In vitro assembly of human H/ACA small nucleolar RNPs reveals unique features of U17 and telomerase RNAs

Abstract

The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. They usually fold into a two-domain hairpin-hinge-hairpin-tail structure, with the conserved motifs H and ACA located in the hinge and tail, respectively. Synthetic RNA transcripts and extracts from HeLa cells were used to reconstitute human U17 and other H/ACA ribonucleoproteins (RNPs) in vitro. Competition and UV cross-linking experiments showed that proteins of about 60, 29, 23, and 14 kDa interact specifically with U17 RNA. Except for U17, RNPs could be reconstituted only with full-length H/ACA snoRNAs. For U17, the 3'-terminal stem-loop followed by box ACA (U17/3'st) was sufficient to form an RNP, and U17/3'st could compete other full-length H/ACA snoRNAs for assembly. The H/ACA-like domain that constitutes the 3' moiety of human telomerase RNA (hTR), and its 3'-terminal stem-loop (hTR/3'st), also could form an RNP by binding H/ACA proteins. Hence, the 3'-terminal stem-loops of U17 and hTR have some specific features that distinguish them from other H/ACA RNAs. Antibodies that specifically recognize the human GAR1 (hGAR1) protein could immunoprecipitate H/ACA snoRNAs and hTR from HeLa cell extracts, which demonstrates that hGAR1 is a component of H/ACA snoRNPs and telomerase in vivo. Moreover, we show that in vitro-reconstituted RNPs contain hGAR1 and that binding of hGAR1 does not appear to be a prerequisite for the assembly of the other H/ACA proteins.

FIG. 1

U17 RNA can form an RNP in vitro. (A) Gel mobility shift assay with radiolabeled U17 RNA. The labeled RNA was incubated in the absence (lane 1) or presence (lane 2) of NE or with NE preparations that had been pretreated with proteinase K (prot. K; lane 3), heat denatured (lane 4), or preincubated with micrococcal nuclease (MNase; lane 5). The arrowhead points to free RNA, and the bracket indicates the RNP complex. (B) Sedimentation of native (endo) and reconstituted (rec) U17 RNPs. HeLa cell nucleoli (top) and the in vitro reconstitution mixture with radiolabeled U17 RNA (bottom) were fractionated by centrifugation through 5 to 20% linear sucrose gradients. To detect native U17 monoparticles (top), each fraction was subjected to RNase A/T₁ mapping (note that fraction 4 was lost during sample preparation). Reconstituted U17 RNPs were analyzed in parallel (bottom), together with sedimentation markers of 8S and 12S that correspond to fragments of Escherichia coli 16S rRNA, i.e., the 3′ domain and the 5′ and central domains, respectively. Fractions are numbered from top to bottom of the gradient. Lanes P and T correspond to undigested antisense probe and U17 transcript, respectively. Cerenkov scintillation counting of reconstituted RNPs indicated that fractions 8 and 9 correspond to peak fractions (not shown). The wider peak observed with reconstituted RNPs could result from loss of certain proteins during centrifugation (smaller particles) and association of some nonspecific proteins (larger particles).

FIG. 2

In vitro-assembled U17 RNP contains a specific subset of proteins. (A) Gel mobility shift assay with competitor RNAs of the H/ACA class. Radiolabeled U17 RNA was incubated in the absence (lane 1) or presence (lane 2) of NE. Increasing amounts (10-, 100-, and 1,000-fold molar excess) of unlabeled competitor RNAs U17 (lanes 3 to 5), U19 (lanes 6 to 8), E2 (lanes 9 to 11), or E3 (lanes 12 to 14) were added to the assays. (B) Gel mobility shift assay with other classes of competitor RNAs. Radiolabeled U17 RNA was incubated in the absence (lane 1) or presence (lane 2) of NE. Box C/D RNAs (lanes 3 to 7), RNAs of RNase MRP (7-2; lane 8) and RNase P (H1; lane 9), and spliceosomal RNAs U2 (lane 10) and U4 (lane 11) were added as competitor at 1,000-fold molar excess. (C and D) UV cross-linking of proteins interacting with U17 RNA. Reconstituted U17 RNPs were subjected to UV light irradiation and RNase A/T₁ digestion, and the cross-linked proteins were fractionated by SDS-PAGE. (C) UV cross-linking in the presence of competitor RNAs of the H/ACA class. Radiolabeled U17 RNA was incubated in the absence (lane 2) or presence (lane 3) of NE. Increasing amounts (10-, 100-, and 1,000-fold molar excess) of unlabeled competitor RNAs U17 (lanes 4 to 6), U19 (lanes 7 to 9), E2 (lanes 10 to 12), or E3 (lanes 13 to 15) were added to the assays. (D) UV cross-linking in the presence of nonspecific competitors. Box C/D RNAs (lanes 3 to 7), 7-2 RNA (lane 8), and RNA H1 (lane 9) were added as unlabeled competitors at 1,000-fold molar excess. Lane 2, cross-links with radiolabeled U17 RNA in the absence of the competitor. Cross-links that progressively disappeared upon increasing the concentration of specific competitor RNAs are indicated by a dot. The ∼17-kDa cross-link, likely to result from the proteolysis of the p23 cross-link, is marked with an asterisk. The masses of protein markers (lanes M) are indicated in kilodaltons.

