The glycine-rich domain of GRP7 plays a crucial role in binding long RNAs and facilitating phase separation.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
11 Jul 2024
11 Jul 2024
Historique:
received:
29
01
2024
accepted:
05
07
2024
medline:
12
7
2024
pubmed:
12
7
2024
entrez:
11
7
2024
Statut:
epublish
Résumé
Microscale thermophoresis (MST) is a well-established method to quantify protein-RNA interactions. In this study, we employed MST to analyze the RNA binding properties of glycine-rich RNA binding protein 7 (GRP7), which is known to have multiple biological functions related to its ability to bind different types of RNA. However, the exact mechanism of GRP7's RNA binding is not fully understood. While the RNA-recognition motif of GRP7 is known to be involved in RNA binding, the glycine-rich region (known as arginine-glycine-glycine-domain or RGG-domain) also influences this interaction. To investigate to which extend the RGG-domain of GRP7 is involved in RNA binding, mutation studies on putative RNA interacting or modulating sites were performed. In addition to MST experiments, we examined liquid-liquid phase separation of GRP7 and its mutants, both with and without RNA. Furthermore, we systemically investigated factors that might affect RNA binding selectivity of GRP7 by testing RNAs of different sizes, structures, and modifications. Consequently, our study revealed that GRP7 exhibits a high affinity for a variety of RNAs, indicating a lack of pronounced selectivity. Moreover, we established that the RGG-domain plays a crucial role in binding longer RNAs and promoting phase separation.
Identifiants
pubmed: 38992080
doi: 10.1038/s41598-024-66955-5
pii: 10.1038/s41598-024-66955-5
doi:
Substances chimiques
RNA-Binding Proteins
0
Glycine
TE7660XO1C
RNA
63231-63-0
ATGRP7 protein, Arabidopsis
0
Arabidopsis Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
16018Subventions
Organisme : European Commission
ID : 810131
Organisme : Deutsche Forschungsgemeinschaft
ID : 433194101 Research Unit 5116
Informations de copyright
© 2024. The Author(s).
Références
Siomi, H., Choi, M., Siomi, M. C., Nussbaum, R. L. & Dreyfuss, G. Essential role for KH domains in RNA binding: Impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome. Cell 77, 33–39 (1994).
pubmed: 8156595
doi: 10.1016/0092-8674(94)90232-1
Burd, C. G. & Dreyfuss, G. Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 615–621 (1994).
pubmed: 8036511
doi: 10.1126/science.8036511
Adam, S. A., Nakagawa, T., Swanson, M. S., Woodruff, T. K. & Dreyfuss, G. mRNA polyadenylate-binding protein: Gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol. Cell. Biol. 6, 2932–2943 (1986).
pubmed: 3537727
pmcid: 367862
Berg, J. M. Zinc finger domains: Hypotheses and current knowledge. Annu. Rev. Biophys. Biophys. Chem. 19, 405–421 (1990).
pubmed: 2114117
doi: 10.1146/annurev.bb.19.060190.002201
Birney, E., Kumar, S. & Krainer, A. R. Analysis of the RNA-recognition motif and RS and RGG domains: Conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Res. 21, 5803–5816 (1993).
pubmed: 8290338
pmcid: 310458
doi: 10.1093/nar/21.25.5803
Kiledjian, M. & Dreyfuss, G. Primary structure and binding activity of the hnRNP U protein: Binding RNA through RGG box. EMBO J. 11, 2655–2664 (1992).
pubmed: 1628625
pmcid: 556741
doi: 10.1002/j.1460-2075.1992.tb05331.x
Geuens, T., Bouhy, D. & Timmerman, V. The hnRNP family: Insights into their role in health and disease. Hum. Genet. 135, 851–867 (2016).
pubmed: 27215579
pmcid: 4947485
doi: 10.1007/s00439-016-1683-5
van Nocker, S. & Vierstra, R. D. Two cDNAs from Arabidopsis thaliana encode putative RNA binding proteins containing glycine-rich domains. Plant Mol. Biol. 21, 695–699 (1993).
pubmed: 8448367
doi: 10.1007/BF00014552
Vernon, R. M. C. et al. Pi-Pi contacts are an overlooked protein feature relevant to phase separation. Elife 7, 1–48 (2018).
doi: 10.7554/eLife.31486
Chong, P. A., Vernon, R. M. & Forman-Kay, J. D. RGG/RG motif regions in RNA binding and phase separation. J. Mol. Biol. 430, 4650–4665 (2018).
pubmed: 29913160
doi: 10.1016/j.jmb.2018.06.014
Leder, V. et al. Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy. Biochem. Biophys. Res. Commun. 453, 69–74 (2014).
pubmed: 25251471
doi: 10.1016/j.bbrc.2014.09.056
Ziemienowicz, A., Haasen, D., Staiger, D. & Merkle, T. Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AfGRP7. Plant Mol. Biol. 53, 201–212 (2003).
