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

Bernstein, Kara A.; Granneman, Sander; Lee, Alicia V.; Manickam, Swarnameenakshi; Baserga, Susan J.

doi:10.1128/mcb.26.4.1195-1208.2006

Cited by 64 publications

(83 citation statements)

References 63 publications

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“…The Dbp6 and Drs1 Q motif mutants also conferred a dominant negative growth defect (1). Although our analysis of the Q motif was not exhaustive because we did not test other substitutions, our results suggest that an intact glutamine in the Q motif in most cases is not essential for function of the DEAD box RNA helicases involved in ribosome biogenesis.…”

Section: Vol 26 2006 Mutational Analysis Of Dexd/h Box Rna Helicasementioning

confidence: 84%

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Required for Small Ribosomal Subunit Synthesis

Granneman¹,

Bernstein²,

Bleichert³

et al. 2006

Molecular and Cellular Biology

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The 17 putative RNA helicases required for pre-rRNA processing are predicted to play a crucial role in ribosome biogenesis by driving structural rearrangements within preribosomes. To better understand the function of these proteins, we have generated a battery of mutations in five putative RNA helicases involved in 18S rRNA synthesis and analyzed their effects on cell growth and pre-rRNA processing. Our results define functionally important residues within conserved motifs and demonstrate that lethal mutations in predicted ATP binding-hydrolysis motifs often confer a dominant negative phenotype in vivo when overexpressed in a wild-type background. We show that dominant negative mutants delay processing of the 35S pre-rRNA and cause accumulation of pre-rRNA species that normally have low steady-state levels. Our combined results establish that not all conserved domains function identically in each protein, suggesting that the RNA helicases may have distinct biochemical properties and diverse roles in ribosome biogenesis.In the nucleolus, RNA polymerase I transcribes a single polycistronic pre-rRNA that is processed to obtain the mature 18S, 5.8S, and 25S-28S rRNAs. In the yeast Saccharomyces cerevisiae, the primary 35S pre-rRNA is packaged into a large RNA-protein complex (RNP) termed the small ribosomal subunit (SSU) processome-90S preribosome (8,15,16). The SSU processome is essential for processing at sites A 0 , A 1 , and A 2 , thereby releasing the 20S and 27SA 2 pre-rRNAs from the primary transcript ( Fig. 1) (8). The U3 small nucleolar RNA (U3 snoRNA) is an integral component of the SSU processome and base pairs with the pre-rRNA for assembly and function of the SSU processome (8). Processing at sites A 0 , A 1 , and A 2 initiates the formation of 43S and 66S preribosomal particles (12,32,46). Alternatively, cleavage at site A 3 occurs first, leading to the formation of the 23S and 27SA 3 prerRNAs. Subsequent processing at sites A 0 (22S), A 1 (21S), and A 2 yields the 20S pre-rRNA (43S preribosome) (Fig. 1).The 27SA 2 and 27SA 3 precursors, already packaged in 66S large ribosomal subunit (LSU) preribosomes, are then processed to the mature 5.8S and 25S rRNAs. The 20S precursor, part of the 43S preribosomes, is transported to the cytoplasm where it is cleaved at site D, generating the mature 18S rRNA and 40S small ribosomal subunits.Ribosome biogenesis in yeast requires over 150 trans-acting protein factors, not including the small nucleolar RNAs that are also involved (12,32,46). As might be expected, many of the nonribosomal proteins harbor motifs that are characteristic for enzymes. These include numerous P-loop-type NTPases, protein kinases, and putative remodeling enzymes, like DEXD/H box proteins and AAA-ATPases, underscoring the complexity of ribosome biogenesis (12,13,17,46). The DEXD/H box proteins function in a variety of aspects of RNA metabolism, including pre-mRNA splicing, nuclear transcription, translation, and pre-rRNA processing (45). These proteins are often referred to as RNA helicases ...

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Section: Vol 26 2006 Mutational Analysis Of Dexd/h Box Rna Helicasementioning

confidence: 84%

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Required for Small Ribosomal Subunit Synthesis

Granneman¹,

Bernstein²,

Bleichert³

et al. 2006

Molecular and Cellular Biology

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“…Yet, alanine substitution in motif Ia of the DEAH-box RNA helicase Prp22 resulted in a minor or no effect on yeast growth (Schneider et al 2004). Indeed, helicases often display variability in their sensitivity to mutation of their conserved motifs (Bernstein et al 2006;Granneman et al 2006a), including the DEAD-box itself FIGURE 7. The helicase-like domain of Utp25 mediates protein-protein interactions with Utp3.…”

