![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Departments of Molecular Biophysics and Biochemistry,1 Genetics,2 Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 065203
Received 25 July 2005/ Returned for modification 19 August 2005/ Accepted 14 November 2005
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
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 or unwindases, since they can unwind small duplexes of RNA in an NTP-dependent, generally ATP-dependent, manner in vitro. However, many DEXD/H box proteins appear to have more diverse or distinct functions than just unwinding RNA (7, 45) and are therefore generally referred to as DEXD/H box NTPases or DEXD/H box proteins. For simplicity, we will refer to these proteins as RNA helicases.
In pre-mRNA splicing, RNA helicases play a pivotal role either by unwinding or weakening RNA duplexes or by altering RNA-protein interactions (41, 45). In addition, they may also function as "RNPases." Active and processive unwinding of RNA duplexes can result in dissociation of RNA-bound proteins, as has been demonstrated in vitro (20). Like pre-mRNA splicing, ribosome biogenesis is a highly dynamic process that involves numerous RNA-RNA and RNA-protein interactions and structural rearrangements. The RNA helicases may mediate or disrupt base-pairing interactions between the large number of snoRNAs and the pre-rRNA or initiate structural rearrangements within the pre-rRNP to facilitate protein binding or dissociation. Thus, it is very likely that they play a central regulatory role in ribosome biogenesis.
A total of 17 RNA helicases have been implicated in ribosome biogenesis, each with a unique function; many of these are conserved in humans (45). Seven of these proteins (Dhr1, Dhr2, Dbp8, Rok1, Fal1, Rrp3, and Dbp4) (4, 6, 21, 24, 27, 47) have been shown to be specifically involved in synthesis of the 18S rRNA and thus the assembly of the SSU. Has1 is the only putative RNA helicase that is required for both SSU and LSU synthesis (11). These proteins belong to the class of DEAD (Dbp8, Rok1, Fal1, Rrp3, and Dbp4) and DEAH (Dhr1 and Dhr2) box RNA helicases and are essential for cell growth. A characteristic feature of these proteins is the presence of a conserved core within the polypeptides, which generally consists of seven to eight motifs (Fig. 1B). Motif I (Walker A), motif II (Walker B; DEXD or DEXH), and motif III (SAT) have been implicated in binding of ATP and the cation cofactor (motifs I and II) and in coupling ATP hydrolysis with the mechanical function (3, 45). The recently discovered Q motif, which appears to be specific for DEAD box-type RNA helicases, has been proposed to be required for ATP binding, ATP hydrolysis, and binding of the RNA substrate (5, 44).
To gain more insight into the role of the conserved motifs, we have performed a comprehensive characterization of almost all the DEXD/H box RNA helicases involved in ribosome biogenesis. Based on previous in vivo and in vitro studies with RNA helicases involved in pre-mRNA splicing, our study focused on generating dominant negative mutations to freeze the biochemical reactions in which these proteins are involved, thereby leading to accumulation of preribosomal complexes. The function of the endogenous helicase is thus inhibited by overexpression of an inhibitory variant of the same protein (18). For example, a mutant enzyme with an inactivated catalytic site retains its ability to bind substrate but can no longer release it, thereby sequestering the limited substrate. Such an approach has been successfully applied to precisely determine the function of many RNA helicases in energy-dependent steps of spliceosome assembly-disassembly. In almost all cases, these proteins were trapped in inactive spliceosomes (2, 10, 19, 26, 31, 39, 40). This strategy has also been adapted to characterize the recently discovered Q motif (44).
Here, we present our results on the RNA helicases involved in SSU biogenesis. We have found that mutations in domains required for ATP binding (motifs I and II) generally caused a dominant negative growth defect, while mutations in the Q motif and motif III did not. For each dominant negative protein, we have determined the effects of the mutations on pre-rRNA processing and found that yeast strains expressing these mutants accumulate pre-rRNA species that under normal conditions are barely detectable. Our results suggest that overexpression of dominant negative helicases stalls processing of pre-rRNA, leading to defects in processing at sites A0, A1, and A2. Since all the tested RNA helicases are essential for growth, we also addressed whether the mutants can support growth. Collectively, our results show that conserved motifs do not function identically in each protein and imply that these RNA helicases may have distinct or diverse biochemical properties. Our results also define functionally important residues in the SSU RNA helicases.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
63
his3-200 leu2-1) was used as the
parental strain to construct the strains described here.
