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

  1. Christian Trahan and
  2. François Dragon
  1. Département des Sciences Biologiques and Centre de Recherche BioMed, Université du Québec à Montréal, Montréal, Québec, H3C 3P8, Canada

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 32P-labeled RNAs and 35S-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 Δ378-451 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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Footnotes

  • Reprint requests to: François Dragon, Département des Sciences Biologiques and Centre de Recherche BioMed, Université du Québec à Montréal, Montréal, Québec, H3C 3P8, Canada; e-mail: dragon.francois{at}uqam.ca; fax: (514) 987-4647.

  • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1354009.

    • Received September 9, 2008.
    • Accepted October 30, 2008.
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