Discovering the RNA-Binding Proteome of Plant Leaves with an Improved RNA Interactome Capture Method

Abstract

RNA-binding proteins (RBPs) play a crucial role in regulating RNA function and fate. However, the full complement of RBPs has only recently begun to be uncovered through proteome-wide approaches such as RNA interactome capture (RIC). RIC has been applied to various cell lines and organisms, including plants, greatly expanding the repertoire of RBPs. However, several technical challenges have limited the efficacy of RIC when applied to plant tissues. Here, we report an improved version of RIC that overcomes the difficulties imposed by leaf tissue. Using this improved RIC method in Arabidopsis leaves, we identified 717 RBPs, generating a deep RNA-binding proteome for leaf tissues. While 75% of these RBPs can be linked to RNA biology, the remaining 25% were previously not known to interact with RNA. Interestingly, we observed that a large number of proteins related to photosynthesis associate with RNA in vivo, including proteins from the four major photosynthetic supercomplexes. As has previously been reported for mammals, a large proportion of leaf RBPs lack known RNA-binding domains, suggesting unconventional modes of RNA binding. We anticipate that this improved RIC method will provide critical insights into RNA metabolism in plants, including how cellular RBPs respond to environmental, physiological and pathological cues.

Proteome-wide Profiling of RNA-Binding Protein Responses to flg22 Reveals Novel Components of Plant Immunity

“…S1f). In addition, from the 1,135 RBPs identified, 743 were previously reported in RIC studies in plants 9,12 , whereas 392 are newly discovered here and represent novel putative RBPs. Hence, we provide a deep RBPome generated in plant tissues, which contains hundreds of putative RBPs previously unknown to bind RNA.…”

“…To capture the dynamics of the plant RBPome during immune responses, Arabidopsis leaves were infiltrated with either H2O (mock) or the immune elicitor flg22 and leaf tissue was harvested at 2 h and 12 h post treatment (hpt). Protein-RNA interactions were 'frozen' by UV irradiation and RBPs covalently linked to RNA were isolated following a recently developed, plant-optimised version of RIC, referred to here as ptRIC 12 (Fig. 1a,c).…”

“…Before studying the dynamics of the RBPome upon flg22 treatment, the individual leaf RBPomes for each of the 4 different conditions (mock 2 hpt, flg22 2 hpt, mock 12 hpt and flg22 12 hpt) were determined by analysing the enrichment of the proteins identified in crosslinked (CL; +UV) over not crosslinked (NoCL; -UV) samples as previously described 12 (Fig. S1a-d).…”

“…Several groups have recently applied RIC to plant samples, discovering hundreds of novel plant RBPs [12][13][14][15] . Building on these advances, a recent study applied RIC to profile RBP dynamics in plant suspension cultures in response to drought stress 16 .…”

“…Here we applied our recent plantoptimised RIC protocol (ptRIC) 12 to leaves treated with the immunogenic peptide flg22. Strikingly, our study uncovers 186 flg22-responsive RBPs that display differential association with RNA upon immunity activation.…”