Galactose-inducible, triple hemagglutinin (3HA)-tagged genes and HA-
and tandem affinity purification (TAP) carboxyl-tagged
genes (Kanr marker and Kluyveromyces lactis TRP
marker, respectively) were generated as previously described
(25,
34). Strains with
doxycycline (DOX)-repressible DEXD/H box RNA helicases alleles
(tetO7; Kanr marker) were generated as described
previously (14). Strains
expressing 3HA carboxyl-tagged proteins were grown in YPD (1% yeast
extract, 2% peptone, and 2% dextrose) at 30°C. Strains
expressing 3HA amino-terminal-tagged proteins
(GAL::3HA-DHR1;
GAL::3HA-FAL1)
were grown in YPG/R (1% yeast extract, 2% peptone, 2% galactose, and 2%
raffinose). For the serial dilution experiment (see Fig.
5), 2-µg/ml
doxycycline (Sigma) was used. Strains carrying pYES2 plasmids (pYES2 or
pYES2.1, 2µm URA; Invitrogen) were grown in synthetic
complete medium lacking uracil (-URA; Clontech), supplemented with 2%
dextrose (SD) or with 2% galactose and 2% raffinose
(SG/R).
|
RNA manipulations and immunoprecipitations. For the Northern blot analyses, cells transformed with pYES2 RNA helicase-DNA constructs were grown in SD-URA at 23°C to exponential phase and shifted to SG/R-URA, and growth was monitored for 24 or 48 h. Depending on when the rate of growth changed, RNA was extracted from cells harvested at either 0, 10, or 24 h (Dbp8, Dhr1, and Dhr2) after the shift or 0, 24, or 48 h after the shift (Fal1 and Rrp3). Northern blot analysis of high-molecular-weight RNA species on formaldehyde-1.25% agarose gels was performed as described previously (9). Immunoprecipitations and analysis of coimmunoprecipitated RNAs were performed as described previously (23).
Serial dilutions. To determine whether proteins were dominant negative, YPH499 strains carrying pYES2 RNA helicase plasmids were grown in SD-URA to exponential phase and subsequently shifted to SG/R-URA for 6 h to induce protein expression. Ten-fold dilutions were made and spotted on SD-URA and SG/R-URA solid media. For the complementation experiments, tetO7 strains carrying pYES2 plasmids were grown in SD-URA. Ten-fold dilutions were made and spotted on SD-URA and doxycycline-supplemented SG/R-URA. Plates were incubated at 17°C, 23°C, and 30°C.
Western blot analysis. Mpp10 was detected using a rabbit polyclonal antibody (9). For the Western blot analysis (see Fig. 3B and C), strains harboring pYES2 RNA helicase plasmids were grown in SD-URA to exponential phase. Protein expression was induced for 6 h in SG/R-URA. TAP-tagged proteins were detected with the peroxidase antiperoxidase antibody (PAP; Sigma) using procedures described previously (23).
|
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
To determine the degree of overexpression of the mutated helicases, we compared the helicase protein levels in strains expressing the wild-type TAP-tagged RNA helicases from the pYES2 plasmid to strains expressing the genomically encoded TAP-tagged RNA helicases. Strikingly, expression from the pYES2 plasmid yielded approximately 5- to 50-fold-higher expression levels than expression from the endogenous promoter (Fig. 3A; Genomic versus pYES2). In this exposure, the genomically expressed Rrp3-TAP and Fal1-TAP were not detectable and only became visible in longer exposures (data not shown). We next analyzed the expression levels of the mutant RNA helicases and compared them to the wild-type proteins expressed from the pYES2 plasmids (Fig. 3B). Protein expression was induced for 6 h by growing the cells in galactose-containing medium (SG/R). The results show that, except for Dbp8 Q29L, Dbp8 K52R, and Dhr2 D203A, the expression levels of the mutant RNA helicases were comparable to those of the wild-type proteins, demonstrating that the mutations did not dramatically affect their accumulation in the cell.
