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

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.

Experiences with a training DSW knowledge model for early-stage researchers.

Background: Data management is fast becoming an essential part of scientific practice, driven by open science and FAIR (findable, accessible, interoperable, and reusable) data sharing requirements. Whilst data management plans (DMPs) are clear to data management experts and data stewards, understandings of their purpose and creation are often obscure to the producers of the data, which in academic environments are often PhD students. Methods: Within the RNAct EU Horizon 2020 ITN project, we engaged the 10 RNAct early-stage researchers (ESRs) in a training project aimed at formulating a DMP. To do so, we used the Data Stewardship Wizard (DSW) framework and modified the existing Life Sciences Knowledge Model into a simplified version aimed at training young scientists, with computational or experimental backgrounds, in core data management principles. We collected feedback from the ESRs during this exercise. Results: Here, we introduce our new life-sciences training DMP template for young scientists. We report and discuss our experiences as principal investigators (PIs) and ESRs during this project and address the typical difficulties that are encountered in developing and understanding a DMP. Conclusions: We found that the DS-wizard can also be an appropriate tool for DMP training, to get terminology and concepts across to researchers. A full training in addition requires an upstream step to present basic DMP concepts and a downstream step to publish a dataset in a (public) repository. Overall, the DS-Wizard tool was essential for our DMP training and we hope our efforts can be used in other projects.

Frag4Lead: growing crystallographic fragment hits by catalog using fragment-guided template docking.

In recent years, crystallographic fragment screening has matured into an almost routine experiment at several modern synchrotron sites. The hits of the screening experiment, i.e. small molecules or fragments binding to the target protein, are revealed along with their 3D structural information. Therefore, they can serve as useful starting points for further structure-based hit-to-lead development. However, the progression of fragment hits to tool compounds or even leads is often hampered by a lack of chemical feasibility. As an attractive alternative, compound analogs that embed the fragment hit structurally may be obtained from commercial catalogs. Here, a workflow is reported based on filtering and assessing such potential follow-up compounds by template docking. This means that the crystallographic binding pose was integrated into the docking calculations as a central starting parameter. Subsequently, the candidates are scored on their interactions within the binding pocket. In an initial proof-of-concept study using five starting fragments known to bind to the aspartic protease endothiapepsin, 28 follow-up compounds were selected using the designed workflow and their binding was assessed by crystallography. Ten of these compounds bound to the active site and five of them showed significantly increased affinity in isothermal titration calorimetry of up to single-digit micromolar affinity. Taken together, this strategy is capable of efficiently evolving the initial fragment hits without major synthesis efforts and with full control by X-ray crystallography.