FIG. 3

The 3′ domain RNA fragments of U17 can compete for RNP assembly. (A) Secondary structure model of human U17a RNA. Boxes H (AAAUAA) and ACA are shown in boldface. The arrow indicates the end of U17/5′D (positions 1 to 117), the 5′ domain RNA fragment that contains box H. The 3′ domain RNA fragment U17/3′D (positions 117 to 207) contains the ACA motif. The nucleotide sequence of the fragment corresponding to the 3′-terminal stem-loop of U17 (U17/3′st) is shown and boxed with a solid line, and the deleted portion of this fragment (23 nts replaced by a UUCG tetraloop) is indicated by a dashed line. The secondary structure model was adapted from references and . (B) Gel mobility shift assay with U17 RNA and its derived RNA fragments used as competitors. As a control (C), radiolabeled U17 RNA was incubated with NE in the absence of competitor RNA (lane 1). Increasing amounts (10-, 100-, and 1,000-fold molar excess) of unlabeled competitor RNAs U17 (lanes 2 to 4), U17/5′D (lanes 5 to 7), U17/3′D (lanes 8 to 10), U17/3′st (lanes 11 to 13), and its derived mutated versions U17/3′stΔ23 (lanes 14 to 16), U17/3′stΔACA (lanes 17 to 19), and U17/3′stA→G (lanes 10 to 22) were added to the assays. (C) UV cross-linking with U17 RNA fragments. Radiolabeled U17 (lane 2) and its derived RNA fragments U17/5′D (lane 3), U17/3′D (lane 4), and U17/3′st (lane 5) were separately incubated with NE. The mixtures were subjected to UV light irradiation and RNase A/T₁ digestion, and cross-linked proteins were fractionated by SDS-PAGE. The previously identified specific cross-links (Fig. 2C) are indicated by a dot. Positions of size markers (lane M) are indicated in kilodaltons.

FIG. 4

RNA fragments of U19 and U64 cannot compete for snoRNP assembly. The gel mobility shift assay was performed with radiolabeled U19 RNA, which was incubated with NE in the absence of competitor RNA (control [c]; lane 1) or in the presence of increasing concentrations (10-, 100-, and 1,000-fold molar excess) of unlabeled competitor RNAs U17 (lanes 2 to 4), U17/3′st (lanes 5 to 7), U19 (lanes 8 to 10), U19 3′ domain fragment (U19/3′D; lanes 11 to 13), U19 3′-terminal stem-loop fragment (U19/3′st; lanes 14 to 16), U64 (lanes 17 to 19), and U64 3′ domain fragment (U64/3′D; lanes 20 to 22).

