Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins

Abstract

Mammalian H/ACA small nucleolar RNAs and telomerase RNA share common sequence and secondary structure motifs that form ribonucleoprotein particles (RNPs) with the same four core proteins, NAP57 (also dyskerin or in yeast Cbf5p), GAR1, NHP2, and NOP10. The assembly and molecular interactions of the components of H/ACA RNPs are unknown. Using in vitro transcription/translation in combination with immunoprecipitation of core proteins, UV-crosslinking, and electrophoretic mobility shift assays, we demonstrate the following. NOP10 associates with NAP57 as a prerequisite for NHP2 binding. Although NHP2 on its own binds RNA nonspecifically, this NAP57-NOP10-NHP2 core trimer specifically recognizes H/ACA RNAs. GAR1 associates independently with NAP57 near the pseudouridylase core of mature H/ACA RNPs. In contrast to other RNPs whose assembly is initiated by protein-RNA interactions, the four H/ACA core proteins form a protein-only particle that associates with H/ACA RNAs. Nonetheless, functional H/ACA snoRNPs assembled in cytosolic extracts are stable and do not exchange their RNA components, suggesting that new particle formation requires de novo synthesis.

Figure 1

Pseudouridylation target uridine UV-crosslinks to NAP57 and GAR1. (A) Immunopurified box H/ACA snoRNPs were incubated with a site-specifically ³²P-labeled and 4-thiouridine-substituted short rRNA substrate (corresponding to the site targeted by snoRNA E3) and UV irradiated. The proteins and crosslinked RNAs were eluted from protein A–sepharose beads and analyzed by 15% Tricine SDS–PAGE and autoradiography before (lane 1) and after RNase A treatment (lane 2). (B) After elution with SDS (lane 1), the proteins were re-precipitated in the presence of Triton X-100 with NAP57 (lanes 2 and 3) and GAR1 antibodies (lanes 4 and 5). The supernatants (lanes 2 and 4) and pellets (lanes 3 and 5) were analyzed as in (A). (C) The same as lane 1 in (B), but with a substrate corresponding to the site targeted by snoRNA U65. The migration positions of NAP57, GAR1, an NAP57 breakdown product (asterisk), and the molecular weight markers are indicated.

Figure 2

Immunoprecipitation of in vitro-translated snoRNP core proteins. (A) The individual cDNAs for the indicated H/ACA core proteins were transcribed and translated in rabbit reticulocyte lysate in the presence of ³⁵S-methionine and analyzed by 15% Tricine SDS–PAGE and fluorography. (B) All four H/ACA core proteins were co-translated (lane 1) and precipitated with NAP57 peptide antibodies in the absence (lanes 2 and 4) and presence (lanes 3 and 5) of free competing peptide. Compared to the input (lane 1), twice the amount of pellets (lanes 2 and 3) and supernatants (lanes 4 and 5) were loaded. Note the anomalous migration of immunoprecipitated NAP57 (vertical bar). (C) H/ACA core proteins and fibrillarin as control were co-translated in various combinations and precipitated with NAP57 antibodies. The input (lanes 1–5) and the precipitates from 10-fold more (lanes 6–10) are shown. (D) Immunoprecipitations of various combinations of NAP57, NHP2, and HA-NOP10 with HA antibodies. The input (odd lanes) and precipitates (even) are shown, respectively. (E) NAP57 precipitates of different combinations of H/ACA core proteins including GAR1. (F) NAP57 precipitation in the presence of all core proteins was performed before (lanes 1 and 2) or after RNase A treatment (lanes 3 and 4). The input (odd lanes) and precipitates (even lanes) are shown. The migrating positions of molecular weight markers are indicated for each panel.

Figure 3

Co-immunoprecipitation of H/ACA core proteins with HA-NAP57 and derivatives. (A) Schematic representation of HA-tagged (HA) NAP57 and derived constructs with the highly conserved pseudouridylase (TruB) and pseudouridine synthase/archaeosine transglycosylase domains (PUA), the lysine clusters (KK), and point mutations (asterisks) highlighted. (B) HA-NAP57 and derivatives were co-translated with the other H/ACA core proteins and fibrillarin as described in the legend to Figure 2 and precipitated with anti-HA antibodies. Odd lanes correspond to input and even lanes to precipitates. The HA-tagged NAP57 derivatives are (amino acids in parentheses): HA-NAP57 (full-length, 1–509); -ΔC (1–466); -N half (1–259); C half (252–466); F37V, point mutation detected in a family with X-linked DC; D126A, point mutation of the aspartate required for pseudouridylase activity. The positions of the HA-NAP57 constructs are marked (asterisks).

