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Resonance assignment of human LARP4A La module

Resonance assignment of human LARP4A La module Human LARP4A belongs to a superfamily of RNA binding proteins called La-related proteins (LARPs). Whilst being a posi- tive regulator of protein synthesis and a promoter of mRNA stability, LARP4A also controls cell morphology and motility in human breast and prostate cancer cells. All LARPs share a characteristic RNA binding unit named the La–module, which despite a high level of primary structure conservation exhibits a great versatility in RNA target selection. Human LARP4A La–module is the most divergent compared with other LARPs and its RNA recognition properties have only recently started to be revealed. Given the key role of LARP4A protein in cancer cell biology, we have initiated a complete NMR characterisa- 1 15 13 tion of its La-module and here we report the assignment of H, N and C resonances resulting from our studies. Keywords LARP4A · La–module · LARPs · RNA binding protein Biological context been suggested to play a role in the stress response (Gilbert- son et al. 2018; Yang et al. 2011). LARP4A also regulates Human LARP4A is an RNA binding protein (RBP) involved cancer cell morphology and motility: its siRNA-mediated in mRNA stabilisation and translation enhancement, 3′UTR depletion has been shown to increase cell migration and polyA lengthening and miRNA processing (Maraia et al. invasion, whereas its overexpression promotes cell circular- 2017; Mattijssen et al. 2017; Nussbacher and Yeo 2018; ity in breast and prostate cancers (Seetharaman et al. 2016). Yang et al. 2011). As it localises to stress granules, mem- LARP4A binds to the 3′polyA tail of mRNAs, and associ- braneless structures associated with mRNA turnover and ates to translating ribosomes and protein partners including protection of mRNA during stress conditions, LARP4A has RACK1 (Receptor for Activated C Kinase) and PolyA bind- ing protein (PABP) (Maraia et al. 2017; Yang et al. 2011). How the cellular functions of LARP4A in RNA and tumour * Maria R. Conte biology are mediated by its molecular associations to RNA sasi.conte@kcl.ac.uk targets and/or other proteins remains unclear. LARP4A possesses a La–module, a unique RNA binding Randall Centre for Cell and Molecular Biophysics, King’s unit conserved across all the members of the La-related pro- College London, London SE1 1UL, UK teins (LARPs) superfamily and consisting of two domains, Department of Pharmacy, University of Naples Federico II, the La motif (LaM) and an RNA recognition motif (RRM1) Naples, Italy (Bousquet-Antonelli and Deragon 2009; Maraia et  al. MRC Biomedical NMR Centre, The Francis Crick Institute, 2017). Despite sequence conservation, the RNA targets and London NW1 1AT, UK functions of the La–modules in different LARPs are quite Centre for Biomolecular Spectroscopy, King’s College diverse, but the molecular bases of this versatility remain London, London SE1 1UL, UK poorly understood (Maraia et al. 2017). The La–module of Present Address: Department of Chemistry, King’s College the human La protein has been extensively studied at the London, 7 Trinity Street, London SE1 1DB, UK molecular level and its interactions with the 3′UUU tail OH Present Address: The Francis Crick Institute, 1 Midland of the nascent RNA polymerase III transcripts well charac- Road, London NW1 1AT, UK terised: the LaM and RRM1 act in synergy to accommodate Present Address: Institute of Protein Biochemistry, National the 3′UUU target, with the LaM establishing the majority Research Council, Via Pietro Castellino 111, 80131 Naples, OH Italy Vol.:(0123456789) 1 3 170 I. Cruz-Gallardo et al. of the intermolecular contacts with the RNA (Alfano et al. with a KCl gradient from 0 to 1 M. The pure protein was dia- 