Arabidopsis thaliana GLYCINE RICH RNA‐BINDING PROTEIN 7 interaction with its iCLIP target LHCB1.1 correlates with changes in RNA stability and circadian oscillation

• Published in: The Plant journal : for cell and molecular biology 2024-04, Vol.118 (1), p.203-224
• Main Authors: Lewinski, Martin, Steffen, Alexander, Kachariya, Nitin, Elgner, Mareike, Schmal, Christoph, Messini, Niki, Köster, Tino, Reichel, Marlene, Sattler, Michael, Zarnack, Kathi, Staiger, Dorothee
• Format: Article
• Language: English
• Subjects: Arabidopsis; Arabidopsis thaliana; Binding sites; Bioinformatics; Biological clocks; Chlorophyll; circadian; Circadian rhythm; Circadian rhythms; Crosslinking; Environmental effects; Glycine; iCLIP; Immunoprecipitation; Mutants; NMR; Nuclear magnetic resonance; Nucleotides; Oscillations; Proteins; PureCLIP score; Ribonucleic acid; RNA; RNA-binding protein; RNA‐protein interaction; Stability; Uridine
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/tpj.16601

SUMMARY The importance of RNA‐binding proteins (RBPs) for plant responses to environmental stimuli and development is well documented. Insights into the portfolio of RNAs they recognize, however, clearly lack behind the understanding gathered in non‐plant model organisms. Here, we characterize binding of the circadian clock‐regulated Arabidopsis thaliana GLYCINE‐RICH RNA‐BINDING PROTEIN 7 (AtGRP7) to its target transcripts. We identified novel RNA targets from individual‐nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) data using an improved bioinformatics pipeline that will be broadly applicable to plant RBP iCLIP data. 2705 transcripts with binding sites were identified in plants expressing AtGRP7‐GFP that were not recovered in plants expressing an RNA‐binding dead variant or GFP alone. A conserved RNA motif enriched in uridine residues was identified at the AtGRP7 binding sites. NMR titrations confirmed the preference of AtGRP7 for RNAs with a central U‐rich motif. Among the bound RNAs, circadian clock‐regulated transcripts were overrepresented. Peak abundance of the LHCB1.1 transcript encoding a chlorophyll‐binding protein was reduced in plants overexpressing AtGRP7 whereas it was elevated in atgrp7 mutants, indicating that LHCB1.1 was regulated by AtGRP7 in a dose‐dependent manner. In plants overexpressing AtGRP7, the LHCB1.1 half‐life was shorter compared to wild‐type plants whereas in atgrp7 mutant plants, the half‐life was significantly longer. Thus, AtGRP7 modulates circadian oscillations of its in vivo binding target LHCB1.1 by affecting RNA stability. Significance Statement We develop an advanced bioinformatics pipeline to identify RBP targets and their RNA binding motifs from iCLIP data which will be broadly applicable to plant RBP iCLP data. We show that AtGRP7 binds to the LHCB1.1 transcript and decreases its stability with concomitant dampened peak abundance.