The Argonaute‐binding platform of NRPE1 evolves through modulation of intrinsically disordered repeats

Abstract

Summary Argonaute proteins are important effectors in RNA silencing pathways, but they must interact with other machinery to trigger silencing. Ago hooks have emerged as a conserved motif responsible for interaction with Argonaute proteins, but little is know about the sequence surrounding Ago hooks that must restrict or enable interaction with specific Argonautes.Here we investigated the evolutionary dynamics of an Argonaute-binding platform in NRPE1, the largest subunit of RNA Polymerase V. We compared NRPE1 sequences from more than 50 species, including dense sampling of two plant lineages.This study demonstrates that the Argonaute-binding platform of NRPE1 retains Ago-hooks, intrinsic disorder, and repetitive character while being highly labile at the sequence level. We reveal that loss of sequence conservation is due to relaxed selection and frequent expansions and contractions of tandem repeat arrays. These factors allow a complete restructuring of the Ago-binding platform over 50-60 million years. This evolutionary pattern is also detected in a second Ago-binding platform, suggesting it is a general mechanism.The presence of labile repeat arrays in all analyzed NRPE1 Ago-binding platforms indicates that selection maintains repetitive character, potentially to retain the ability to rapidly restructure the Ago-binding platform.

The RNAs of RNA-directed DNA methylation

“…Other Pol II domains can also interact with RNA processing factors, including the foot domain of the largest subunit, which interacts with the capping complex (Suh et al 2010), and the flap loop domain of the second-largest subunit, which interacts with both the cleavage/polyadenylation and capping machineries (Pearson and Moore 2014; Martinez-Rucobo et al 2015). Intriguingly, the Pol IV and Pol V largest subunits, NRPD1 and NRPE1, have CTDs whose sequences have completely diverged from the CTD of the Pol II largest subunit, NRPB1 (Pontier et al 2005; Haag and Pikaard 2011; Huang et al 2015; Trujillo et al 2016). Moreover, NRPD1 and NRPE1 are missing the foot domain (Pontier et al 2005; Luo and Hall 2007).…”

“…The Pol II CTD plays a critical role in all aspects of Pol II transcription (Hocine et al 2010; Hsin and Manley 2012; Bentley 2014). Although the long Pol V CTD has no amino acid sequence similarity with the Pol II CTD , structural features reminiscent of the Pol II CTD are present, including a (predicted) unstructured series of peptide repeats and multiple amino acid residues that could potentially be subjected to post-translational modification, such as phosphorylation (Pontier et al 2005; Haag and Pikaard 2011; Huang et al 2015; Trujillo et al 2016). Targeted investigations of Pol IV and Pol V CTD domains to explore their roles in RdDM and Pol V transcription, their post-translational modification, or their interactions with other proteins seems likely to provide new insights into the unique functions of these fascinating and enigmatic enzymes.…”

Evidence for a unique DNA-dependent RNA polymerase in cereal crops

“…Published NRPE1 ortholog sequences (Trujillo et al 2016) were retrieved from 80 Phytozome versions 11 and 12 (Goodstein et al 2012). Additional sequences, including 81 homologs of NRPE1, NRPD1, NRPF1, NRPB2, NRPD/E2, NRPB/D5, NRPE5, NRPB9, 82 AGO4, SPT5/SPT5L, and DRD1/CLSY1-like, were obtained through BLAST or 83 TBLASTX queries against whole genome sequences in Phytozome, CoGE ( Phylogenetic analysis of these sequences reveals a single NRPE1 ortholog in non-142…”

“…The 64 second, fourth, fifth, and seventh subunits have also duplicated and specialized for Pol 65 IV and V at different times during land plant evolution (Tucker et al 2010;Huang et al 66 2015). In addition, the Argonaute-binding platform in the CTD of NRPE1 is evolving 67 more rapidly than other regions of the protein (Trujillo et al 2016). 68…”

Functional Dissection of the Pol V Largest Subunit CTD in RNA-Directed DNA Methylation

“…Despite originating from a duplication of the Pol II largest subunit, NRPB1, the CTD of the Pol V largest subunit shares no sequence similarity with the CTD of NRPB1 (Haag and Pikaard, 2011; Huang et al, 2015; Pontier et al, 2005; Trujillo et al, 2016). Pol II largest subunit CTDs consist of repeats of the heptad consensus sequence, YSPTSPS.…”