2004; Kotik-Kogan et al. 2008; Teplova et al. 2006). An lysed into a buffer containing 20 mM Tris pH 7.25, 100 mM analogous mechanism has been reported for human LARP7 KCl, 0.2 mM EDTA and 1 mM DTT. and LARP6 (Maraia et al. 2017; Martino et al. 2015; Uchi- kawa et al. 2015). The La–motifs (LaM) of LARPs exhibit a high degree NMR spectroscopy of sequence conservation across the superfamily, particu- 15 15 13 larly in six key residues identified in human La as prime The N and N, C-labeled samples of LARP4A La–mod- mediators of RNA recognition, namely Q20, Y23, Y24, ule (111–287) were concentrated to 600 µM in 20 mM Tris D33, F35, F55 (human La protein numbering). Intriguingly, pH 7.25, 100 mM KCl, 0.2 mM EDTA, 1 mM DTT in 99.8% in human LARP4A Y24 and F55 are replaced by Cys and D O or 10%D O/90%H O as appropriate. All the NMR 2 2 2 Met respectively (Merret et al. 2013). Moreover, primary experiments were performed at 25 °C on Bruker Avance sequence analysis suggests that LARP4A lacks the otherwise III or NEO NMR spectrometers operating at 700, 800 and conserved wing 2 loop at the C-terminus of the LaM and 950 MHz, equipped with triple resonance cryoprobes. NMR contains a short inter domain linker between the LaM and data were processed with Topspin 3.5pl7 software (Bruker) the RRM (Maraia et al. 2017; Martino et al. 2015; unpub- and NMRPipe/NMRDraw (Delaglio et al. 1995). Assign- lished). These distinctive characteristics, divergent from ment was performed with CcpNmr Analysis (Vranken et al. other LARPs, may impact on the RNA binding properties of 2005) and/or CARA/NEASY (Bartels et  al. 1995)  soft- LARP4A. We therefore set out to unveil the structure and the wares. For the assignment of the backbone resonances a set 1 15 determinants of RNA recognition of LARP4A, to understand of experiments including H- N HSQC, HNCO, HNCA, its cellular functions and roles in cancer biology. Here, we HN(CO)CA, HNCACB and CBCA(CO)NH was used. The report the chemical shift assignments of the backbone and side-chain resonance assignments were determined using 1 15 1 15 1 13 side-chain resonances of LARP4A La–module. H- N HSQC, H/ N- and H/ C-edited NOESY-HSQC and HCCH-TOCSY spectra (Fesik et al. 1988). Methods and experiments Extent of assignment and data deposition Protein expression and purification The chemical shift assignment for LARP4A La–module has LARP4A La–module, spanning residues 111–287, was been deposited in the Biological Magnetic Resonance Bank cloned in a pET-Duet1 vector (Novagen) with a hexa-His- (http://www.bmrb.wisc.edu/), accession number 27666. tidine tag at the N-terminus, followed by a TEV protease Human LARP4A La–module displays a well-resolved 1 15 cleavage site. The recombinant protein was expressed in H- N HSQC indicating that the protein is folded (Fig. 1a). Escherichia coli Rosetta II cells (Novagen) and uniformly It comprises 177 residues, with 4 glycine and 8 proline 15 15 13 labelled with N or N/ C in minimal media containing residues. An almost complete backbone assignment (93%) 15 13 NH Cl (1 g/L) and C glucose (2 g/L). The cells were was achieved, identifying 93% of NH (156/168), 87% of Hα grown to an OD value of 0.6 and induced at 18 °C with (154/177), 97% of Cα (171/177) and 95% of Cβ (165/173) 1 mM IPTG (Isopropyl β-d -1-thiogalactopyranoside) over- resonances unambiguously. For side chains, 87% of the ali- 1 13 night. The harvested cells were resuspended in a buffer con - phatic and 48% of the aromatic side-chains ( H and C reso- taining 50 mM Tris pH 8, 300 mM NaCl, 10 mM imidazole, nances beyond the Cγ position) were assigned. Resonances 5% glycerol, one Complete protease inhibitor cocktail tablet of Asn111, Ser132, His234, Asn275, Thr276 and the linker (Roche), 2 mM phenylmethylsulfonyl fluoride and lysozyme. residues (His196–Arg198) could not be