While we had wanted to include the SSU helicases Dbp4 and Rok1 in our analyses, we were unable to do so for the following reasons. In contrast to the other helicases, TAP-tagged Dbp4 was expressed only at low levels from the pYES2 plasmid (data not shown) and so was not a good candidate for the generation of dominant negative mutants. Strains expressing a genomically encoded Rok1-TAP protein grew much slower than the parental strain (approximately twofold; data not shown), suggesting that the tag alone impaired Rok1 function. We therefore focused on the other putative RNA helicases required for SSU biogenesis.
RNA helicases carrying mutations in motifs I and II are generally dominant negative. To determine whether the mutations conferred a dominant negative growth defect when overexpressed in the presence of the endogenous protein, we made 10-fold serial dilutions of strains carrying pYES2 plasmids harboring the wild-type or mutant RNA helicases. Yeasts were spotted on galactose-containing medium to express the helicases and on dextrose-containing medium to ascertain equal spotting of cells. Growth was monitored at 17°C, 23°C, and 30°C (Fig. 4 and Table 1). For most RNA helicases, overexpression of motif I and motif II mutants, which we predicted would block ATP binding and/or hydrolysis, caused reduced growth rates, although to varying degrees. This was frequently most prominent at 17°C and indicates that the RNA helicases harboring mutations in ATP binding-hydrolysis motifs are dominant negative in vivo. There were, however, some exceptions including Dhr1 K420R, Dbp8 K52R (at 17°C and 23°C), and Dhr2 D203A. Furthermore, motif I and II mutants in Rrp3 and Fal1 caused only very moderate dominant negative growth defects. However, in general (also based on results discussed below) (Table 1), we conclude that mutations in motifs I and II are dominant negative.
|
|
In summary, our results show that for most of the RNA helicases tested, mutations in motif I and motif II caused dominant negative growth defects, whereas mutations in motif III and the Q motif did not.
Highly conserved residues in motif I and motif II are essential for function; however, substitutions in the Q motif and motif III are not always deleterious. All of the RNA helicases required for SSU biogenesis are essential for viability. To assess the importance of the predicted motifs for function in vivo, we tested whether the mutated proteins could support growth in strains where the endogenous proteins were depleted. For this purpose, the chromosomally encoded genes were placed under the control of the tetracycline-doxycycline-regulatable promoter (tetO7). When these conditional strains transformed with the pYES2 RNA helicase constructs are grown on DOX-supplemented galactose-containing plates, they were dependent on the RNA helicase protein expressed from the pYES2 plasmid. In this way, we could test whether the RNA helicase mutants were required for cell viability and thus whether they were functional in vivo. The results of the complementation assays are shown in Fig. 5 and summarized in Table 1. The first panel on the left in Fig. 5 shows that the addition of DOX to solid medium caused a significantly reduced growth rate in tetO7 conditional strains, although with some variability, demonstrating that the antibiotic causes efficient RNA helicase depletion. As expected, we observed that mutations in RNA helicases that conferred a dominant negative growth defect were generally not functional in vivo. Conversely, a large number of the RNA helicase mutants that were not dominant negative sustained growth as well as their wild-type counterpart and were therefore functional. Exceptions were the Dhr2 D203A and Dhr1 K420R mutants, which either partially (Dhr2 D203A) or completely (Dhr1 K420R) (Table 1) failed to support growth but were not dominant negative.
In contrast, mutations in the Q motif had little effect on growth. Both the glutamine-to-alanine or glutamine-to-leucine mutations in the Q motif supported growth, indicating that an intact Q motif is not essential for function in SSU RNA helicases. However, mutation of the Q in Dbp8 and Rrp3 did cause slight temperature sensitivity (Fig. 5; Table 1), suggesting that it is important to a limited extent. In contrast to previously published work (44), the glutamine-to-leucine mutation in the Fal1 Q motif did not appear dramatically impair its function (Fal1 Q50L) (Fig. 5).