Section: Discussionmentioning

confidence: 99%

The DEAD-box RNA helicase-like Utp25 is an SSU processome component

Charette

Baserga²

2010

RNA

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The SSU processome is a large ribonucleoprotein complex consisting of the U3 snoRNA and at least 43 proteins. A database search, initiated in an effort to discover additional SSU processome components, identified the uncharacterized, conserved and essential yeast nucleolar protein YIL091C/UTP25 as one such candidate. The C-terminal DUF1253 motif, a domain of unknown function, displays limited sequence similarity to DEAD-box RNA helicases. In the absence of the conserved DEAD-box sequence, motif Ia is the only clearly identifiable helicase element. Since the yeast homolog is nucleolar and interacts with components of the SSU processome, we examined its role in pre-rRNA processing. Genetic depletion of Utp25 resulted in slowed growth. Northern analysis of pre-rRNA revealed an 18S rRNA maturation defect at sites A 0 , A 1 , and A 2 . Coimmunoprecipitation confirmed association with U3 snoRNA and with Mpp10, and with components of the t-Utp/UtpA, UtpB, and U3 snoRNP subcomplexes. Mutation of the conserved motif Ia residues resulted in no discernable temperaturesensitive or cold-sensitive growth defects, implying that this motif is dispensable for Utp25 function. A yeast two-hybrid screen of Utp25 against other SSU processome components revealed several interacting proteins, including Mpp10, Utp3, and Utp21, thereby identifying the first interactions among the different subcomplexes of the SSU processome. Furthermore, the DUF1253 domain is required and sufficient for the interaction of Utp25 with Utp3. Thus, Utp25 is a novel SSU processome component that, along with Utp3, forms the first identified interactions among the different SSU processome subcomplexes.

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“…Seven (Dhr1,Dhr2,Dbp8,Rok1,Fal1,Rrp3,Dbp4) are required for SSU biogenesis (Song et al 1995;O'Day et al 1996;Liang et al 1997;Venema et al 1997;Colley et al 2000;Granneman et al 2006a), while eight (Dbp2, Dbp6, Dbp9, Mak5, Drs1, Dbp10, Spb4, Mtr4) are required for LSU biogenesis (Sachs and Davis 1990;Ripmaster et al 1993;de la Cruz et al 1998ade la Cruz et al ,b, 2004Kressler et al 1998;Burger et al 2000;Bond et al 2001;Bernstein et al 2006). Prp43 and Has1 associate with, and are required for, both SSU and LSU biogenesis at stages after these subunits have entered separate processing pathways, suggesting that these proteins may have more general functions in the ribosome assembly process (Emery et al 2004;Lebaron et al 2005;Bernstein et al 2006;Combs et al 2006;Leeds et al 2006;Liang and Fournier 2006). Interestingly, DEAD-box proteins are also among the identified bacterial assembly factors, pointing to conserved roles.…”

Section: Dexh/d Proteinsmentioning

confidence: 99%

“…The size, complexity, and dynamic nature of maturing pre-ribosomal particles, the host of potential cofactors, as well as the possibility that these proteins may act at stages that differ from where maturation stalls in their absence, all pose a tremendous challenge to investigators seeking to understand the role(s) of these proteins in ribosome biogenesis. In addition, a subset of these proteins may cooperate to some extent as they demonstrate synthetic lethal interactions and are associated in coimmunoprecipitation experiments Gavin et al 2002;Bernstein et al 2006). Work in the Baserga laboratory has probed the function of DExH/D proteins involved in both SSU and LSU biogenesis using systematic mutagenesis of conserved residues in motifs responsible for ATP hydrolysis in each helicase (Bernstein et al 2006;Granneman et al 2006a).…”

Section: Dexh/d Proteinsmentioning

confidence: 99%

“…In addition, a subset of these proteins may cooperate to some extent as they demonstrate synthetic lethal interactions and are associated in coimmunoprecipitation experiments Gavin et al 2002;Bernstein et al 2006). Work in the Baserga laboratory has probed the function of DExH/D proteins involved in both SSU and LSU biogenesis using systematic mutagenesis of conserved residues in motifs responsible for ATP hydrolysis in each helicase (Bernstein et al 2006;Granneman et al 2006a). Different, although sometimes similar, rRNA processing defects were observed for each of the mutants, and most were unable to support growth (Bernstein et al 2006;Granneman et al 2006a).…”

Section: Dexh/d Proteinsmentioning

confidence: 99%

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Powering through ribosome assembly

Strunk¹,

Karbstein²

2009

RNA

193

168

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Ribosome assembly is required for cell growth in all organisms. Classic in vitro work in bacteria has led to a detailed understanding of the biophysical, thermodynamic, and structural basis for the ordered and correct assembly of ribosomal proteins on ribosomal RNA. Furthermore, it has enabled reconstitution of active subunits from ribosomal RNA and proteins in vitro. Nevertheless, recent work has shown that eukaryotic ribosome assembly requires a large macromolecular machinery in vivo. Many of these assembly factors such as ATPases, GTPases, and kinases hydrolyze nucleotide triphosphates. Because these enzymes are likely regulatory proteins, much work to date has focused on understanding their role in the assembly process. Here, we review these factors, as well as other sources of energy, and their roles in the ribosome assembly process. In addition, we propose roles of energy-releasing enzymes in the assembly process, to explain why energy is used for a process that occurs largely spontaneously in bacteria. Finally, we use literature data to suggest testable models for how these enzymes could be used as targets for regulation of ribosome assembly.

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

Cited by 64 publications

References 63 publications

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Required for Small Ribosomal Subunit Synthesis

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Required for Small Ribosomal Subunit Synthesis

The DEAD-box RNA helicase-like Utp25 is an SSU processome component

Powering through ribosome assembly

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