assigned. Non- After sonication and clarification, the lysate was loaded on a native residues derived from the vector sequence after the 5 mL His-Trap (GE Healthcare) affinity column and the His- His-tag cleavage, a serine and a valine preceding Asn111, tagged protein was eluted with a gradient from 0 to 300 mM were not assigned. of imidazole. The protein was dialyzed into a buffer com- An analysis of the backbone chemical shifts performed prising 50 mM Tris pH 7.25, 100 mM KCl, 0.2 mM EDTA, with TALOS+ (Shen et al. 2009) revealed that LARP4A 1 mM DTT and digested with TEV protease at 4 °C over- La–module contains eight α-helices and seven β-strands dis- night. To isolate the un-tagged protein from the protease, tributed between the LaM and RRM1 (Fig. 1b). LARP4A tags and non-digested protein, the mixture was applied onto LaM displays the same secondary structure topology a Nickel affinity column (Generon). To eliminate any nucleic found in other LaMs previously described (Alfano et al. acid from the sample, the protein was further purified using 2004; Mar tino et  al. 2015): α1(115–129)–α1′(133–137) a 5 mL Hi-Trap heparin column (GE Healthcare) and eluted –α2(139–145)–β1(151–153)–α3(155–158)–α4(161–165) 1 3 Resonance assignment of human LARP4A La module 171 Acknowledgements ICG and MRC acknowledge support from a New- ton Royal Society Fellowship ref. NF140482 and a H2020 actions: Marie Sklodowska Curie Fellowship ref. 655341; LM acknowledges support by an EMBO Long Term Fellowship ref. ALT400-2010. RT and SDT were visiting PhD students from the Department of Phar- macy, University of Naples “Federico II”. We thank the Centre for Biomolecular Spectroscopy for access to biophysical infrastruc- ture. The Centre was funded by the Wellcome Trust and British Heart Foundation (Grant Nos. 202767/Z/16/Z and IG/16/2/32273 respectively). This work was supported by the Francis Crick Insti- tute through provision of access to the MRC Biomedical NMR Centre. The Francis Crick Institute receives its core funding from Can- cer Research UK (Grant No. FC001029), the UK Medical Research Council (FC001029), and the Wellcome Trust (Grant No. FC001029). Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat iveco mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. References Alfano C, Sanfelice D, Babon J, Kelly G, Jacks A, Curry S, Conte MR (2004) Structural analysis of cooperative RNA binding by the La motif and central RRM domain of human La protein. Nat Struct Mol Biol 11:323–329 Bartels C, Xia TH, Billeter M, Guntert P, Wuthrich K (1995) The pro- gram XEASY for computer-supported NMR spectral analysis of biological macromolecules. J Biomol NMR 6:1–10 Bousquet-Antonelli C, Deragon JM (2009) A comprehensive analysis of the La-motif protein superfamily. RNA 15:750–764 Clery A, Blatter M, Allain FH (2008) RNA recognition motifs: boring? Not quite. Curr Opin Struct Biol 18:290–298 Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293 Fig. 1 LARP4A La–module NH amide assignment and secondary Fesik SW, Luly JR, Erickson JW, Abad-Zapatero C (1988) Isotope- 1 15 structure. a H- N HSQC spectrum of human LARP4A recorded at edited proton NMR study on the structure of a pepsin/inhibitor 800  MHz and 25  °C. Amide group peaks are labelled with the resi- complex. Biochemistry 27:8297–8301 due type and numbered corresponding to the protein sequence. A Gilbertson S, Federspiel JD, Hartenian E, Cristea IM, Glaunsinger closer view of the central part of the spectrum is shown for clarity. B (2018) Changes in mRNA abundance drive shuttling of RNA b TALOS+ prediction of secondary structure elements for LARP4A binding proteins, linking cytoplasmic RNA degradation to tran- La–module. The secondary structure probabilities (red, α-helices; scription. eLife https ://doi.org/10.7554/eLife .37663 blue, β-strands), plotted