1OWX), (e) hLARP7 (PDB 5KNW), and (f) p65 (PDB 4EYT) all contain a long C‐terminal helix (α3) that obscures the β‐sheet platform. (g) In p65, the unstructured C‐terminal of the helix (α3x) refolds upon RNA binding. (h) Close‐up view of the interaction between p65 and the S4 RNA. Selected residues are highlighted in stick representation. (i) Sequence alignment of the α3 region for hLa, hLARP7, and p65 performed with Clustal Omega in Uniprot portal (http://www.uniprot.org/align/) and edited and analyzed with Jalview. Residues colored in dark red indicate conservation. Boxed residues denote amino acids that interact with RNA in p65.
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RRM2α and short basic motif (SBM) regions of La proteins representing early eukaryotes. (a) Results of secondary structure prediction for sequence of putative RRM2 region of T. brucei La protein (GenBank: AAF34598.1) performed by Jpred‐4. The secondary structure elements are indicated below in the sequence: α‐helices depicted by red cylinders and β‐strands by green arrows. (b) Solution structure of hLa RRM2α shown for reference (PDB 1OWX), with α3 colored to match underlying (c): red to coincide with boundaries of red rectangle, magenta to match conserved positions demarcated by asterisks, and green to coincide with rectangle downstream sequence boundary in C. (c) Alignment of La protein sequences beginning from the approximate middle of RRM2 extending toward their C‐termini. The overlapping green and orange rectangles represent the sequences aligning with hLa α3 region of RRM2α and a conserved sequence block, respectively. Asterisks above the hLa sequence demarcate conserved residues, the first three of which reside in the tract connecting β4′ to the α3 helix, colored in magenta in the structure in (b). The blue rectangle encompasses the SBM region described for hLa (see text).
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Comparison of the wing 2 motifs and interdomain linkers in La‐related proteins (LARPs). (a) Sequence alignment focusing on LaM wing 2, interdomain linker, and the beginning of RRM1 of LARPs. Human (Hs) La sequence was aligned with 32 LARPs as for Figure . Black vertical rectangles indicate conserved signatures characterizing wing 2; the dark red horizontal arrows denote the beginning of RRM1 strand β1 determined experimentally for hLa, LARP7, LARP6, and LARP4 (I. Cruz‐Gallardo and M.R. Conte, unpublished). The potential extent of the variable linker regions is indicated with a dotted line above the sequences. (b, c) Close‐up views of the wing 2 region of hLa superimposed with hLARP7 (b) and hLARP6 (c). Selected residues involved in the interaction of wing 2 with the rest of the domain are highlighted as sticks. hLa (PDB 2VOS) is depicted in yellow, hLARP7 in light orange (PDB 4WKR), and hLARP6 in cyan (PDB 2MTF).
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Sequence alignment of the LaM and their N‐terminal regions in La and La‐related proteins (LARPs). For each family, the sequence of the human LaM was aligned with proteins of different species using Clustal Omega in Uniprot portal (http://www.uniprot.org/align/). The alignments were edited and analyzed with Jalview software. Residues were colored in gray scale according to the extent of conservation. Species selected include vertebrates‐eutherians (Hs, Homo sapiens, Mm, Mus musculus), vertebrates (Gg, Gallus gallus), invertebrates (Dm, Drosophila melanogaster; Nv, Nematostella vectensis), plants (At, Arabidopsis thaliana), and protists (Dd, Dictyostelium discoideum; Ps, Phytophora sojae). The secondary structure elements for human LaMs appear at the top of the sequence for each family, where α‐helices are depicted by cylinders and β‐strands by arrows. For La, and LARPs 7, 6, and 4, these structured elements have been determined experimentally for the human proteins (I. Cruz‐Gallardo and M.R. Conte, unpublished). For HsLARP1a, a prediction of secondary structure was performed with Jpred‐4 server (http://www.compbio.dundee.ac.uk/jpred/). The conserved residues of the hydrophobic pocket of the LaM are labeled with asterisks and boxed in orange.
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The La module of human La protein in complex with UUU 3′OH RNA. (a) The La module of La (PDB 2VOS) is composed of the LaM (yellow), an interdomain linker (pale green), and the RRM1 (brown). The UUU 3′OH RNA is shown as sticks, color coded by atom type. For clarity, the bases beyond U‐3 have been omitted. Cartoon under the structure shows schematic V‐shaped model with U‐2 in the cleft between the LaM and RRM1 (see text). (b) Close‐up view of La–RNA interaction showing the three 3′ terminal bases in the same orientation as (a). Selected side chains are shown as sticks; dashed lines indicate hydrogen bonds. (c) Close‐up view of the interaction, rotated by 90°.
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