The ATPase and helicase activities of Prp43p are stimulated by the G-patch protein Pfa1p during yeast ribosome biogenesis

Lebaron, Simon; Papin, Christophe; Capeyrou, Régine; Chen, Yanling; Froment, Carine; Monsarrat, Bernard; Caizergues‐Ferrer, Michèle; Grigoriev, Mikhaïl; Henry, Y.

doi:10.1038/emboj.2009.335

Cited by 92 publications

(199 citation statements)

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“…It may be that formation of the final beak structure is necessary for 20S pre-rRNA cleavage and that the dominant-negative mutations in Ltv1 prevent this step, either by blocking the conformational change or by inhibiting the release of factors necessary for the conformational change. In this context, two reports published during the preparation of this manuscript showing a synthetic genetic interaction between LTV1 and PRP43 and PAF1 are intriguing (Lebaron et al 2009;Pertschy et al 2009). Prp43, an RNA helicase, and Paf1, a G-patch protein that stimulates the helicase activity of Prp43, are both required in a Dltv1 background for cytoplasmic 20S rRNA cleavage.…”

Section: Resultsmentioning

confidence: 99%

Dominant Mutations in the Late 40S Biogenesis Factor Ltv1 Affect Cytoplasmic Maturation of the Small Ribosomal Subunit in Saccharomyces cerevisiae

et al. 2010

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In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus from rRNAs and ribosomal proteins, exported as premature complexes, and processed in final maturation steps in the cytoplasm. Ltv1 is a conserved 40S ribosome biogenesis factor that interacts with pre-40S complexes in vivo and is proposed to function in yeast in nuclear export. Cells lacking LTV1 grow slowly and are significantly impaired in mature 40S subunit production. Here we show that mutation or deletion of a putative nuclear export sequence in LTV1 is strongly dominant negative, but the protein does not accumulate in the nucleus, as expected for a mutation affecting export. In fact, most of the mutant protein is cytoplasmic and associated with pre-40S subunits. Cells expressing mutant Ltv1 have a 40S biogenesis defect, accumulate 20S rRNA in the cytoplasm as detected by FISH, and retain the late-acting biogenesis factor Tsr1 in the cytoplasm. Finally, overexpression of mutant Ltv1 is associated with nuclear retention of 40S subunit marker proteins, RpS2-GFP and RpS3-GFP. We suggest that the proximal consequence of these LTV1 mutations is inhibition of the cytoplasmic maturation of 40S subunits and that nuclear retention of pre-40S subunits is a downstream consequence of the failure to release and recycle critical factors back to the nucleus. R IBOSOME biogenesis is a major biosynthetic activity of eukaryotic cells and a significant ratelimiting factor in cell growth and proliferation. The pathway appears to be conserved in most respects in eukaryotes from yeast to humans and has been extensively analyzed in the model organism, Saccharomyces cerevisiae. Ribosome biogenesis begins cotranscriptionally in the nucleolus, continues in the nucleoplasm, and is completed in the cytoplasm where final maturation events must occur (for reviews, see More than 150 trans-acting factors have been identified by genetic and proteomic studies with roles in ribosome biogenesis. Generally, these factors are well conserved through eukaryotic evolution, but the specific function of many of the proteins remains largely unknown.In yeast, the 18S, 5.8S, and 25S ribosomal RNAs (rRNAs) are transcribed as a single 35S precursor rRNA. About 40 ribosome biogenesis factors, U3 snoRNA, and ribosomal proteins assemble cotranscriptionally on the nascent 35S pre-rRNA to form the 90S pre-ribosome (Dragon et al. 2002;Grandi et al. 2002;Schafer et al. 2003). Cleavage of the 35S pre-rRNA at site A2 releases a pre-40S particle containing 20S pre-rRNA, which then sheds most of the processing factors associated with it (Schafer et al. 2003). Pre-60S biogenesis factors and ribosomal proteins then assemble on the remaining transcript. The pre-60S subunit undergoes extensive remodeling and processing in the nucleolus and nucleoplasm, acquiring new processing factors and incorporating the independently transcribed 5S pre-rRNA before being exported through the nuclear pore to the cytoplasm (reviewed in Fromont-Racine et al. 2003;Tschochner and Hurt 2003). The pre-40S an...

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Section: Resultsmentioning

confidence: 99%

Dominant Mutations in the Late 40S Biogenesis Factor Ltv1 Affect Cytoplasmic Maturation of the Small Ribosomal Subunit in Saccharomyces cerevisiae

et al. 2010

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“…Like DEAD-box proteins, DEAH-box proteins are part of the SF2 family of RNA helicases. The DEAH-box protein Prp43 has roles in pre-mRNA splicing as well as ribosome biogenesis (22,(76)(77)(78). Interestingly, these roles are mediated by different cofactors (22,23,25,71,79), suggesting that cofactor-mediated specificity could even hold true for other members of the SF2 helicases besides DEAD-box proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Of the 25 DEAD-box proteins in Saccharomyces cerevisiae, five (eIF4A, Fal1, Ded1, Dbp5, and Dbp8) are known to be regulated by cofactors (12,19,21,28,34,35,(65)(66)(67). Similarly, three related SF2-helicases from the DEAH and Ski2-like subfamilies (Prp43, Mtr4, and Brr2) are also regulated by cofactors (22,23,25,(68)(69)(70)(71). These cofactors can increase or decrease the rates of ATP hydrolysis, RNA unwinding, or phosphate or RNA release.…”