against the residue number, are based on Kotik-Kogan O, Valentine ER, Sanfelice D, Conte MR, Curry S (2008) backbone HN, N, C′, Cα, and Cβ chemical shifts. Residues for which Structural analysis reveals conformational plasticity in the rec- backbone amide resonance assignments are missing are indicated by ognition of RNA 3’ ends by the human La protein. Structure asterisks 16:852–862 Maraia RJ, Mattijssen S, Cruz-Gallardo I, Conte MR (2017) The La and related RNA-binding proteins (LARPs): structures, functions, –α5(170–178)–β2(183–185)–β3(190–193). Likewise, the and evolving perspectives. Wiley Interdiscip Rev RNA. https :// RRM1 harbours the canonical topology for this class of doi.org/10.1002/wrna.1430 Martino L, Pennell S, Kelly G, Busi B, Brown P, Atkinson RA, Salis- domains (Clery et  al. 2008): β1(200–204)–α1(212–218) bury NJH, Ooi ZH, See KW, Smerdon SJ, Alfano C, Bui TTT, –β2(229–233)–β3(236–242)–α2(247–259) β4(267–274). Conte MR (2015) Synergic interplay of the La motif, RRM1 and The backbone and side-chains chemical shifts of the the interdomain linker of LARP6 in the recognition of collagen isolated LaM (111–196) and RRM1 (196–287) were also mRNA expands the RNA binding repertoire of the La module. Nucleic Acids Res 43:645–660 analysed, revealing that they remained largely unchanged Mattijssen S, Arimbasseri AG, Iben JR, Gaidamakov S, Lee J, Hafner in the context of the La–module, and suggesting that the M, Maraia RJ (2017) LARP4 mRNA codon-tRNA match con- two domains do not adopt a rigid orientation relative to one tributes to LARP4 activity for ribosomal protein mRNA poly(A) other in solution. tail length protection. eLife https ://doi.org/10.7554/eLife .28889 1 3 172 I. Cruz-Gallardo et al. Merret R, Martino L, Bousquet-Antonelli C, Fneich S, Descombin J, Uchikawa E, Natchiar KS, Han X, Proux F, Roblin P, Zhang E, Durand Billey E, Conte MR, Deragon JM (2013) The association of a A, Klaholz BP, Dock-Bregeon AC (2015) Structural insight into La module with the PABP-interacting motif PAM2 is a recurrent the mechanism of stabilization of the 7SK small nuclear RNA by evolutionary process that led to the neofunctionalization of La- LARP7. Nucleic Acids Res 43:3373–3388 related proteins. RNA 19:36–50 Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, Nussbacher JK, Yeo GW (2018) Systematic discovery of RNA binding Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN proteins that regulate microRNA levels. Mol Cell 69:1005–1016. data model for NMR spectroscopy: development of a software e7 pipeline. Proteins 59:687–696 Seetharaman S, Flemyng E, Shen J, Conte MR, Ridley AJ (2016) The Yang R, Gaidamakov SA, Xie J, Lee J, Martino L, Kozlov G, Craw- RNA-binding protein LARP4 regulates cancer cell migration and ford AK, Russo AN, Conte MR, Gehring K, Maraia RJ (2011) invasion. Cytoskeleton 73:680–690 La-related protein 4 binds poly(A), interacts with the poly(A)- Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS+: a hybrid binding protein MLLE domain via a variant PAM2w motif, and method for predicting protein backbone torsion angles from NMR can promote mRNA stability. Mol Cell Biol 31:542–556 chemical shifts. J Biomol NMR 44:213–223 Teplova M, Yuan YR, Phan AT, Malinina L, Ilin S, Teplov A, Patel Publisher’s Note Springer Nature remains neutral with regard to DJ (2006) Structural basis for recognition and sequestration of jurisdictional claims in published maps and institutional affiliations. UUUOH 3’ temini of nascent RNA polymerase III transcripts by La, a rheumatic disease autoantigen. Molecular cell 21:75–85 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biomolecular NMR Assignments Springer Journals

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Springer Journals
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Copyright © 2019 by The Author(s)
