Pre-programming (‘cell-fating’) has been explored extensively in eukaryotic cells, including developmental biology and stem-cell differentiation. It is heavily regulated through post-transcriptional regulation (PTR), and particularly translational repression. RNA-binding proteins (RBPs) are essential in PTR, including in cell-fating and translation repression. Despite significant conservation of RBPs across the tree of life, there are notable periods of transition, particularly with eukaryogenesis. As eukaryotic lineages evolved, they expanded on bacterial and archaeal RNA Binding domains (RBDs) and formulated “novel” domains, notably including the emergence of pumilio homology proteins (PUF). The emergence of PUFs was a major innovation underpinning “cell fate” decisions that guide eukaryotic stage-transition, cellular development and embryogenesis. Intriguingly, RBP biology and functionality appears almost entirely conserved from yeast through to humans. This leaves open questions as to when eukaryotic RBP regulation evolved, what functionality these early RBP systems had, and what the early eukaryotic RBPome looked like. Such systems are essential to study basic elements of RBP biology and regulation in, presumably, their simplest form.
The phylum Metamonada (Protista), which includes human gastrointestinal parasite Giardia duodenalis, is one of the earliest diverging extant lineages of the Eukaryota and appears to be the oldest lineage in which many eukaryotic RBP systems, include PUF proteins, first appear. As part of a broader exploration of the earliest eukaroytic RBPome, we explored these PUFs for complex eukaryotic functions. We undertook bioinformatic mining and 3D protein structural homology modelling of Giardia PUF proteins followed by molecular simulations to map homologous RNA interacting residues within PUFs. We used transcriptomic and proteomic data to explore the kinetic behaviour of PUFs through Giardia’s stage transitions, combined with interactome capture of poly-A mRNA-bound proteins to confirm that the giardial PUFs interact with RNA. We further employed PAR-CLIP strategies to identify the diverse target RNAs bound to Giardia PUFs and assessed the functional impact of CRISPR-based PUF-silencing on Giardia stage transition. Lastly, we used in vitro phase separation assays to test if Giardia PUF can mediate RNA granule formation, which is an essential step in RBP-based regulation of cell-fate. This first, comprehensive study of the earliest eukaryotic PUFs show that the RBP systems are as complex at the base of the eukaryotic tree as they are at the tips. We propose that the evolution of eukaryotic RBPs likely occurred at, and may have played a crucial role in, the emergence of eukaryotes.