The RNase XRN2 is essential in RNA metabolism. In Caenorhabditis elegans, XRN2 functions with PAXT-1, which shares a putative XRN2-binding domain (XTBD) with otherwise unrelated mammalian proteins. Here, we characterize the structure and function of an XTBD–XRN2 complex. Although XTBD stably interconnects two XRN2 domains through numerous interacting residues, mutation of a single critical residue suffices to disrupt XTBD–XRN2 complexes in vitro and to recapitulate paxt-1–null mutant phenotypes in vivo. Demonstrating conservation of function, vertebrate XTBD-containing proteins bind XRN2 in vitro, and human CDKN2AIPNL (HsC2AIL) can substitute for PAXT-1 in vivo. In vertebrates, which express three distinct XTBD-containing proteins, XRN2 may partition into distinct stable heterodimeric complexes, which probably differ in subcellular localization or function. In C. elegans, complex formation with PAXT-1, the sole XTBD protein, serves to preserve the stability of XRN2 in the absence of substrate.
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We thank J. Keusch for technical support, D. Hess for mass spectrometry analysis, M. de la Mata for assistance with cell transfections, M. Zou (Friedrich Miescher Institute for Biomedical Research) for zebrafish lysate and J.A. Chao and N. Thomä for critical comments on the manuscript. We are grateful to the staff of the Swiss Light Source, Paul Scherrer Institute, Villingen, where parts of the experiments were performed. The research leading to these results received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 241985 (European Research Council 'miRTurn'; to H. Großhans), the Swiss National Science Foundation (SNF 31003A_143313; to H. Großhans) and the Novartis Research Foundation through the Friedrich Miescher Institute (to I.K., H. Gut and H. Großhans).
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Integrated supplementary information
(a) A Coomassie-stained SDS-PAGE gel of PAXT-1 truncation mutants (asterisks) co-expressed with XRN2 (arrowhead). All mutant proteins bind XRN2 to similar extents. For each PAXT-1 mutant, two clones were expressed and pulled on their His6-tag to co-elute XRN2. (b) Structural representation of the XTBD – XRN2 complex highlighting the tower domain and the PBS-protruding loop. XTBD and the remaining parts of XRN2 are in light colors. (c) Structural representation of XTBD bound to XRN2, with helices and loops indicated. (d) Superposition of C. elegans XTBD – XRN2 complex and S. pombe Rai1p – Rat1p complex reveals that XTBD and Rai1p bind to distinct sites on XRN2. (e) Superposition of C. elegans XTBD – XRN2 complex and D. melanogaster XRN1 reveals potential structural clashes of XRN1 with XTBD (dashed red circles in close-up, top).
(a) SEC-MALS analysis of the XTBD –XRN2ΔZLC complex. The blue line indicates the gel filtration elution profile, monitored by UV light absorption at 280 nm. Red dots indicate molar mass values under the peak. The measured molar mass for the complex (80.9 kDa) was calculated over peak fractions covering the elution volume 14.8 ml – 16.3 ml, which represent a completely monodisperse sample (polydispersity = 1.000). (b) Close-up of the binding interface of XTBD (yellow) and XRN2ΔZLC (cyan/gray). Key residues mediating interaction are represented as sticks with oxygens and nitrogens colored in red and blue (atom colors), respectively. (c) Zoom-in on residue Cys54, which forms hydrophobic interactions with Pro656 and Cα / Cβ of Phe659 as well as a weak hydrogen bond with Leu675. (d) Biological replicate of the western blot shown in Fig. 2g, detecting XRN2 and PAXT-1 from worms grown at 26°C. (e), (f) Small scale expression of His6-PAXT-1, -PAXT-1_Y56A and- PAXT-1_C54G. (e) For each construct, cleared lysate, unbound and Ni-NTA pull-down fraction were loaded. (f) Western blot using antibodies against XRN2 and PAXT-1 was performed on the cleared lysates.
(a) A human C2AIL homology model (magenta) superimposed onto the XTBD - XRN2 complex (yellow and cyan, respectively). (b) Pfam alignment of selected XTBDs from PAXT-1 of C. elegans (Uniprot identifier Q21738) and C. briggsae (A8XFX8), respectively, and CDKN2AIP from D. rerio (F1QZX8), D. melanogaster (Q9VF83), H. sapiens (Q9NXV6), and M. musculus (Q8BI72), respectively, on which the homology model is based. The critical residue Tyr56 is found at position 104 in this alignment. (c) Alignment based on HMM-search of XRN2 PBS. Shown are representative sequences from C. elegans (Q9U299), C. briggsae (Q60SG7), D. rerio (Q802V7), D. melanogaster (Q9VM71), H. sapiens (Q9H0D6) and M. musculus (Q9DBR1) XRN2.
(a) Michaelis-Menten kinetic curves of XRN2 and the PAXT-1 – XRN2 complex, respectively. XRN2’s catalytic activity was determined on a broad range of substrate concentrations using an assay based on a FAM fluorophore-coupled RNA substrate and a quencher-coupled DNA primer (Sinturel et al., RNA, 15, 2057-2062, 2009). As the substrate range accessible by this assay is limited by the dynamic range of measurable FAM fluorescence by the qPCR machine, we extended it by adding a four-fold excess of unlabeled 22 nt let-7 miRNA to the 30 nt RNA – DNA duplex. The curves are extrapolations from measurements shown in the inset. The corresponding KM values are marked as colored squares. At 0.013 s-1, the kcat of XRN2 alone is comparable to that of the PAXT-1 – XRN2 complex (table), and turnover rates are identical at substrate concentrations up to 0.5 · 10-6 M (inset). A small difference in Michaelis-Menten constants KM (table) suggests a modestly increased affinity of PAXT-1-bound XRN2 to its substrate relative to XRN2 alone. We conclude that PAXT-1 has little or no stimulatory activity on XRN2, at least under the conditions of this assay. (b) Replicates of the assay shown in Figure 4d.
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Richter, H., Katic, I., Gut, H. et al. Structural basis and function of XRN2 binding by XTB domains. Nat Struct Mol Biol 23, 164–171 (2016). https://doi.org/10.1038/nsmb.3155
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