Cbf5p, the putative pseudouridine synthase of H/ACA-type snoRNPs, can form a complex with Gar1p and Nop10p in absence of Nhp2p and box H/ACA snoRNAs

Henras, Anthony K.; Capeyrou, Régine; Henry, Y.; Caizergues‐Ferrer, Michèle

doi:10.1261/rna.7770604

Cited by 49 publications

(40 citation statements)

References 82 publications

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“…Disruption of these protein complexes with various harsher conditions prior to PAGE failed (data not shown), suggesting that H/ACA proteins can form highly stable complexes, in line with previous observations (Henras et al 2004;Leulliot et al 2007). …”

Section: Resultssupporting

confidence: 89%

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

Trahan

Dragon²

2008

RNA

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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 D378-451 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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Section: Resultssupporting

confidence: 89%

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

Trahan

Dragon²

2008

RNA

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“…Therefore, yeast H/ACA RNPs, like their archaeal counterparts, also possess the intrinsic ability to self-assemble in the absence of assembly factors. The observed interaction patterns among the four proteins are consistent with previous results (Henras et al 2004;Wang and Meier 2004).…”

Section: Discussionsupporting

confidence: 92%

“…1A). It should be noted that Nhp2 tended to dissociate from Cbf5 complexes during storage, likely because of a weak interaction (Henras et al 2004). The availability of large amounts of soluble and pure Cbf5 complexes in various compositions allowed us to analyze the assembly, activity, and structure of yeast H/ACA RNP in detail.…”

Section: Purification Of Recombinant Yeast H/aca Rnp Proteinsmentioning

confidence: 99%

Reconstitution and structural analysis of the yeast box H/ACA RNA-guided pseudouridine synthase

Li¹,

Duan²,

Li³

et al. 2011

Genes Dev.

150

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Box H/ACA ribonucleoprotein particles (RNPs) mediate pseudouridine synthesis, ribosome formation, and telomere maintenance. The structure of eukaryotic H/ACA RNPs remains poorly understood. We reconstituted functional Saccharomyces cerevisiae H/ACA RNPs with recombinant proteins Cbf5, Nop10, Gar1, and Nhp2 and a two-hairpin H/ACA RNA; determined the crystal structure of a Cbf5, Nop10, and Gar1 ternary complex at 1.9 Å resolution; and analyzed the structure-function relationship of the yeast complex. Although eukaryotic H/ACA RNAs have a conserved two-hairpin structure, isolated single-hairpin RNAs are also active in guiding pseudouridylation. Nhp2, unlike its archaeal counterpart, is largely dispensable for the activity, reflecting a functional adaptation of eukaryotic H/ACA RNPs to the variable RNA structure that Nhp2 binds. The N-terminal extension of Cbf5, a hot spot for dyskeratosis congenita mutation, forms an extra structural layer on the PUA domain. Gar1 is distinguished from the assembly factor Naf1 by containing a C-terminal extension that controls substrate turnover and the Gar1-Naf1 exchange during H/ACA RNP maturation. Our results reveal significant novel features of eukaryotic H/ACA RNPs.

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“…Gar1 binds to one end of the Cbf5 catalytic domain without contacting Nop10. This is in good agreement with biochemical data demonstrating an independent interaction of archaeal, yeast, and mammalian Gar1 with Cbf5 homologs [14][15][16][17]. However, Gar1, which has been tied to the catalytic core by crosslinking in mammalian H/ACA RNPs [14], is situated too far from the active site aspartate to make contact with it.…”

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confidence: 90%

How a single protein complex accommodates many different H/ACA RNAs

Meier

2006

Trends in Biochemical Sciences

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Over 100 mammalian H/ACA RNAs form an equal number of ribonucleoproteins (RNPs) by associating with the same four core proteins. The function of these H/ACA RNPs is essential for ribosome biogenesis, pre-mRNA splicing, telomere maintenance, and, likely, for additional cellular processes. Recent crystal structures of archaeal H/ACA protein complexes illustrate how the same four proteins accommodate more than 100 distinct though related H/ACA RNAs and reveal a spatial mutation cluster underlying the bone marrow failure syndrome dyskeratosis congenita (DC).Keywords H/ACA RNPs; H/ACA RNAs; crystal structure; core proteins; dyskeratosis congenita; RNAprotein interaction The vast majority of mammalian H/ACA RNPs engage in isomerization of uridines to pseudouridines (pseudouridylation) in ribosomal and spliceosomal small nuclear RNAs. Although the function of most pseudouridines is unknown, some are essential for optimal translation and for pre-mRNA splicing [1]. Perhaps the most intriguing species of H/ACA RNPs are defined by the snoRNA U17/E1 (snR30 in yeast), telomerase RNA (which ends in an H/ACA RNA structure), and a growing number of orphan H/ACA RNAs (without complementarity to any stable RNAs) [2]. U17 is the only essential H/ACA RNA and is required for pre-ribosomal RNA processing; telomerase RNA is required for the replication of chromosome ends; and the orphan H/ACA RNAs are by definition of unknown function but potentially involved in similar important processes as U17 and telomerase RNA (Fig. 1) [3]. Recently, three groups solved the crystal structures of archaeal H/ACA RNP core complexes consisting of two or three core proteins (Fig. 1) [4][5][6]. For the first time, these structures provide molecular details of protein-protein interactions in the H/ACA core complex and of the pseudouridylase itself. meier@aecom.yu Fig. 1), the other class being C/D RNAs. On average 130-140 nucleotides in length, H/ACA RNAs conform to a consensus 5'-hairpin-hinge-hairpin-tail-3' secondary structure with the characteristic ACA trinucleotide exactly three residues from the 3' end ( Fig. 1). H/ACA RNAs identify the ~130 known mammalian pseudouridylation sites by base pairing to a few nucleotides flanking the target uridines. Complementarity to target RNAs lies in the upper half of a bulge (pseudouridylation pocket) of one or both of the hairpins placing the unpaired target uridine at the bottom of a helix [7,8]. Pseudouridylation is catalyzed by one of the four H/ACA core proteins, the pseudouridylase NAP57 (also dyskerin or, in yeast and archaea, Cbf5) [9]. The small basic proteins GAR1, NOP10, and NHP2 (L7Ae in archaea) round out H/ACA RNPs. All four core proteins, except GAR1, are essential for the structural integrity of H/ACA RNPs [3]. The structuresThe structure of the archaeal pseudouridylase Cbf5 was solved complexed with Nop10 [5,6] or with Nop10 and Gar1 [4]. Based on structural and sequence homologies, pseudouridylases are classified into 5 families, RluA, RsuA, TruA, TruB, and TruD [10].Crystal structur...

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Cbf5p, the putative pseudouridine synthase of H/ACA-type snoRNPs, can form a complex with Gar1p and Nop10p in absence of Nhp2p and box H/ACA snoRNAs

Cited by 49 publications

References 82 publications

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

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

Reconstitution and structural analysis of the yeast box H/ACA RNA-guided pseudouridine synthase

How a single protein complex accommodates many different H/ACA RNAs

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