Dbp9p, a putative ATP-dependent RNA helicase involved in 60S-ribosomal-subunit biogenesis, functionally interacts with Dbp6p

Daugeron, Marie‐Claire; Kressler, Dieter; Linder, Patrick

doi:10.1017/s1355838201010640

Cited by 52 publications

(53 citation statements)

References 57 publications

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“…We used coimmunoprecipitation experiments to determine if RNA helicases required for similar pre-rRNA processing steps interact. Our results are in agreement with and expand upon previous results from TAP tag purifications, synthetic lethality screens, and yeast two-hybrid experiments (8,10,14,22,26). For example, Daugeron and Lindner found synthetic lethal interactions between yeast with mutations made in Dbp7, Dbp6, and Dbp9 but not Spb4, suggesting that these three helicases function at the same prerRNA processing steps (10).…”

Section: Discussionsupporting

confidence: 91%

“…One RNA helicase is required for both LSU and SSU biogenesis. Of the 10 helicases involved in LSU biogenesis, Dbp6, Dbp7, Dbp9, Mak5, and Drs1 have previously been described as involved in both 25S and 5.8S rRNA maturation, whereas Dbp3, Dbp10, Spb4, and Mtr4 are necessary for processing of the 5.8S rRNA alone (5,8,10,12,13,28,40,54,56). Has1 has previously been shown to be required for SSU biogenesis; here, we confirm that it has an additional role in LSU biogenesis (18).…”

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confidence: 99%

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Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Involved in Large Ribosomal Subunit Biogenesis

Bernstein

Granneman²,

Lee³

et al. 2006

Molecular and Cellular Biology

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DEXD/H box putative RNA helicases are required for pre-rRNA processing in Saccharomyces cerevisiae, although their exact roles and substrates are unknown. To characterize the significance of the conserved motifs for helicase function, a series of five mutations were created in each of the eight essential RNA helicases (Has1, Dbp6, Dbp10, Mak5, Mtr4, Drs1, Spb4, and Dbp9) involved in 60S ribosomal subunit biogenesis. Each mutant helicase was screened for the ability to confer dominant negative growth defects and for functional complementation. Different mutations showed different degrees of growth inhibition among the helicases, suggesting that the conserved regions do not function identically in vivo. Mutations in motif I and motif II (the DEXD/H box) often conferred dominant negative growth defects, indicating that these mutations do not interfere with substrate binding. In addition, mutations in the putative unwinding domains (motif III) demonstrated that conserved amino acids are often not essential for function. Northern analysis of steady-state RNA from strains expressing mutant helicases showed that the dominant negative mutations also altered pre-rRNA processing. Coimmunoprecipitation experiments indicated that some RNA helicases associated with each other. In addition, we found that yeasts disrupted in expression of the two nonessential RNA helicases, Dbp3 and Dbp7, grew worse than when either one alone was disrupted.Virtually every cellular process involving RNA requires the function of an ATP-dependent DEXD/H box RNA helicase. These enzymes are known to be involved in mRNA splicing, transcription, RNA editing, and ribosome biogenesis. RNA helicases are conserved from bacteria to humans and require eight conserved motifs for ATP binding and hydrolysis, substrate binding, and conformational changes related to their functions ( Fig. 1A) (11,15,16,42,45,48). In particular, motif I, motif II (DEXD/H box), and motif VI are required for ATPase activity; motif III (SAT) couples ATP hydrolysis with helicase function and motif VI is also required for RNA interactions. Recently, it has been shown that RNA helicases, in addition to their ability to bind and unwind RNA, can also modify ribonucleoprotein complexes (RNPs) (19,25).Pre-rRNA processing begins with transcription of the 35S pre-rRNA in the nucleolus. Through a stepwise series of cleavage events, this transcript is processed into the mature 18S, 5.8S, and 25S rRNAs (Fig. 1B). The 5S rRNA is transcribed and processed separately. The 5S, 5.8S, and 25S rRNAs become incorporated into the large ribosomal subunit (LSU), while the 18S becomes incorporated into the small ribosomal subunit (SSU). Processing in internal transcribed spacer 1 separates the small and large ribosomal precursors and forms the 20S and 27SA 2 pre-rRNAs (Fig. 1B). The 20S pre-rRNA is subsequently processed at site D to form the mature 18S rRNA. Processing of the 5.8S and 25S rRNAs occurs through two distinct pathways. In the major pathway, the 27SA 2 prerRNA is subsequently cleaved into the...