Section: Discussionmentioning

confidence: 99%

“…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 also function to provide specificity for DEAD-box and DEAH-box proteins (1, 18).…”

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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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“…Prp43 activity contributes to the maintenance of spliceosome integrity because reduced Prp43 function promotes the use of structurally aberrant spliceosomes and the splicing of suboptimal premRNA substrates (Pandit et al 2006;Koodathingal et al 2010;Mayas et al 2010;Chen et al 2013). In addition to features of the postcatalytic spliceosome, specific changes in spliceosome composition linked to ATP hydrolysis by the Prp2, Prp16, and Prp22 DExD/H-box proteins render defective splicing complexes sensitive to Prp43 recruitment and ATP-dependent dissociation (Chen et al 2013).Data from several groups implicate three Prp43-interacting factors in the regulation of this protein's role in pre-mRNA splicing (Spp382/Ntr1) and pre-rRNA processing (Sqs1/Pfa1 and Pxr1/Gno1) Lebaron et al 2005;Tsai et al 2005;Boon et al 2006;Pandit et al 2006;Tanaka et al 2007;Tsai et al 2007;Lebaron et al 2009;Pertschy et al 2009;Walbott et al 2010;Christian et al 2014). Spp382 is an essential pre-mRNA splicing factor required for Prp43 recruitment to the spliceosome.…”

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confidence: 99%

Limited Portability of G-Patch Domains in Regulators of the Prp43 RNA Helicase Required for Pre-mRNA Splicing and Ribosomal RNA Maturation in Saccharomyces cerevisiae

Banerjee¹,

McDaniel²,

Rymond

2015

Genetics

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The Prp43 DExD/H-box protein is required for progression of the biochemically distinct pre-messenger RNA and ribosomal RNA (rRNA) maturation pathways. In Saccharomyces cerevisiae, the Spp382/Ntr1, Sqs1/Pfa1, and Pxr1/Gno1 proteins are implicated as cofactors necessary for Prp43 helicase activation during spliceosome dissociation (Spp382) and rRNA processing (Sqs1 and Pxr1). While otherwise dissimilar in primary sequence, these Prp43-binding proteins each contain a short glycine-rich G-patch motif required for function and thought to act in protein or nucleic acid recognition. Here yeast two-hybrid, domain-swap, and site-directed mutagenesis approaches are used to investigate G-patch domain activity and portability. Our results reveal that the Spp382, Sqs1, and Pxr1 G-patches differ in Prp43 two-hybrid response and in the ability to reconstitute the Spp382 and Pxr1 RNA processing factors. G-patch protein reconstitution did not correlate with the apparent strength of the Prp43 two-hybrid response, suggesting that this domain has function beyond that of a Prp43 tether. Indeed, while critical for Pxr1 activity, the Pxr1 G-patch appears to contribute little to the yeast two-hybrid interaction. Conversely, deletion of the primary Prp43 binding site within Pxr1 (amino acids 102-149) does not impede rRNA processing but affects small nucleolar RNA (snoRNA) biogenesis, resulting in the accumulation of slightly extended forms of select snoRNAs, a phenotype unexpectedly shared by the prp43 loss-of-function mutant. These and related observations reveal differences in how the Spp382, Sqs1, and Pxr1 proteins interact with Prp43 and provide evidence linking G-patch identity with pathway-specific DExD/H-box helicase activity.KEYWORDS Saccharomyces cerevisiae; spliceosome; rRNA processing; DExD/H-box protein; pre-mRNA splicing N UMEROUS genome-wide interaction and gene expression studies identify ribosome biogenesis as a central integrative feature of Saccharomyces cerevisiae metabolism (see, e.g ., Mnaimneh et al. 2004;Davierwala et al. 2005;Gavin et al. 2006;Collins et al. 2007;Hu et al. 2007;Magtanong et al. 2011). In rapidly dividing organisms such as this budding yeast, ribosomal RNAs (rRNAs) make up the lion's share of cellular nucleic acid by mass, while ribosomal protein transcripts can account for more than half the transcribed messenger RNA (mRNA). Because ribosome biogenesis is so energetically costly, eukaryotes have evolved multiple means to regulate rRNA and ribosomal protein production in response to changes in cellular demand and surveillance systems to remove aberrant ribosomal protein complexes formed during assembly or after environmental insult (Jorgensen et al. 2002;Fingerman et al. 2003; FromontRacine et al. 2003;Jorgensen et al. 2004;Marion et al. 2004;Henras et al. 2008;Kressler et al. 2010;Lafontaine 2010). The coordination of pre-mRNA processing with ribosome biogenesis is especially relevant in the intron-poor environment of the yeast genome, where the highly expressed ribosomal protein transcr...

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The ATPase and helicase activities of Prp43p are stimulated by the G-patch protein Pfa1p during yeast ribosome biogenesis

Cited by 92 publications

References 38 publications

Dominant Mutations in the Late 40S Biogenesis Factor Ltv1 Affect Cytoplasmic Maturation of the Small Ribosomal Subunit in Saccharomyces cerevisiae

Dominant Mutations in the Late 40S Biogenesis Factor Ltv1 Affect Cytoplasmic Maturation of the Small Ribosomal Subunit in Saccharomyces cerevisiae

Cofactor-dependent specificity of a DEAD-box protein

Limited Portability of G-Patch Domains in Regulators of the Prp43 RNA Helicase Required for Pre-mRNA Splicing and Ribosomal RNA Maturation in Saccharomyces cerevisiae

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