Mechanism of the AAA+ ATPases pontin and reptin in the biogenesis of H/ACA RNPs

Abstract

The AAA+ ATPases pontin and reptin function in a staggering array of cellular processes including chromatin remodeling, transcriptional regulation, DNA damage repair, and assembly of macromolecular complexes, such as RNA polymerase II and small nucleolar (sno) RNPs. However, the molecular mechanism for all of these AAA+ ATPase associated activities is unknown. Here we document that, during the biogenesis of H/ACA RNPs (including telomerase), the assembly factor SHQ1 holds the pseudouridine synthase NAP57/dyskerin in a viselike grip, and that pontin and reptin (as components of the R2TP complex) are required to pry NAP57 from SHQ1. Significantly, the NAP57 domain captured by SHQ1 harbors most mutations underlying X-linked dyskeratosis congenita (X-DC) implicating the interface between the two proteins as a target of this bone marrow failure syndrome. Homing in on the essential first steps of H/ACA RNP biogenesis, our findings provide the first insight into the mechanism of action of pontin and reptin in the assembly of macromolecular complexes.

FIGURE 1.

The CS domain of SHQ1 alone binds to NAP57 only in trans to the SSD. (A) Schematic of linear NAP57 constructs used in the study. NAP57 contains a central catalytic (cat) domain that is excised in the NAPΔcat construct. (B) Schematic of a structure model of NAP57 without its unstructured N- and C-terminal extremities (NAP 31–422). N- and C-terminal parts wrap around each other to form a separate domain (light green) from the catalytic part of the enzyme (dark green). (C) Model of full-length NAP57 that highlights the major X-DC mutation cluster and the unstructured C-terminal tail of the NAPΔcat domain (the short N-terminal extremity is not shown). (D) Schematic of linear SHQ1 constructs used in the study. SHQ1 has two major domains, the CS domain (deep red) and the SHQ1-specific domain (SSD, orange). (E) Schematic of a model of the R2TP complex with the heterohexameric ring of the AAA+ ATPases pontin and reptin, and with PIH1D1 and RPAP3. Note all schematics are roughly drawn to scale to each other. (F) Growth of a yeast strain deleted for shq1 and complemented with the yeast SHQ1 constructs indicated on the left, individually (rows 1–4) or combined (row 5). Dilutions (1:1) were spotted left to right. (G–K) Coomassie blue stained SDS-PAGE of recombinant proteins retained on amylose resin by their maltose binding protein (MBP) tags and input (1/10th). Note in all figures, MBP-tagged proteins are highlighted in bold and the added/bound proteins are indicated in regular print. Also, the MBP-tag is omitted when the migration position of the fusion proteins is marked on the side of the gel. As reported previously, all MBP-NAP57 constructs containing its charged and unstructured C terminus migrate as a doublet (Grozdanov et al. 2009a,b; Walbott et al. 2011). (G) Binding of the CS domain of SHQ1 and its SSD in trans to full-length NAP57. The contrast in the boxed area is enhanced. Migrating positions of molecular weight markers (kDa) are indicated on the right. (H) Addition of SSD to MBP-CS and MBP-MS2 phage coat protein (MCP). (I) Binding of the CS domain of SHQ1 and its SSD in trans to NAP57Δcat. (J) Binding of SHQ1 to MBP-NAP57 after RNase treatment of either protein or (K) the same with the CS domain of SHQ1 and its SSD in trans.

FIGURE 2.

The CS domain of SHQ1 binds to the major X-DC mutation cluster of NAP57, forming a tight clamp together with the SSD. Amylose resin pull-down assays as in Figure 1G–K. (A) Binding of the CS domain of SHQ1 in trans to its SSD to wild-type and X-DC mutant NAP57. The hypomorphic M350T mutation abolishes binding of the CS domain of SHQ1, but not of the SSD. A contrast-enhanced image of the area right above where the CS domain migrates is outlined (boxed). (B) The SSD alone binds to NAP57 with X-DC mutations. (C) Increasing salt beyond physiological levels abolishes SHQ1 binding to NAP57 (lanes 2–5) but even 2 M salt is unable to release SHQ1 once bound (lanes 6–9). (D) As in C, but binding to NAP57 of the CS domain of SHQ1 in trans to its SSD. Only binding of the CS domain, but not that of the SSD, is salt sensitive (lanes 2–5) and neither is released once bound (lanes 6–9). (E) As in D, but binding to NAPΔcat.

FIGURE 3.

SHQ1 can be released from NAP57 by cytosolic S100 extracts in an ATP- and HSP90-independent fashion. (A,C–E) Amylose resin pull-down assays as in Figure 1G–K. (A) SHQ1 bound to MBP-NAP57 (lane 2) was incubated with recombinant pontin, reptin, or both (2.5 μg each) in the presence (+) and absence (−) of 1 mM ATP. (B) Western blot of recombinant pontin (lane 1) and cytosolic S100 extract (lane 2) probed with pontin antibodies and developed with enhanced chemiluminescence (ECL). (C) SHQ1 bound to MBP-NAP57 (lane 1) was incubated with 1 μL and 10 μL of S100 extract in the presence (+) and absence (−) of 1 mM ATP. (D) SHQ1 bound to MBP-NAP57 was incubated with S100 that was pretreated with (+) and without (−) Apyrase (140 mU/μL) for 30 min at 37°C. (E) SHQ1 bound to MBP-NAP57 was incubated with S100 that was pretreated with the HSP90 inhibitor geldanamycin (GA) at 0, 4, and 8 μM for 60 min at 37°C.

FIGURE 4.