pubmed: 14756317
doi: 10.1023/B:PLAN.0000009288.46713.1f
Yan, Y., Ham, B. K., Chong, Y. H., Yeh, S. D. & Lucas, W. J. A plant SMALL RNA-BINDING PROTEIN 1 family mediates cell-to-cell trafficking of RNAi signals. Mol. Plant 13, 321–335 (2020).
pubmed: 31812689
doi: 10.1016/j.molp.2019.12.001
Giavalisco, P., Kapitza, K., Kolasa, A., Buhtz, A. & Kehr, J. Towards the proteome of Brassica napus phloem sap. Proteomics 6, 896–909 (2006).
pubmed: 16400686
doi: 10.1002/pmic.200500155
Schöning, J. C. et al. Auto-regulation of the circadian slave oscillator component AtGRP7 and regulation of its targets is impaired by a single RNA recognition motif point mutation. Plant J. 52, 1119–1130 (2007).
pubmed: 17924945
doi: 10.1111/j.1365-313X.2007.03302.x
Staiger, D., Zecca, L., Wieczorek-Kirk, D. A., Apel, K. & Eckstein, L. The circadian clock regulated RNA-binding protein AtGRP7 autoregulates its expression by influencing alternative splicing of its own pre-mRNA. Plant J. 33, 361–371 (2003).
pubmed: 12535349
doi: 10.1046/j.1365-313X.2003.01629.x
Schmal, C., Reimann, P. & Staiger, D. A circadian clock-regulated toggle switch explains AtGRP7 and AtGRP8 oscillations in Arabidopsis thaliana. PLoS Comput. Biol. 9, 3 (2013).
doi: 10.1371/journal.pcbi.1002986
Kim, J. S. et al. Glycine-rich RNA-binding protein7 affects abiotic stress responses by regulating stomata opening and closing in Arabidopsis thaliana. Plant J. 55, 455–466 (2008).
pubmed: 18410480
doi: 10.1111/j.1365-313X.2008.03518.x
Schüttpelz, M. et al. Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy. J. Am. Chem. Soc. 130, 9507–9513 (2008).
pubmed: 18576621
doi: 10.1021/ja801994z
Jeong, B. R. et al. Structure function analysis of an ADP-ribosyltransferase type III effector and its RNA-binding target in plant immunity. J. Biol. Chem. 286, 43272–43281 (2011).
pubmed: 22013065
pmcid: 3234823
doi: 10.1074/jbc.M111.290122
Xiao, J. et al. JACALIN-LECTIN LIKE1 regulates the nuclear accumulation of GLYCINE-RICH RNA-BINDING PROTEIN7, influencing the RNA processing of FLOWERING LOCUS C antisense transcripts and flowering time in Arabidopsis. Plant Physiol. 169, 2102–2117 (2015).
pubmed: 26392261
pmcid: 4634062
Seidel, S. A. I. et al. Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59, 301–315 (2013).
pubmed: 23270813
doi: 10.1016/j.ymeth.2012.12.005
Hellman, L. M. & Fried, M. G. Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat. Protoc. 2, 1849–1861 (2007).
pubmed: 17703195
pmcid: 2757439
doi: 10.1038/nprot.2007.249
Doyle, M. L. Characterization of binding interactions by isothermal titration calorimetry. Curr. Opin. Biotechnol. 8, 31–35 (1997).
pubmed: 9013658
doi: 10.1016/S0958-1669(97)80154-1
Wiseman, T., Williston, S., Brandts, J. F. & Lin, L. N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131–137 (1989).
pubmed: 2757186
doi: 10.1016/0003-2697(89)90213-3
Schuck, P. Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules. Annu. Rev. Biophys. Biomol. Struct. 26, 541–566 (1997).
pubmed: 9241429
doi: 10.1146/annurev.biophys.26.1.541
Jerabek-Willemsen, M. et al. MicroScale thermophoresis: Interaction analysis and beyond. J. Mol. Struct. 1077, 101–113 (2014).
doi: 10.1016/j.molstruc.2014.03.009
Perzanowska, O., Smietanski, M., Jemielity, J. & Kowalska, J. Chemically modified Poly(A) analogs targeting PABP: Structure activity relationship and translation inhibitory properties. Chem. A Eur. J. 28, e202201115 (2022).
doi: 10.1002/chem.202201115
Ji, D. et al. Discovery of G-quadruplex-forming sequences in SARS-CoV-2. Brief. Bioinform. 22, 1150–1160 (2021).
pubmed: 32484220
doi: 10.1093/bib/bbaa114
Streitner, C. et al. An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with transcripts in Arabidopsis thaliana. Nucleic Acids Res. 40, 11240–11255 (2012).
pubmed: 23042250
pmcid: 3526319
doi: 10.1093/nar/gks873
Meyer, K. et al. Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7. Genome Biol. 18, 1–22 (2017).
doi: 10.1186/s13059-017-1332-x
Phan, A. T. et al. Structure-function studies of FMRP RGG peptide recognition of an RNA duplex-quadruplex junction. Nat. Struct. Mol. Biol. 18, 796–804 (2011).
pubmed: 21642970
pmcid: 3130835
doi: 10.1038/nsmb.2064
Arribas-Hernández, L. & Brodersen, P. Occurrence and functions of m6A and other covalent modifications in plant mRNA. Plant Physiol. 182, 79–96 (2020).
pubmed: 31748418
doi: 10.1104/pp.19.01156
Reichel, M., Köster, T. & Staiger, D. Marking RNA: M6A writers, readers, and functions in Arabidopsis. J. Mol. Cell Biol. 11, 899–910 (2019).
pubmed: 31336387
pmcid: 6884701
doi: 10.1093/jmcb/mjz085
Zand-Karimi, H. et al. Arabidopsis apoplastic fluid contains sRNA- and circular RNA-protein complexes that are located outside extracellular vesicles. Plant Cell 34, 1863–1881 (2022).
pubmed: 35171271
pmcid: 9048913
doi: 10.1093/plcell/koac043
Yang, L. et al. m5C methylation guides systemic transport of messenger RNA over graft junctions in plants. Curr. Biol. 29, 2465-2476.e5 (2019).
pubmed: 31327714
doi: 10.1016/j.cub.2019.06.042
Tripet, B. P. et al. Structural and biochemical analysis of the Hordeum vulgare L. Hv GR-RBP1 protein, a glycine-rich RNA-binding protein involved in the regulation of barley plant development and stress response. Biochemistry 53, 7945–7960 (2014).
pubmed: 25495582
doi: 10.1021/bi5007223
Wang, L. et al. RALF1-FERONIA complex affects splicing dynamics to modulate stress responses and growth in plants. Sci. Adv. 6, 1–13 (2020).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
doi: 10.1038/s41586-021-03819-2
pubmed: 34265844
pmcid: 8371605
Ye, Y. & Godzik, A. Flexible structure alignment by chaining aligned fragment pairs allowing twists. Bioinformatics 19, ii246–ii255 (2003).
pubmed: 14534198
doi: 10.1093/bioinformatics/btg1086
Li, Z., Jaroszewski, L., Iyer, M., Sedova, M. & Godzik, A. FATCAT 2.0: Towards a better understanding of the structural diversity of proteins. Nucleic Acids Res. 48, W60–W64 (2020).
pubmed: 32469061
pmcid: 7319568
doi: 10.1093/nar/gkaa443
Lin, Y., Protter, D. S. W., Rosen, M. K. & Parker, R. Formation and maturation of phase-separated liquid droplets by RNA-binding proteins. Mol. Cell 60, 208–219 (2015).
pubmed: 26412307
pmcid: 4609299
doi: 10.1016/j.molcel.2015.08.018
McBride, A. E., Conboy, A. K., Brown, S. P., Ariyachet, C. & Rutledge, K. L. Specific sequences within arginine-glycine-rich domains affect mRNA-binding protein function. Nucleic Acids Res. 37, 4322–4330 (2009).
pubmed: 19454603
pmcid: 2715232
doi: 10.1093/nar/gkp349
Xu, F. et al. Phase separation of GRP7 that is facilitated by FERONIA-mediated phosphorylation inhibits mRNA translation to modulate plant temperature resilience. Mol. Plant 17, 460–477 (2024).
pubmed: 38327052
doi: 10.1016/j.molp.2024.02.001
Ryan, V. H. et al. Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration. EMBO J. 40, 1–22 (2021).
doi: 10.15252/embj.2020105001
Ostendorp, A. et al. Intrinsically disordered plant protein PARCL co-localizes with RNA in phase-separated condensates whose formation can be regulated by mutating the PLD. J. Biol. Chem. 2022, 102631 (2022).
doi: 10.1016/j.jbc.2022.102631
Lin, M. K., Lee, Y. J., Lough, T. J., Phimmey, B. S. & Lucas, W. J. Analysis of the pumpkin phloem proteome provides insights into angiosperm sieve tube function. Mol. Cell. Proteom. 8, 343–356 (2009).
doi: 10.1074/mcp.M800420-MCP200
Gruber, A. R., Lorenz, R., Bernhart, S. H., Neuböck, R. & Hofacker, I. L. The Vienna RNA Websuite. Nucleic Acids Res. 36, W70–W74 (2008).
pubmed: 18424795
pmcid: 2447809
doi: 10.1093/nar/gkn188
Lorenz, R. et al. ViennaRNA package 20. Algor. Mol. Biol. 6, 1–14 (2011).
