Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Dec 1;18(23):4546-51.
doi: 10.1093/hmg/ddp416. Epub 2009 Sep 4.

Pathogenic NAP57 Mutations Decrease Ribonucleoprotein Assembly in Dyskeratosis Congenita

Affiliations
Free PMC article

Pathogenic NAP57 Mutations Decrease Ribonucleoprotein Assembly in Dyskeratosis Congenita

Petar N Grozdanov et al. Hum Mol Genet. .
Free PMC article

Abstract

X-linked dyskeratosis congenita (DC) is a rare bone marrow failure syndrome caused by mostly missense mutations in the pseudouridine synthase NAP57 (dyskerin/Cbf5). As part of H/ACA ribonucleoproteins (RNPs), NAP57 is important for the biogenesis of ribosomes, spliceosomal small nuclear RNPs, microRNAs and the telomerase RNP. DC mutations concentrate in the N- and C-termini of NAP57 but not in its central catalytic domain raising questions as to their impact. We demonstrate that the N- and C-termini together form the binding surface for the H/ACA RNP assembly factor SHQ1 and that DC mutations modulate the interaction between the two proteins. Pinpointing impaired interaction between NAP57 and SHQ1 as a potential molecular basis for X-linked DC has implications for therapeutic approaches, e.g. by targeting the NAP57-SHQ1 interface with small molecules.

Figures

Figure 1.
Figure 1.
Models of human H/ACA core complex protein structures. (A) Entire human H/ACA protein core complex modeled as described in the text. The protein only complex consists of NHP2 (yellow), NOP10 (dark blue), GAR1 (magenta) and NAP57 (red–green–cyan for the N-terminal, catalytic and C-terminal domain, respectively). Space filling models identify the amino acids mutated in this study according to mutations observed in X-linked DC (orange) and the catalytic aspartate (black). To indicate how much of the actual protein sequences are missing, the starting and ending positions are indicated (including the number of residues of the full-length proteins). (B) Spliced N- (red) and C-termini (cyan) of NAP57 representing the NAP57Δcat construct generated for this study. It concentrates 32 of 35 mutation sites identified in NAP57 of X-linked DC-patients (Fig. 2A); 21 are present in the structure (orange). The splice junction between N- and C-termini is indicated (arrow).
Figure 2.
Figure 2.
SHQ1 binds to the spliced N- (NT) and C-termini (CT) of NAP57 (NAP57Δcat) that concentrates most DC mutation sites. (A) Schematic of constructs. The location of DC mutations is indicated above NAP57 (bars) including those that are mutated to two different amino acids (double height) and a C-terminal deletion (Δ). Note the Δcat construct with the catalytic domain (cat) excised but retaining the RNA binding pseudouridylase and archaeosine transglycosylase (PUA) domain. Positively charged stretches that double as nuclear localization signals are indicated (K). (B and C) Recombinant proteins fused to the maltose binding protein (MBP) were mixed with SHQ1 (arrows), retained on amylose resin and analyzed on Coomassie blue stained SDS–polyacrylamide gels before (input, 10%) and after retention (bound). As previously observed for full-length NAP57 (13), every construct containing its CT migrated as two bands (in the case of MBP-NT, the lower bands were breakdown products). The bacteriophage MS2 coat protein (MCP) was included as a control. (D, inset) Enhanced contrast of region identified by dashed box below demonstrating increased binding of SHQ1 with the NT present. (E and F) Nuclear tethering assay: double immunofluorescence of constructs fused to LacI, which are transfected and tethered to lac operator repeats stably integrated into the genome (arrows) and which do (E) or do not (F) recruit endogenous SHQ1. Bar = 10 µm.
Figure 3.
Figure 3.
The CS domain of SHQ1 is not required for NAP57Δcat binding. (A) Schematic of SHQ1 highlighting its HSP20-like CS domain. (B and C) MBP-fusion protein retention assays as in Figure 2B–D but using SHQ1ΔCS (B, arrows) and the SHQ1-CS domain alone (C, arrows). Conditions and abbreviations are in Figure 2D–F. Nuclear tethering assays (Fig. 2E and F) with the indicated constructs. Bar = 10 µm.
Figure 4.
Figure 4.
Amino acid changes observed in NAP57 of DC patients modulate its interaction with SHQ1. MBP retention assays with mutant NAP57 (see legend to Fig. 2B–D). (A) Wild-type (wt) and an A353R mutant NAP57 (mt) fused to MBP were used to retain SHQ1 under increasing salt concentrations. Note the increased binding of the mutant. (B) The fractions retained with wild-type (left) or mutant MBP-NAP57 (A353V, right) were analyzed by SDS–PAGE and the Coomassie blue stained lanes scanned and enlarged (left and right of the gel). Note the increased binding of the mutant accounting for ∼40% of all X-linked DC cases. (C) The fractions retained with the NAP57 variants indicated above as in (B). Note the increased (lanes 2 and 5) and reduced binding (lanes 3 and 6) of certain mutants relative to wild-type NAP57 (lane 1).

Cited by 20 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback