Functional Characterization the Group Three Pumilio/FBF RNA-Binding Proteins of Arabidopsis thaliana

Plants encode over 1800 RNA binding proteins (RBPs) that modulate a myriad of steps in gene regulation from chromatin organization through translation, yet only a small number of these proteins and their target transcripts have been functionally characterized. Two classes of RBPs, Pentatricopeptide Repeats (PPR) and Pumilio/fem-3 binding factor (PUF) proteins, recognize and bind linear RNA through a unique, modular, sequence-specific method determined by the amino acid sequence of key residues. PPRs have been well studied due to their abundance in plant genomes and critical roles in organellar RNA editing, while very little is known about plant PUFs, despite extensive work done in animal systems. Animal PUFs are important in a variety of important biological processes including embryo and tissue development, cell patterning, viral RNA sensing, and cancer. Arabidopsis thaliana encodes for 26 PUFs (APUM), the majority of which have not be functionaly characterized. To fill this gap, we set out to better understand the conservation and structure of all APUMs. Using phylogenetic analysis, predictive protein structural modeling, and PUF binding element (PBE) prediction we were able to define the conservation of PUFs within plant species, as well as determine the PBE for all APUMs, illustrating the potential for unique PBEs in two Groups of APUMs. Group (G) 3 APUMs were selected for further functional and molecular characterization. Thus, we conducted the first global determination of in vivo APUM target RNAs via RIPseq and identified a core subset of RNA targets that contained an enriched, 3’UTR localized, G3 APUM PBE, 5’-UGUAYAUA-3’. Overexpression of G3 APUMs caused a cascade of direct and indirect effects that resulted in a global change in transcript abundance. The binding of G3 APUMs led to a decrease in transcripts involved in development and anatomical structure that resulted in slow growth and a delay in flowering, illustrating the role of G3 APUMs in both post-transcriptional gene regulation and plant development