Bhattacharyya, D., Arachchilage, G. M. & Basu, S. Metal cations in G-quadruplex folding and stability. Front. Chem. 4, 207258 (2016).
doi: 10.3389/fchem.2016.00038
Campbell, N. H. & Neidle, S. G-Quadruplexes and metal ions. Met. Ions Life Sci. 10, 119–134 (2012).
pubmed: 22210337
Rogelj, B. et al. Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain. Sci. Rep. 2, 1–10 (2012).
doi: 10.1038/srep00603
Hoell, J. I. et al. RNA targets of wild-type and mutant FET family proteins. Nat. Struct. Mol. Biol. 18, 1428–1431 (2011).
pubmed: 22081015
pmcid: 3230689
doi: 10.1038/nsmb.2163
Wang, X., Schwartz, J. C. & Cech, T. R. Nucleic acid-binding specificity of human FUS protein. Nucleic Acids Res. 43, 7535–7543 (2015).
pubmed: 26150427
pmcid: 4551922
doi: 10.1093/nar/gkv679
Loughlin, F. E. et al. The solution structure of FUS bound to RNA reveals a bipartite mode of RNA recognition with both sequence and shape specificity. Mol. Cell 73, 490-504.e6 (2019).
pubmed: 30581145
doi: 10.1016/j.molcel.2018.11.012
Huelga, S. C. et al. Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins. Cell Rep. 1, 167–178 (2012).
pubmed: 22574288
pmcid: 3345519
doi: 10.1016/j.celrep.2012.02.001
Uren, P. J. et al. High-throughput analyses of hnRNP H1 dissects its multi-functional aspect. RNA Biol. 13, 400–411 (2016).
pubmed: 26760575
pmcid: 4841607
doi: 10.1080/15476286.2015.1138030
Rossbach, O. et al. Crosslinking-immunoprecipitation (iCLIP) analysis reveals global regulatory roles of hnRNP L. RNA Biol. 11, 146–155 (2014).
pubmed: 24526010
pmcid: 3973733
doi: 10.4161/rna.27991
Thandapani, P., O’Connor, T. R., Bailey, T. L. & Richard, S. Defining the RGG/RG motif. Mol. Cell 50, 613–623 (2013).
pubmed: 23746349
doi: 10.1016/j.molcel.2013.05.021
Ozdilek, B. A. et al. Intrinsically disordered RGG/RG domains mediate degenerate specificity in RNA binding. Nucleic Acids Res. 45, 7984–7996 (2017).
pubmed: 28575444
pmcid: 5570134
doi: 10.1093/nar/gkx460
Ghisolfi, L., Joseph, G., Amalric, F. & Erard, M. The glycine-rich domain of nucleolin has an unusual supersecondary structure responsible for its RNA-helix-destabilizing properties. J. Biol. Chem. 267, 2955–2959 (1992).
pubmed: 1737751
doi: 10.1016/S0021-9258(19)50679-2
Ghisolfi, L., Kharrat, A., Joseph, G., Amalric, F. & Erard, M. Concerted activities of the RNA recognition and the glycine-rich C-terminal domains of nucleolin are required for efficient complex formation with pre-ribosomal RNA. Eur. J. Biochem. 209, 541–548 (1992).
pubmed: 1425660
doi: 10.1111/j.1432-1033.1992.tb17318.x
Takahama, K. & Oyoshi, T. Specific binding of modified rgg domain in tls/fus to g-quadruplex rna: Tyrosines in rgg domain recognize 2′-oh of the riboses of loops in g-quadruplex. J. Am. Chem. Soc. 135, 18016–18019 (2013).
pubmed: 24251952
doi: 10.1021/ja4086929
Pype, S., Slegers, H., Moens, L., Merlevede, W. & Goris, J. Tyrosine phosphorylation of a M(r) 38,000 A/B-type hnRNP protein selectively modulates its RNA binding. J. Biol. Chem. 269, 31457–31465 (1994).
pubmed: 7527388
doi: 10.1016/S0021-9258(18)31716-2
Monahan, Z. et al. Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity. EMBO J. 36, 2951–2967 (2017).
pubmed: 28790177
pmcid: 5641905
doi: 10.15252/embj.201696394
Pahlow, S., Ostendorp, A., Krüßel, L. & Kehr, J. Phloem sap sampling from Brassica napus for 3D-PAGE of protein and ribonucleoprotein complexes. JoVE J. Vis. Exp. 2018, e57097 (2018).
Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).
pubmed: 15915565
doi: 10.1016/j.pep.2005.01.016
Cazenave, C. & Uhlenbeck, O. C. RNA template-directed RNA synthesis by T7 RNA polymerase. Proc. Natl. Acad. Sci. U. S. A. 91, 6972–6976 (1994).
pubmed: 7518923
pmcid: 44320
doi: 10.1073/pnas.91.15.6972