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Section: Discussionsupporting

confidence: 91%

mentioning

confidence: 99%

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Involved in Large Ribosomal Subunit Biogenesis

Bernstein

Granneman²,

Lee³

et al. 2006

Molecular and Cellular Biology

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“…1B, lane 2), even though cells were still growing at wild-type rates (data not shown). As seen upon depletion of other pre-60S subunit assembly factors, we also observed accumulation of 35S and 33S/32S pre-rRNAs at later time-points, suggesting that a greater fraction of pre-rRNAs are processed post-transcriptionally under these conditions (Sun and Woolford 1994;Zanchin et al 1997;de la Cruz et al 1998de la Cruz et al , 2005Kressler et al 1998Kressler et al , 1999Zanchin and Goldfarb 1999;Burger et al 2000;Dunbar et al 2000;Basu et al 2001;Daugeron et al 2001;Pestov et al 2001;Saveanu et al 2001Saveanu et al , 2003Saveanu et al , 2007Adams et al 2002;Oeffinger et al 2002;Wehner and Baserga 2002;Oeffinger and Tollervey 2003;Dez et al 2004;Emery et al 2004;Galani et al 2004;Horsey et al 2004;Rodriguez-Mateos et al 2009). Furthermore, this was accompanied by a decrease in levels of 20S prerRNA and increased amounts of 23S pre-rRNA (Fig.…”

Section: Resultssupporting

confidence: 53%

Disruption of ribosome assembly in yeast blocks cotranscriptional pre-rRNA processing and affects the global hierarchy of ribosome biogenesis

Talkish

Biedka

Jakovljevic

et al. 2016

RNA

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In higher eukaryotes, pre-rRNA processing occurs almost exclusively post-transcriptionally. This is not the case in rapidly dividing yeast, as the majority of nascent pre-rRNAs are processed cotranscriptionally, with cleavage at the A 2 site first releasing a pre-40S ribosomal subunit followed by release of a pre-60S ribosomal subunit upon transcription termination. Ribosome assembly is driven in part by hierarchical association of assembly factors and r-proteins. Groups of proteins are thought to associate with pre-ribosomes cotranscriptionally during early assembly steps, whereas others associate later, after transcription is completed. Here we describe a previously uncharacterized phenotype observed upon disruption of ribosome assembly, in which normally late-binding proteins associate earlier, with pre-ribosomes containing 35S pre-rRNA. As previously observed by many other groups, we show that disruption of 60S subunit biogenesis results in increased amounts of 35S pre-rRNA, suggesting that a greater fraction of pre-rRNAs are processed post-transcriptionally. Surprisingly, we found that early preribosomes containing 35S pre-rRNA also contain proteins previously thought to only associate with pre-ribosomes after early pre-rRNA processing steps have separated maturation of the two subunits. We believe the shift to post-transcriptional processing is ultimately due to decreased cellular division upon disruption of ribosome assembly. When cells are grown under stress or to high density, a greater fraction of pre-rRNAs are processed post-transcriptionally and follow an alternative processing pathway. Together, these results affirm the principle that ribosome assembly occurs through different, parallel assembly pathways and suggest that there is a kinetic foot-race between the formation of protein binding sites and pre-rRNA processing events.

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“…In agreement with the fact that 5S rRNA forms a stable RNP with the 60S r-protein Rpl5p (Deshmukh et al 1993), levels of 5S rRNA were almost unaffected in the npa1-1 strain. Similar pre-rRNA processing defects have been described on mutation in or depletion of several factors involved in 60S r-subunit synthesis, among them Dbp6p, Dbp7p, Dbp9p, and Nop8p (Daugeron and Linder 1998;Kressler et al 1998;Zanchin and Goldfarb 1999;Daugeron et al 2001). Pre-rRNA processing analysis also suggests that the rsa3 null allele leads to a mild reduction in both 27SB pre-rRNAs.…”

Section: Npa1p Is Required For 60s Biogenesissupporting

confidence: 66%

Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

Rosado

Cruz²

2004

RNA

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Ribosome biogenesis requires >100 nonribosomal proteins, which are associated with different preribosomal particles. The substrates, the interacting partners, and the timing of action of most of these proteins are largely unknown. To elucidate the functional environment of the putative ATP-dependent RNA helicase Dbp6p from Saccharomyces cerevisiae, which is required for 60S ribosomal subunit assembly, we have previously performed a synthetic lethal screen and thereby revealed a genetic interaction network between Dbp6p, Rpl3p, Nop8p, and the novel Rsa3p. In this report, we extended the characterization of this functional network by performing a synthetic lethal screen with the rsa3 null allele. This screen identified the so far uncharacterized Npa1p (YKL014C). Polysome profile analysis indicates that there is a deficit of 60S ribosomal subunits and an accumulation of half-mer polysomes in the slowly growing npa1-1 mutant. Northern blotting and primer extension analysis shows that the npa1-1 mutation negatively affects processing of all 27S pre-rRNAs and the normal accumulation of both mature 25S and 5.8S rRNAs. In addition, 27SA 2 pre-rRNA is prematurely cleaved at site C 2 . Moreover, GFP-tagged Npa1p localizes predominantly to the nucleolus and sediments with large complexes in sucrose gradients, which most likely correspond to pre-60S ribosomal particles. We conclude that Npa1p is required for ribosome biogenesis and operates in the same functional environment of Rsa3p and Dbp6p during early maturation of 60S ribosomal subunits.

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Dbp9p, a putative ATP-dependent RNA helicase involved in 60S-ribosomal-subunit biogenesis, functionally interacts with Dbp6p

Cited by 52 publications

References 57 publications

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Involved in Large Ribosomal Subunit Biogenesis

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Involved in Large Ribosomal Subunit Biogenesis

Disruption of ribosome assembly in yeast blocks cotranscriptional pre-rRNA processing and affects the global hierarchy of ribosome biogenesis

Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

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