Subject
Physics; Biological and Medical Physics, Biophysics; Polymer Sciences; Biochemistry, general
ISSN
1874-2718
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DOI
10.1007/s12104-019-09871-4
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Abstract

Human LARP4A belongs to a superfamily of RNA binding proteins called La-related proteins (LARPs). Whilst being a posi- tive regulator of protein synthesis and a promoter of mRNA stability, LARP4A also controls cell morphology and motility in human breast and prostate cancer cells. All LARPs share a characteristic RNA binding unit named the La–module, which despite a high level of primary structure conservation exhibits a great versatility in RNA target selection. Human LARP4A La–module is the most divergent compared with other LARPs and its RNA recognition properties have only recently started to be revealed. Given the key role of LARP4A protein in cancer cell biology, we have initiated a complete NMR characterisa- 1 15 13 tion of its La-module and here we report the assignment of H, N and C resonances resulting from our studies. Keywords LARP4A · La–module · LARPs · RNA binding protein Biological context been suggested to play a role in the stress response (Gilbert- son et al. 2018; Yang et al. 2011). LARP4A also regulates Human LARP4A is an RNA binding protein (RBP) involved cancer cell morphology and motility: its siRNA-mediated in mRNA stabilisation and translation enhancement, 3′UTR depletion has been shown to increase cell migration and polyA lengthening and miRNA processing (Maraia et al. invasion, whereas its overexpression promotes cell circular- 2017; Mattijssen et al. 2017; Nussbacher and Yeo 2018; ity in breast and prostate cancers (Seetharaman et al. 2016). Yang et al. 2011). As it localises to stress granules, mem- LARP4A binds to the 3′polyA tail of mRNAs, and associ- braneless structures associated with mRNA turnover and ates to translating ribosomes and protein partners including protection of mRNA during stress conditions, LARP4A has RACK1 (Receptor for Activated C Kinase) and PolyA bind- ing protein (PABP) (Maraia et al. 2017; Yang et al. 2011). How the cellular functions of LARP4A in RNA and tumour * Maria R. Conte biology are mediated by its molecular associations to RNA sasi.conte@kcl.ac.uk targets and/or other proteins remains unclear. LARP4A possesses a La–module, a unique RNA binding Randall Centre for Cell and Molecular Biophysics, King’s unit conserved across all the members of the La-related pro- College London, London SE1 1UL, UK teins (LARPs) superfamily and consisting of two domains, Department of Pharmacy, University of Naples Federico II, the La motif (LaM) and an RNA recognition motif (RRM1) Naples, Italy (Bousquet-Antonelli and Deragon 2009; Maraia et  al. MRC Biomedical NMR Centre, The Francis Crick Institute, 2017). Despite sequence conservation, the RNA targets and London NW1 1AT, UK functions of the La–modules in different LARPs are quite Centre for Biomolecular Spectroscopy, King’s College diverse, but the molecular bases of this versatility remain London, London SE1 1UL, UK poorly understood (Maraia et al. 2017). The La–module of Present Address: Department of Chemistry, King’s College the human La protein has been extensively studied at the London, 7 Trinity Street, London SE1 1DB, UK molecular level and its interactions with the 3′UUU tail OH Present Address: The Francis Crick Institute, 1 Midland of the nascent RNA polymerase III transcripts well charac- Road, London NW1 1AT, UK terised: the LaM and RRM1 act in synergy to accommodate Present Address: Institute of Protein Biochemistry, National the 3′UUU target, with the LaM establishing the majority Research Council, Via Pietro Castellino 111, 80131 Naples, OH Italy Vol.:(0123456789) 1 3 170 I. Cruz-Gallardo et al. of