Acting on the CS domain of SHQ1, all components of the R2TP complex are required for SHQ1 removal from NAP57. (A) SHQ1 bound to MBP-NAP57 (lanes 1–3) or to control MBP-MCP (lanes 4,5) in amylose resin pull-down assays as in Figure 3C–E, except that the SDS-PAGE was transferred to nitrocellulose and stained with amido black (upper panel) and probed with pontin antibodies (lower panel). The bound proteins were incubated with S100 that was pretreated with (+) and without (−) pontin antibodies. (B–H) Same as A but coomassie blue stained gels. (B) SHQ1 bound to MBP-NAP57 (lane 1) was released with S100 extract (lane 2), which was pretreated with pontin antibodies (lane 3) that were incubated with 0.5, 1.5, and 2.5 μg of recombinant pontin (lanes 4–6) or reptin (lanes 7–9). (C) Same as B but with reptin antibodies. (D) MBP-NAP57 was incubated with the SHQ1 constructs indicated above the gel and treated with (+) and without (−) S100. Note the SSD bound to MBP-NAP57 was not removed by S100 (lanes 3,4). (E) The CS domain of SHQ1 bound in trans to its SSD to MBP-NAP57 was removed by S100 (lane 2), which was inhibited by pontin and reptin antibodies (lanes 3,4). (F) PIH1D1 antibodies inhibited S100-mediated removal of SHQ1 from MBP-NAP57 (lane 3). The inhibition was relieved by recombinant PIH1D1 (lane 4). (G) Same as F but with RPAP3 antibodies and protein. (H) Control. Unlike pontin antibodies (lane 6), antibodies directed against NAP57, SHQ1, and NAF1 did not inhibit the release activity of S100 extracts (lanes 3–5, respectively).

FIGURE 5.

Mapping of direct interactions of NAP57 and SHQ1 with the components of the R2TP complex. All panels, except G (which is a glutathione bead pull-down), are amylose resin pull-downs as in Figure 1G–K. (A) Incubation of pontin and reptin alone and together with MBP-NAP57 and control MBP-MCP. Note recombinant reptin (lane 2) includes a minor band that migrates closely to pontin and is apparently a read-through product because it also binds to NAP57 (lane 8). (B) Incubation of pontin and reptin combined with MBP-NAP57 constructs identifies the NAPΔcat domain as docking site (lane 9). (C) Pontin and reptin bind also individually to NAPΔcat. (D) Pontin and reptin, alone and together, bind to NAP57 without its charged and unstructured terminal extremities, MBP-NAP 31–422. (E) Pontin and reptin alone and together bind to MBP-SHQ1. (F) Pontin and reptin alone and together bind to the CS domain of SHQ1. Note MBP-CS migrates between pontin and reptin (arrow). (G) Glutathione-S-transferase (GST) fusions of pontin and reptin combined incubated with the CS domain of SHQ1 and its SSD individually (lanes 1–4) and together (lanes 5,6). A minor band from the fusion proteins that migrates right below the SSD is marked (black square). (H) Incubation of PIH1D1 with MBP-NAP57 constructs identifies NAPΔcat as the binding domain (lane 7). (I) RPAP3 fails to bind to MBP-NAP57 alone (lane 2) and in the context of the other three R2TP components (R2P), which are retained (lane 4). Note a bacterial heat shock protein that sometimes copurifies with MBP-NAP57 and PIH1D1 migrates below RPAP3 (asterisk), and two lower bands contaminate the RPAP3 preparations (black dots). (J) Neither PIH1D1 (lane 3) nor RPAP3 (lane 4) bind to MBP-SHQ1. Note equal background level binding of PIH1D1 to control MBP-MCP (lane 6). (K) Unlike S100 (lane 8), neither pontin and reptin combined, PIH1D1, RPAP3, nor altogether (R2TP) release SHQ1 from MBP-NAP57, irrespective of 1 mM ATP addition. However, pontin, reptin, and PIH1D1, when present, are retained by the complex (lanes 3,4,6,7).

FIGURE 6.

Pontin and reptin are required for H/ACA RNP accumulation in vivo. (A) Western blots of low and high salt cell extracts from HeLa cells treated with the siRNAs to the targets indicated on top (in bold) and probed with antibodies to the proteins indicated on the right. Pontin and reptin knockdowns deplete pontin, fibrillarin, NAP57, and NHP2, but not SHQ1 nor tubulin or Nopp140 (lanes 3,4), whereas fibrillarin knockdown only depletes fibrillarin (lane 2). (B) Northern blots of total RNA extracted from cells treated with siRNAs to the targets indicated on top (in bold) and probed for RNAs indicated on the right. The amounts of the snoRNAs are each expressed as percent relative to the mock treated sample and were quantified relative to snRNA U1.

FIGURE 7.

Mode of action of the R2TP complex on the NAP57•SHQ1 complex. (A) The NAP57 extremities are required for S100-mediated release from SHQ1. SHQ1 bound to full-length (1–514, lane 3) or to NAP57 without its extremities (31–422, lane 4) was only released by S100 from the former (lane 5) but not the latter (lane 6). Histogram of the quantification of SHQ1 release relative to bound protein (below). (B) The C-terminal tail of NAP57 alone is required for S100-mediated SHQ1 release. SHQ1 bound to full-length NAP57 (1–514, lane 1), to NAP57 without N-terminal extremity (31–514, lane 2), without C-terminal tail (1–422, lane 3), and without both extremities (31–422, lane 4). S100 only released SHQ1 from NAP57 constructs with the C-terminal tail (lanes 5,6) but not from those without it (lanes 7,8). Note MBP-NAP57 lacking its C-terminal tail migrates as a single band (e.g., lanes 7,8). (C) Schematic summary of the results. See text for details.