Two mutant forms of the S1/TPR-containing protein Rrp5p affect the 18S rRNA synthesis in Saccharomyces cerevisiae

Torchet, Claire; Jacq, Claude; Denmat, Sylvie Hermann‐Le

doi:10.1017/s1355838298981511

Cited by 57 publications

(75 citation statements)

References 55 publications

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“…Both proteins are required for early, nucleolar 40S ribosome maturation steps (40,41). In vitro pull-down experiments demonstrate that Rrp5 directly binds Rok1, consistent with previously described genetic interactions (42). Rrp5 is bound near a pre-rRNA duplex that undergoes a conformational change to regulate cleavage steps during ribosome assembly (39,43).…”

supporting

confidence: 83%

“…Previous data implicate the RNA-binding protein Rrp5 as a likely cofactor for the DEAD-box protein Rok1, and Rok1 overexpression suppresses the temperature-sensitive phenotype of a mutation in the first tetratricopeptide (TPR) motif of Rrp5 (42). TPR motifs are common protein-protein interaction modules (44), suggesting that this genetic interaction could reflect a direct interaction.…”

Section: Resultsmentioning

confidence: 99%

“…Rrp5 is a 192-kDa protein containing 12 S1 RNA-binding domains toward its N terminus and seven TPR motifs toward its C terminus. Because expression and purification of full-length Rrp5 do not produce large yields of protein (39), and because the N and C termini of Rrp5 can function independently (39,42,46), we used the C-terminal fragment of Rrp5, called Rrp5C hereafter (39), for in vitro protein binding experiments to test for a direct interaction with Rok1. This fragment contains the last 3 S1 RNA-binding domains as well as all seven TPR motifs, and when combined with the N-terminal piece, it fully complements the Rrp5 deletion (39).…”

Section: Resultsmentioning

confidence: 99%

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Cofactor-dependent specificity of a DEAD-box protein

Young

Khoshnevis

Karbstein

2013

Proc. Natl. Acad. Sci. U.S.A.

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DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.RNA helicases | annealing D EAD-box proteins are RNA-binding ATPases that are involved in all aspects of RNA metabolism: translation initiation, pre-mRNA splicing, mRNA export and decay, and ribosome biogenesis. In these processes, their functions include RNA duplex unwinding, RNA-protein complex remodeling, RNA duplex annealing, and ATP-dependent RNA binding (1-5).In vivo, these enzymes have specific functions that often involve the recognition of specific RNAs and the discrimination against a myriad of nonsubstrates. Nevertheless, only DbpA, a DEAD-box protein involved in bacterial ribosome assembly (6-8), has sequence-specific ATPase activity (6, 9), which arises from unique sequences in its C terminus (10). Such sequence specificity has not been demonstrated for any other DEAD-box protein and is consistent with their highly conserved structures and the almost universal conservation of residues that contact the RNA. Furthermore, analysis of the structures of RNA-bound DEAD-box proteins reveals that RNA contacts are made with the sugarphosphate backbone (11-17), largely precluding sequence-specific interactions between DEAD-box proteins and RNA.Many DEAD-box and related DEAH-box proteins function in conjunction with cofactors (ref. 18 and references therein). These cofactors can regulate the activity of DEAD-box or DEAH-box proteins by stimulating or inhibiting their ATPase, helicase, RNAbinding, or nucleotide-binding activities. Interestingly, cofactors are often RNA-binding proteins (19)(20)(21)(22)(23)(24)(25). We and others therefore postulated that these cofactors could als...

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supporting

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Cofactor-dependent specificity of a DEAD-box protein

Young

Khoshnevis

Karbstein

2013

Proc. Natl. Acad. Sci. U.S.A.

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“…of both the 18S and the 25S/5.8S rRNAs (Venema and Tollervey 1996;Torchet et al 1998;Eppens et al 1999). Here, we report that Rrp12p is required for the late maturation of the 18S and 5.8S rRNA.…”

Section: Discussionmentioning

confidence: 64%

A pre-ribosome-associated HEAT-repeat protein is required for export of both ribosomal subunits

Oeffinger¹,

Dlakić²,

Tollervey³

2004

Genes Dev.

114

137

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Rrp12p (Ypl012w) is unusual among characterized ribosome synthesis factors in being associated with late precursors to both the 40S and 60S subunits. Rrp12p is predominately nuclear with nucleolar enrichment at steady state, but shuttled between the nucleus and cytoplasm in a heterokaryon assay. Strains depleted of Rrp12p are impaired in the nuclear export of both ribosomal subunits. Sequence analysis combined with fold recognition and modeling showed that Rrp12p is a member of a family of pre-ribosome-associated HEAT-repeat proteins. Like other HEAT-repeat transport factors, Rrp12p binds in vitro to nucleoporin FG-repeats of both the GLFG and FXFG families and to the GTPase Gsp1p (yeast RAN). Rrp12p also showed robust in vitro binding to a pre-rRNA transcript, in addition to poly(A) and poly(U) . We propose that Rrp12p binds to the RNA components of the pre-ribosomes and promotes export of both subunits via its interactions with the nucleoporins and Gsp1p.[Keyword: Ribosome export] Supplemental material is available at http://www.genesdev.org.

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“…Cleavage at A3 initiates the formation of the 5 ′ end of 5.8S rRNA (Chu et al 1994;Henry et al 1994). However, cleavage of 35S rRNA by RNase MRP at A3 can presumably still continue even in the absence of A1/A2 cleavage (Henry et al 1994;Torchet et al 1998;Torchet and Hermann-Le Denmat 2000). A bud23Δ mutant by itself is severely compromised for A2 cleavage and is probably largely dependent on cotranscriptional A3 cleavage by RNase MRP to release the pre-40S particle.…”

Section: Genetic Interaction With Rnase Mrpmentioning

confidence: 99%

The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP

Sardana

White

Johnson

2013

RNA

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Bud23 is responsible for the conserved methylation of G1575 of 18S rRNA, in the P-site of the small subunit of the ribosome. bud23Δ mutants have severely reduced small subunit levels and show a general failure in cleavage at site A2 during rRNA processing. Site A2 is the primary cleavage site for separating the precursors of 18S and 25S rRNAs. Here, we have taken a genetic approach to identify the functional environment of BUD23. We found mutations in UTP2 and UTP14, encoding components of the SSU processome, as spontaneous suppressors of a bud23Δ mutant. The suppressors improved growth and subunit balance and restored cleavage at site A2. In a directed screen of 50 ribosomal trans-acting factors, we identified strong positive and negative genetic interactions with components of the SSU processome and strong negative interactions with components of RNase MRP. RNase MRP is responsible for cleavage at site A3 in pre-rRNA, an alternative cleavage site for separating the precursor rRNAs. The strong negative genetic interaction between RNase MRP mutants and bud23Δ is likely due to the combined defects in cleavage at A2 and A3. Our results suggest that Bud23 plays a role at the time of A2 cleavage, earlier than previously thought. The genetic interaction with the SSU processome suggests that Bud23 could be involved in triggering disassembly of the SSU processome, or of particular subcomplexes of the processome.

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Two mutant forms of the S1/TPR-containing protein Rrp5p affect the 18S rRNA synthesis in Saccharomyces cerevisiae

Cited by 57 publications

References 55 publications

Cofactor-dependent specificity of a DEAD-box protein

Cofactor-dependent specificity of a DEAD-box protein

A pre-ribosome-associated HEAT-repeat protein is required for export of both ribosomal subunits

The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP

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