the intermolecular contacts with the RNA (Alfano et al. with a KCl gradient from 0 to 1 M. The pure protein was dia- 2004; Kotik-Kogan et al. 2008; Teplova et al. 2006). An lysed into a buffer containing 20 mM Tris pH 7.25, 100 mM analogous mechanism has been reported for human LARP7 KCl, 0.2 mM EDTA and 1 mM DTT. and LARP6 (Maraia et al. 2017; Martino et al. 2015; Uchi- kawa et al. 2015). The La–motifs (LaM) of LARPs exhibit a high degree NMR spectroscopy of sequence conservation across the superfamily, particu- 15 15 13 larly in six key residues identified in human La as prime The N and N, C-labeled samples of LARP4A La–mod- mediators of RNA recognition, namely Q20, Y23, Y24, ule (111–287) were concentrated to 600 µM in 20 mM Tris D33, F35, F55 (human La protein numbering). Intriguingly, pH 7.25, 100 mM KCl, 0.2 mM EDTA, 1 mM DTT in 99.8% in human LARP4A Y24 and F55 are replaced by Cys and D O or 10%D O/90%H O as appropriate. All the NMR 2 2 2 Met respectively (Merret et al. 2013). Moreover, primary experiments were performed at 25 °C on Bruker Avance sequence analysis suggests that LARP4A lacks the otherwise III or NEO NMR spectrometers operating at 700, 800 and conserved wing 2 loop at the C-terminus of the LaM and 950 MHz, equipped with triple resonance cryoprobes. NMR contains a short inter domain linker between the LaM and data were processed with Topspin 3.5pl7 software (Bruker) the RRM (Maraia et al. 2017; Martino et al. 2015; unpub- and NMRPipe/NMRDraw (Delaglio et al. 1995). Assign- lished). These distinctive characteristics, divergent from ment was performed with CcpNmr Analysis (Vranken et al. other LARPs, may impact on the RNA binding properties of 2005) and/or CARA/NEASY (Bartels et  al. 1995)  soft- LARP4A. We therefore set out to unveil the structure and the wares. For the assignment of the backbone resonances a set 1 15 determinants of RNA recognition of LARP4A, to understand of experiments including H- N HSQC, HNCO, HNCA, its cellular functions and roles in cancer biology. Here, we HN(CO)CA, HNCACB and CBCA(CO)NH was used. The report the chemical shift assignments of the backbone and side-chain resonance assignments were determined using 1 15 1 15 1 13 side-chain resonances of LARP4A La–module. H- N HSQC, H/ N- and H/ C-edited NOESY-HSQC and HCCH-TOCSY spectra (Fesik et al. 1988). Methods and experiments Extent of assignment and data deposition Protein expression and purification The chemical shift assignment for LARP4A La–module has LARP4A La–module, spanning residues 111–287, was been deposited in the Biological Magnetic Resonance Bank cloned in a pET-Duet1 vector (Novagen) with a hexa-His- (http://www.bmrb.wisc.edu/), accession number 27666. tidine tag at the N-terminus, followed by a TEV protease Human LARP4A La–module displays a well-resolved 1 15 cleavage site. The recombinant protein was expressed in H- N HSQC indicating that the protein is folded (Fig. 1a). Escherichia coli Rosetta II cells (Novagen) and uniformly It comprises 177 residues, with 4 glycine and 8 proline 15 15 13 labelled with N or N/ C in minimal media containing residues. An almost complete backbone assignment (93%) 15 13 NH Cl (1 g/L) and C glucose (2 g/L). The cells were was achieved, identifying 93% of NH (156/168), 87% of Hα grown to an OD value of 0.6 and induced at 18 °C with (154/177), 97% of Cα (171/177) and 95% of Cβ (165/173) 1 mM IPTG (Isopropyl β-d -1-thiogalactopyranoside) over- resonances unambiguously. For side chains, 87% of the ali- 1 13 night. The harvested cells were resuspended in a buffer con - phatic and 48% of the aromatic side-chains ( H and C reso- taining 50 mM Tris pH 8, 300 mM NaCl, 10 mM imidazole, nances beyond the Cγ position) were assigned. Resonances 5% glycerol, one Complete protease inhibitor cocktail tablet of Asn111, Ser132, His234, Asn275, Thr276 and the linker (Roche), 2 mM phenylmethylsulfonyl fluoride and lysozyme. residues (His196–Arg198) could not be assigned. Non- After sonication and clarification, the lysate was loaded on a native residues derived from the vector sequence after the 5 mL His-Trap (GE Healthcare) affinity column and the His- His-tag cleavage, a serine and a valine preceding Asn111, tagged protein was eluted with a gradient from 0 to 300 mM were not assigned. of imidazole. The protein was dialyzed into a buffer com- An analysis of the backbone chemical shifts performed prising 50 mM Tris pH 7.25, 100 mM KCl, 0.2 mM EDTA, with TALOS+ (Shen et al. 2009) revealed that LARP4A 1 mM DTT and digested with TEV protease at 4 °C over- La–module contains eight α-helices and seven β-strands dis- night. To isolate the un-tagged protein from the protease, tributed between the LaM and RRM1 (Fig. 1b). LARP4A tags and non-digested protein, the mixture was applied onto LaM displays the same secondary structure topology a Nickel affinity column (Generon). To eliminate any nucleic found in other LaMs previously described (Alfano et al. acid from the sample, the protein was further purified using 2004; Mar tino et  al. 2015): α1(115–129)–α1′(133–137) a 5 mL Hi-Trap heparin column (GE Healthcare) and eluted –α2(139–145)–β1(151–153)–α3(155–158)–α4(161–165) 1 3 Resonance assignment of human LARP4A La module 171 Acknowledgements ICG and MRC acknowledge support from a New- ton Royal Society Fellowship ref. NF140482 and a H2020 actions: Marie Sklodowska Curie Fellowship ref. 655341; LM acknowledges support by an EMBO Long Term Fellowship ref. ALT400-2010. RT and SDT were visiting PhD students from the Department of Phar- macy, University of Naples “Federico II”. We thank the Centre for Biomolecular Spectroscopy for access to biophysical infrastruc- ture. The Centre was funded by the Wellcome Trust and British Heart Foundation (Grant Nos. 202767/Z/16/Z and IG/16/2/32273 respectively). This work was supported by the Francis Crick Insti- tute through provision of access to the MRC Biomedical NMR Centre. The Francis Crick Institute receives its core funding from Can- cer Research UK (Grant No. FC001029), the UK Medical Research Council (FC001029), and the Wellcome Trust (Grant No. FC001029). Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat iveco mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. References Alfano C, Sanfelice D, Babon J, Kelly G, Jacks A, Curry S, Conte MR (2004) Structural analysis of cooperative RNA binding by the La motif and central RRM domain of human La protein. Nat Struct Mol Biol 11:323–329 Bartels C, Xia TH, Billeter M, Guntert P, Wuthrich K (1995) The pro- gram XEASY for computer-supported NMR spectral analysis of biological macromolecules. J Biomol NMR 6:1–10 Bousquet-Antonelli C, Deragon JM (2009) A comprehensive analysis of the La-motif protein superfamily. RNA 15:750–764 Clery A, Blatter M, Allain FH (2008) RNA recognition motifs: boring? Not quite. Curr Opin Struct Biol 18:290–298 Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293 Fig. 1 LARP4A La–module NH amide assignment and secondary Fesik SW, Luly JR, Erickson JW, Abad-Zapatero C (1988) Isotope- 1 15 structure. a H- N HSQC spectrum of human LARP4A recorded at edited proton NMR study on the structure of a pepsin/inhibitor 800  MHz and 25  °C. Amide group peaks are labelled with the resi- complex. Biochemistry 27:8297–8301 due type and numbered corresponding to the protein sequence. A Gilbertson S, Federspiel JD, Hartenian E, Cristea IM, Glaunsinger closer view of the central part of the spectrum is shown for clarity. B (2018) Changes in mRNA abundance drive shuttling of RNA b TALOS+ prediction of secondary structure elements for LARP4A binding proteins, linking cytoplasmic RNA degradation to tran- La–module. The secondary