Figure 4

Electrophoretic mobility shift assay of ³²P-labeled RNA with recombinant NHP2 (A–C) and co-immunoprecipitation of ³²P-labeled RNA with in vitro-translated H/ACA core proteins (D–G). (A) Radiolabeled H/ACA snoRNA E3 was incubated with increasing amounts of purified, bacterially expressed GST-tagged NHP2 and analyzed by 4% native polyacrylamide gel electrophoresis and autoradiography. (B) Same as in (A) but in the presence of either unlabeled H/ACA snoRNA E3 (lanes 1 and 2) or C/D snoRNA U3 (lanes 3 and 4) at a 200- and 1000-fold molar excess (odd and even lanes, respectively). (C) Gel shift analysis of H/ACA snoRNA E3 (lanes 1–3) and of the stem–loop forming 3′ UTR of the Ash1 mRNA (lanes 4 and 5) in the absence of protein (lanes 1 and 4), the presence of recombinant GST–NHP2 (lanes 2 and 5), or of GST–NOP10 (lane 3). (D) The H/ACA core proteins and fibrillarin were co-translated in the presence of ³²P-labeled H/ACA snoRNA E3 (lanes 1–3) and C/D snoRNA U3 (lanes 4–6) and precipitated with NAP57 antibodies as described in the legend to Figure 2. The input (lanes 1 and 4), the precipitates (lanes 2 and 5), and the labeled snoRNAs alone (lanes 3 and 6) are shown. (E) Same as in (D), except that only various combinations of the core trimer proteins and fibrillarin were used in the presence of ³²P-labeled E3. The input (lanes 1–4) and the respective NAP57 precipitates (lanes 5–8) are depicted. (F) Co-precipitation of C/D snoRNA U3 (lanes 1 and 2), the H/ACA telomerase RNA hTR (lanes 3–5), and the 3′ UTR (see ^C; lanes 6–8) with the H/ACA core trimer. The input (lanes 1, 3, and 6), the NAP57 precipitates (lanes 2, 4, and 7), and the RNAs alone (lanes 5 and 8) are shown. (G) Co-precipitation of the H/ACA RNAs E3 (lanes 1–4) and hTR (lanes 5–8) with HA-NAP57 and derivatives (see Figure 3A) in the context of the core trimer. The RNAs alone (lanes 1 and 5) and the HA-antibody precipitates (lanes 2–4 and 5–8) are depicted. The position of the HA-NAP57 constructs is indicated (vertical lines).

Figure 5

Assembly of functional E3 H/ACA snoRNPs in cytosolic extracts. (A) In vitro pseudouridylase assay. Autoradiograph of a thin-layer chromatogram of uridine (Up) and pseudouridine (Ψp) liberated from a short site-specifically ³²P-labeled rRNA substrate (corresponding to the sequence modified by E3) after incubation with cytosolic S-100 extracts that had been incubated prior with unlabeled H/ACA snoRNAs E3 (lane 2) and E2 (lane 3) or no RNA (lane 1). The amount of pseudouridine produced is expressed as percent of uridine and pseudouridine combined. (B) Same as in (A), except that all extracts were first incubated with E3 in the presence of H/ACA snoRNA E2 (lanes 2 and 3) and C/D snoRNA U3 (lanes 4 and 5) in 10- and 100-fold molar excess (lanes 2 and 4, and 3 and 5, respectively). (C) Electrophoretic mobility shift analysis of ³²P-labeled H/ACA snoRNA E3 after incubation with cytosolic S-100 extracts (lane 1) in the presence of unlabeled H/ACA snoRNA E2 (lanes 2 and 3) and C/D snoRNA U3 (lanes 4 and 5) in 10- and 1000-fold molar excess (lanes 2 and 4, and 3 and 5, respectively). The migrating position on the native gel of the labeled E3 snoRNA and the newly formed snoRNP is indicated on the autoradiograph. Note that most of E3 is degraded in the extracts unless incorporated into a snoRNP. (D) Same as lane 1 in (C), except that antibodies against the proteins indicated on top were added during snoRNP formation. Note the supershift with antibodies to NHP2 (lane 2). (E) Same as (C), except that unlabeled H/ACA snoRNA E2 was added before (lanes 2 and 3) or after (lanes 4 and 5) snoRNP formation in 10- and 1000-fold molar excess (lanes 2 and 4, and 3 and 5, respectively).

Figure 6

Schematic of H/ACA RNP assembly pathway and intra-RNP molecular interactions. The schematic is based on results from this study as explained in detail in the text. The sizes of proteins are depicted in approximate scale to their molecular weight.