FIG. 5

hTR and its derived 3′-terminal fragments can compete for U17 assembly. (A) Schematic structure of hTR RNA (nt 1 to 451) and secondary structure model of its H/ACA-like domain. Open boxes represent the template region and the H and ACA motifs. Relevant restriction sites are indicated by an arrow. Numbers indicate the first nucleotides of 5′-truncated hTR RNA fragments. The secondary structure model was adapted from reference . (B) Gel mobility shift assay in the presence of competitor RNAs hTR and its derived RNA fragments. Radiolabeled U17 RNA was incubated in the absence (lane 1) or presence (lane 2) of WCE. Increasing amounts (10-, 100-, and 1,000-fold molar excess) of unlabeled competitor RNAs hTR (lanes 3 to 5), hTRΔ23 (lanes 6 to 8), hTR/148 (lanes 9 to 11), hTR/206 (lanes 12 to 14), hTR/268 (lanes 15 to 17), hTR/308 (lanes 18 to 20), hTR/3′st (nt 378 to 451; lanes 21 to 23), and hTR/3′stA→G (lanes 24 to 26), which contains an A-to-G mutation in the ACA motif, were added to the assays. (C) Gel mobility shift assay with radiolabeled hTR/206. The labeled RNA was incubated in the absence (lane 1) or presence (lane 2) of WCE and could form two RNP complexes, designated RNP and RNP^∗ for the faster- and the slower-migrating complexes, respectively. Unlabeled competitor RNAs U17 (lanes 3 to 5), U17/3′st (lanes 6 to 8), U17/3′stA→G (lanes 9 to 11), U19 (lanes 12 to 14), U64 (lanes 15 to 17), hTR (lanes 18 to 20), hTR/3′st (lanes 21 to 23) and hTR/3′stA→G (lanes 24 to 26) were added in increasing concentrations (10-, 100-, and 1,000-fold molar excess).

FIG. 6

(A) hGAR1 cDNA and predicted protein sequences. Nucleotide positions are given on the left, and amino acids are numbered on the right. The open reading frame (nt 265 to 915) encodes a protein of 217 amino acids. Sequences of the GAR domains are in bold. Two in-frame termination codons of the 5′ untranslated region are underlined. A putative polyadenylation signal is double underlined. (B) Increasing dilutions of affinity-purified antipeptide Abs αP795 and αP796 were used for Western blotting of HeLa cell extracts in a multiscreen apparatus. Sizes are indicated in kilodaltons.

FIG. 7

Anti-hGAR1 Abs immunoprecipitate human H/ACA snoRNPs. (A) 3′-end-labeled RNAs isolated from WCE (an aliquot) (Total; lane 2) and from anti-hGAR1 (αG; lanes 3, 5, 7, and 10) and antifibrillarin (αF; lanes 4, 6, 8, and 11) immunoprecipitates were fractionated on an 8% sequencing gels. Sizes of DNA markers obtained from MspI-digested pBR322 (M; lanes 1 and 9) are indicated in nucleotides. Immunoprecipitates were washed with NET-2 buffer containing different concentrations of NaCl (indicated above the lanes). [5′-³²P]pCp-labeled RNAs in lanes 10 and 11 were separated on a gel without prior ethanol precipitation. (B) RNase mappings. Antisense RNA probes (lanes P) specific for U3, U17, U19, and hTR were used for RNase A/T₁ mapping of RNA isolated from WCE (lanes T) and from anti-hGAR1 (lanes G) and antifibrillarin (lanes F) immunoprecipitates.

FIG. 8

Reconstituted RNPs contain hGAR1. Radiolabeled RNAs listed on the left were individually incubated with WCE, and aliquots of the mixtures were later incubated with PAS beads (lanes 1; background control) or PAS beads that had been precoated with anti-hGAR1 peptide Abs in the absence (lanes 2) or presence (lanes 3) of the immunizing peptide.

FIG. 9

Assembly of U17 RNP does not require hGAR1. (A) Gel mobility shift assay with radiolabeled U17 RNA. Radiolabeled U17 RNA was incubated in the absence (lane 1) or presence (lane 2) of NE, mock-depleted NE (lane 3), NE preincubated with PAS beads coated with IgGs from preimmune serum (lane 4), or NE preincubated with PAS beads coated with anti-hGAR1 Abs. (B) UV cross-linking of proteins interacting with U17 RNA. In vitro reconstituted U17 RNPs were subjected to UV light irradiation and RNase A/T₁ digestion, and the cross-linked proteins were fractionated by SDS-PAGE. Four H/ACA-specific proteins of about 60, 29, 23, and 14 kDa (indicated by a dot) could be cross-linked to U17 RNA incubated with NE (control lane 2), mock-depleted NE (lane 3), or NE pretreated with preimmune serum (lane 4). Only the cross-link corresponding to the 29-kDa protein (indicated by the arrowhead) was greatly diminished with the GAR1-depleted NE (lane 5). Preincubation with anti-hGAR1 Abs resulted in depletion of ∼70% of hGAR1, as verified by Western analysis (data not shown). Sizes of markers (M) are indicated in kilodaltons.