structure probabilities (red, α-helices; scription. eLife https ://doi.org/10.7554/eLife .37663 blue, β-strands), plotted against the residue number, are based on Kotik-Kogan O, Valentine ER, Sanfelice D, Conte MR, Curry S (2008) backbone HN, N, C′, Cα, and Cβ chemical shifts. Residues for which Structural analysis reveals conformational plasticity in the rec- backbone amide resonance assignments are missing are indicated by ognition of RNA 3’ ends by the human La protein. Structure asterisks 16:852–862 Maraia RJ, Mattijssen S, Cruz-Gallardo I, Conte MR (2017) The La and related RNA-binding proteins (LARPs): structures, functions, –α5(170–178)–β2(183–185)–β3(190–193). Likewise, the and evolving perspectives. Wiley Interdiscip Rev RNA. https :// RRM1 harbours the canonical topology for this class of doi.org/10.1002/wrna.1430 Martino L, Pennell S, Kelly G, Busi B, Brown P, Atkinson RA, Salis- domains (Clery et  al. 2008): β1(200–204)–α1(212–218) bury NJH, Ooi ZH, See KW, Smerdon SJ, Alfano C, Bui TTT, –β2(229–233)–β3(236–242)–α2(247–259) β4(267–274). Conte MR (2015) Synergic interplay of the La motif, RRM1 and The backbone and side-chains chemical shifts of the the interdomain linker of LARP6 in the recognition of collagen isolated LaM (111–196) and RRM1 (196–287) were also mRNA expands the RNA binding repertoire of the La module. Nucleic Acids Res 43:645–660 analysed, revealing that they remained largely unchanged Mattijssen S, Arimbasseri AG, Iben JR, Gaidamakov S, Lee J, Hafner in the context of the La–module, and suggesting that the M, Maraia RJ (2017) LARP4 mRNA codon-tRNA match con- two domains do not adopt a rigid orientation relative to one tributes to LARP4 activity for ribosomal protein mRNA poly(A) other in solution. tail length protection. eLife https ://doi.org/10.7554/eLife .28889 1 3 172 I. Cruz-Gallardo et al. Merret R, Martino L, Bousquet-Antonelli C, Fneich S, Descombin J, Uchikawa E, Natchiar KS, Han X, Proux F, Roblin P, Zhang E, Durand Billey E, Conte MR, Deragon JM (2013) The association of a A, Klaholz BP, Dock-Bregeon AC (2015) Structural insight into La module with the PABP-interacting motif PAM2 is a recurrent the mechanism of stabilization of the 7SK small nuclear RNA by evolutionary process that led to the neofunctionalization of La- LARP7. Nucleic Acids Res 43:3373–3388 related proteins. RNA 19:36–50 Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, Nussbacher JK, Yeo GW (2018) Systematic discovery of RNA binding Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN proteins that regulate microRNA levels. Mol Cell 69:1005–1016. data model for NMR spectroscopy: development of a software e7 pipeline. Proteins 59:687–696 Seetharaman S, Flemyng E, Shen J, Conte MR, Ridley AJ (2016) The Yang R, Gaidamakov SA, Xie J, Lee J, Martino L, Kozlov G, Craw- RNA-binding protein LARP4 regulates cancer cell migration and ford AK, Russo AN, Conte MR, Gehring K, Maraia RJ (2011) invasion. Cytoskeleton 73:680–690 La-related protein 4 binds poly(A), interacts with the poly(A)- Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS+: a hybrid binding protein MLLE domain via a variant PAM2w motif, and method for predicting protein backbone torsion angles from NMR can promote mRNA stability. Mol Cell Biol 31:542–556 chemical shifts. J Biomol NMR 44:213–223 Teplova M, Yuan YR, Phan AT, Malinina L, Ilin S, Teplov A, Patel Publisher’s Note Springer Nature remains neutral with regard to DJ (2006) Structural basis for recognition and sequestration of jurisdictional claims in published maps and institutional affiliations. UUUOH 3’ temini of nascent RNA polymerase III transcripts by La, a rheumatic disease autoantigen. Molecular cell 21:75–85 1 3

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Published: Jan 10, 2019

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