Functional and structural analysis of the C. elegans PAXT-1-XRN2 complex

XRN2 is an essential nuclear 5’ -> 3’ exoribonuclease that is involved in quality control, processing and degradation of RNAs, such as rRNA, snRNA and miRNAs. Recently, C. elegans XRN2 was discovered to exist in a stable complex with PAXT-1. Binding to XRN2 is mediated by PAXT-1’s DUF3469/XRN2 binding domain (XTBD). Whereas binding of the XTBD confers stability on XRN2 similar to observations made for yeast Rai1 – Rat1 complexes, kinetic analysis using the Michaelis-Menten model does not show any contributions to catalytic activity by PAXT-1. However, XRN2 alone processively degrades small RNAs, like its paralog XRN1, but shows no release activity for miRNAs off AGO, even when bound to PAXT-1. Here we show the XTBD to be a general binding domain for 5’ -> 3’ exoribonucleases 2 and present the crystal structure of the XTBD – XRN2 complex. Whereas the XTBD serves as a general adapter to XRN2, XRN1 binding is excluded due to sterical hindrance. Strikingly, a single point mutation in PAXT-1 Tyr56 completely abrogates binding to XRN2 in vitro and in vivo. Using CRISPR to generate endogenous PAXT-1 Tyr56 to Ala mutations, phenotypes identical to paxt-1 null mutant worms are observed. Remarkably, paxt-1null mutant worms can be rescued by an unrelated XTBD-containing protein from humans, CDKN2AIPNL, which confers stability to XRN2 by formation of a chimeric complex in vivo. We conclude that XRN2 uses a similar mechanism for substrate binding and processive degradation as XRN1 and that PAXT-1 confers stability to the nuclease complex by binding through its XTB domain. Moreover, the XTBD serves as a general binding adapter for the XRN2 nuclease family, yet excluding XRN1 binding.