Dyskeratosis congenita mutations in the H/ACA domain of human telomerase RNA affect its assembly into a pre-RNP - PubMed (original) (raw)

Dyskeratosis congenita mutations in the H/ACA domain of human telomerase RNA affect its assembly into a pre-RNP

Christian Trahan et al. RNA. 2009 Feb.

Abstract

Dyskeratosis congenita (DC) is an inherited disorder that implicates defects in the biology of telomeres, which are maintained by telomerase, a ribonucleoprotein with reverse transcriptase activity. Like all H/ACA RNAs, the H/ACA domain of nascent human telomerase RNA (hTR) forms a pre-RNP with H/ACA proteins NAF1, dyskerin, NOP10, and NHP2 in vivo. To assess the pre-RNP assembly of hTR mutants that poorly accumulate in vivo, we developed an in vitro system that uses components of human origin. Pre-RNPs were reconstituted with synthetic (32)P-labeled RNAs and (35)S-labeled proteins produced in rabbit reticulocyte lysate, and immunoprecipitations were carried out to analyze RNP formation. We show that human NAF1 cannot bind directly to the H/ACA domain of hTR, and requires the core trimer dyskerin-NOP10-NHP2 to be efficiently incorporated into the pre-RNP. This order of assembly seems common to H/ACA RNAs since it was observed with snoRNA ACA36 and scaRNA U92, which are predicted to guide pseudouridylation of 18S rRNA and U2 snRNA, respectively. However, the processing H/ACA snoRNA U17 did not conform to this rule, as NAF1 alone was able to bind it. We also provide the first evidence that DC-related mutations of hTR C408G and Delta378-451 severely impair pre-RNP assembly. Integrity of boxes H and ACA of hTR are also crucial for pre-RNP assembly, while the CAB box is dispensable. Our results offer new insights into the defects caused by some mutations located in the H/ACA domain of hTR.

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Figures

FIGURE 1.

FIGURE 1.

Schematic secondary structure of the H/ACA domain of hTR. Residues are numbered according to full-length hTR. Conserved motifs H and ACA are boxed. Sequence changes made in this study are indicated; they correspond to mutations in conserved box H (A372U), box ACA (A446G), and the CAB box highlighted by a dashed line (G414C and inv413–416), as well as disease-related mutations C408G, G450A, and Δ378–451 deletion. CR7 is enlarged to better show its normal structure, which is altered in the DC mutant C408G.

FIGURE 2.

FIGURE 2.

The tetramer NAF1-dyskerin-NOP10-NHP2 assembles specifically with hTR204. (A) Individual proteins were translated in vitro in the presence of 35S-labeled methionine and analyzed by PAGE (left). For dyskerin IPs (right), 35S-labeled proteins were cotranslated; the presence or absence of each protein in the RRL mixture is indicated by (+) or (−), respectively. Input of translated proteins (in) and immunoprecipitated products (IP) were separated by PAGE. Molecular weight markers (kDa) are shown between the two panels. Control IP with beads alone is shown (lanes 22,23). Note that dyskerin recovered from IPs gives a fuzzy signal. High molecular weight multimers of H/ACA proteins are also observed. (B) Dyskerin IPs (left) and NAF1 IPs (right) of RRL mixtures containing 35S-labeled proteins and 32P-labeled hTR204 (lanes 1-5,11-15) or U3 (lanes 6-10,16-20) RNAs were analyzed by PAGE. Because RNAs co-migrate with dyskerin in these protein gels, input lanes (in) did not contain the 32P-labeled RNAs to better discern the 35S-labeled proteins. IPs with RRL mixtures lacking either dyskerin (-dysk) or NAF1 (-NAF1) were used as negative controls. Labeled hTR204 (lanes 1,11) and U3 (lanes 6,16) RNAs not incubated in RRL are shown. Bands marked by an asterisk (*) correspond to degradation products of dyskerin/NAF1, not to fibrillarin, which was omitted form NAF1 IPs (right). (Bottom) The 32P signal from the protein gels presented in the top panels; they were obtained by blocking the 35S signal with a transparency. (C) Labeled RNAs from input (lanes 1,6,11,16) and IP supernatants (lanes 3,5,8,10,13,15,18,20) were analyzed separately on 8% denaturing polyacrylamide gels. Lanes are numbered in accordance with the corresponding IPs shown in (B).

FIGURE 3.

FIGURE 3.

Binding of NAF1 to hTR204 requires the trimer dyskerin/NOP10/NHP2. (A,C) RRL mixtures containing 35S-labeled proteins and 32P-labeled hTR204 were subjected to (A) dyskerin IP or (C) NAF1 IP and analyzed by (top) PAGE. The presence or absence of each protein in the RRL mixtures is indicated by (+) or (−), respectively. Input lanes (in) did not contain the 32P-labeled hTR204. (C, left,right) Come from the same gel exposition. (Bottom) Only the 32P signal of the protein gel (see legend to Figure 2B for details). (B,D) Labeled hTR204 recovered from supernatants of dyskerin or NAF1 IPs (see A and C, respectively) was analyzed on 8% denaturing polyacrylamide gels. Lanes are numbered in accordance with the corresponding dyskerin and NAF1 IPs shown in (A) and (C), respectively. (E) RRL mixtures containing 35S-labeled proteins and 32P-labeled ACA36, U92, or U17 were subjected to NAF1 IP and analyzed by (top) PAGE. The presence or absence of each protein in the RRL mixtures is indicated by (+) or (−), respectively. Input lanes (in) did not contain the radiolabeled RNAs. Labeled ACA36 (lane 1), U92 (lane 8), and U17 (lane 15) RNAs not incubated in RRL are shown. (Bottom) Only the 32P signal of the protein gels presented in top panels. (F) Labeled ACA36, U92, or U17 recovered from supernatants of NAF1 IPs shown in (E) were analyzed on 8% denaturing polyacrylamide gels. Lanes are numbered in accordance with the corresponding IPs shown in (E).

FIGURE 4.

FIGURE 4.

Some mutations in hTR204 impair pre-RNP assembly. (A) PAGE analysis of NAF1 IPs conducted on RRL mixtures containing 35S-labeled proteins and 32P-labeled hTR204 or its derivatives bearing mutations in conserved motifs (see text for details). Input lanes (in) did not contain the 32P-labeled RNAs. (Bottom) Only the 32P signal of the protein gel (see legend to Figure 2B for details). (B) NAF1 IPs conducted and analyzed as in (A) with 32P-labeled hTR204 or its derivatives bearing disease-related mutations C408G, G450A, and Δ378-451. The double mutant C408G-G412C restores base pairing (see text for details). (C) 32P-labeled hTR204 (arrow) recovered from a NAF1 IP (lane 3) and the IP supernatant (lane 2) was analyzed on an 8% sequencing gel. Untreated 32P-labeled hTR204 (in) was run in parallel (lane 1). Sizes of 32P-labeled DNA markers (lane 4) are indicated in nt on the right.

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