Domain definition and interaction mapping for the endonuclease complex hNob1/hPno1

Abstract

Ribosome biogenesis requires a variety of trans-acting factors in order to produce functional ribosomal subunits. In human cells, the complex formed by the proteins hNob1 and hPno1 is crucial to the site 3 cleavage occurring at the 3'-end of 18S pre-rRNA. However, the properties and activity of this complex are still poorly understood. We present here a detailed characterization of hNob1 organization and its interaction with hPno1. We redefine the boundaries of the endonuclease PIN domain present in hNob1 and we further delineate the precise interacting modules required for complex formation in hNob1 and hPno1. Altogether, our data contributes to a better understanding of the complex biology required during the site 3 cleavage step in ribosome biogenesis.

Keywords: KH domain; PIN domain; Ribosome biogenesis; endonuclease; pre-rRNA; zinc ribbon.

Figure 1.

Human Nob1 domain organization. (a) Sequence alignment and secondary structure prediction of Nob1 homologues from major clades showing conservation along the protein sequence performed with ESPript [30]. Coloring scheme is as follow: Fully conserved residues have a red background, partially conserved amino acids are shown as bold letters over a yellow box. The alignment has the following UniProt entries: human, Q9ULX3; mouse, Q8BW10; zebrafish, F1R4C1; african clawed frog, Q6VEU0; fruit fly, Q9W2Y4; baker’s yeast, Q08444; C. thermophilum, G0S762 and P. horikoshii, O58440. The first 79 amino acids of C. thermophilum sequence have been removed due to non-conservation with the other homologues and space limitation. Secondary structure element prediction from PHYRE is indicated above the alignment and numbered. The sequence corresponding to the proposed first and second half of the PIN domain is boxed in green and salmon, respectively. The proposed PIN domain active site are indicated by red dots while the other residues reported to be involved in the active site are indicated by blue stars. Three black asterisk indicate the mutated residues in panel E. The zinc ribbon domain is outlined in dark blue. (b) PIN domain structural prediction is shown in cartoon representation and colored in green and salmon according to domain limits defined in panel A. Individual secondary structure elements are labelled. Active site residues are shown as colored sticks (cyan, carbon; red, oxygen) and labelled. Mutated residues are shown as sticks and labelled in italic. (c) Schematic view of the endonuclease hNob1 domain organization colored as in panel A. The different hNob1 fragments used for domain boundary definition are shown below. (d) The two halves of the PIN domain interact together. Ni-NTA purification of lysate from cells co-expressing two Nob1 fragments showing complex formation between fragment 1–134 and 206–314. This complex is not observed between fragments 1–134 and 249–314, thus excluding a stable interaction between the zinc ribbon and the first half of the PIN domain. (e) Interface mutations affect PIN domain formation. Ni-NTA purification of lysate from cells co-expressing two Nob1 fragments. The non-histidine tagged fragments was carrying the mutation D83K, L87S or Y91A. Mutation of hNob1 (1–134) L87S abolishes interaction with the fragment 206–314.

Figure 2.

Definition of the minimal fragment of hNob1 or hPno1 required for complex formation. (a) Schematic view of hNob1 fragments (upper part) used for the mapping of the interaction. Co-expression of the indicated hNob1 constructs with or without untagged hPno1 (bottom part). Protein are identified next to the SDS-PAGE. (b) Schematic view of hPno1 fragments (upper part) used for the mapping of the interaction. Co-expression of the indicated untagged hPno1 constructs with histidine-tagged hNob1 (bottom part). Small scale Ni-NTA purifications are described in Material and Methods.

Figure 3.

hNob1 interaction with hPno1 depends on the tryptophan at position 208 in hNob1 and on the KH-like domain of hPno1. (a) Schematic view of the different parts of the hNob1 internal loop used to define the minimal region of interaction with hPno1. (b) Pull-down assay of GST-tagged hNob1 linker constructs with hPno1 showing that the C-terminal part of the linker is sufficient for association. (c) Pull down assay of His-tagged hNob1 (1–314) with or without the mutation W208A. Pull-down experiments shown in panel B and C were performed as described in Material and Methods.