Mutations in motif III had diverse effects on RNA helicase function. Substitutions in motif III (SAT-LAT) affected the function of the DEAH box RNA helicases Dhr1 and Dhr2, while the DEAD box RNA helicase motif III mutants (Dbp8, Rrp3, and Fal1) were capable of supporting growth in depleted cells (Fig. 5; Table 1).
Although we have not tested other substitutions, these results suggest that mutations in highly conserved regions are very often tolerated and that many of these conserved residues are not absolutely essential for function. Furthermore, our results demonstrate that substitutions in motifs I and II generally disrupted SSU RNA helicase function, whereas mutations in the Q motif and motif III were not always deleterious. However, because the mutant proteins were overexpressed in our assay, it is possible that our assay did not allow us to distinguish between fully functional and partially defective enzymes. Nevertheless, considering that the same mutations in different RNA helicases gave different results, we can conclude that not all of the conserved residues are equally important for SSU RNA helicase function in vivo.
Overexpression of dominant negative SSU RNA helicases delays pre-rRNA processing of the 35S pre-rRNA and the synthesis of 18S rRNA. We examined the effect of the dominant negative SSU helicases on pre-rRNA processing. To obtain RNA for analysis, yeast was grown in liquid culture at 23°C, since the growth defect was usually most prominent at low temperatures. At mid-log phase, the cells were shifted to galactose-containing medium to induce expression of the mutant RNA helicases. Growth was monitored for 48 h. In most cases, mutations in helicases that were dominant negative on plates also conferred growth defects in culture, although the effect was sometimes not dramatic (Fig. 6). Yeasts were harvested at several time points after the shift to galactose, depending on when the growth rate changed, and RNA was extracted for Northern blot analysis. For the strains expressing dominant negative mutations that caused a strong growth defect on solid medium (mutations in Dbp8, Dhr1, and Dhr2), we analyzed RNA from cells harvested at 0, 10, and 24 h following the shift to galactose (Fig. 7 and Table 1). For the analysis of strains expressing dominant negative mutants that caused a moderate or weak growth defects on solid medium and in culture (Rrp3 and Fal1), we isolated RNA from cells harvested at 0, 24, and 48 h after the shift (Fig. 7 and Table 1).
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Most of the SSU RNA helicases associate with the SSU processome. Our immunoprecipitation experiments confirmed that almost all of the tested SSU RNA helicases interact with the SSU processome-90S preribosome, as judged by their association with the SSU processome components Mpp10 and U3 snoRNA. Prior to our work, the SSU helicases could be divided into two groups: those that had been frequently copurified with preribosomes in tandem affinity purifications (Dbp8, Dhr1, Dbp4, and Rok1) and those that had been rarely or never detected in preribosomes (Rrp3, Dhr2, and Fal1) (15, 22, 48). Consistent with this, we found that the SSU helicases in the first group all coimmunoprecipitated SSU processome components. Also consistent with this, we found that Fal1 (in the second group) did not coimmunoprecipitate SSU processome components. However, both Rrp3 and Dhr2, which havenever been found in tandem affinity purifications, did coimmunoprecipitate small amounts of SSU processome components. It may be that enzymes such as the SSU RNA helicases transiently interact with preribosomes and thus are not always detectable in purified particles or yield small amounts of coimmunoprecipitating SSU processome components. In addition, while we have found that Dhr2 coimmunoprecipitates SSU processome components Mpp10 and U3 snoRNA, another group found that it did not (4). Consistent with our results, the pattern of pre-rRNAs obtained upon Dhr2 depletion implies a function in the SSU processome-90S preribosome (4).
The DEAD box protein-specific Q motif. It has been proposed that the highly conserved glutamine in the Q motif is required for ATP binding, ATP hydrolysis, and binding of RNA substrate (5, 44). Glutamine-to-alanine or -leucine mutations in the DEAD box proteins eIF4-A, Ded1, Prp5, and Fal1 were previously shown to be lethal, suggesting that this conserved amino acid has an important function in these proteins (44). Therefore, we were surprised that analogous mutations in the Q motifs of the DEAD box SSU RNA helicases Dbp8, Rrp3, and Fal1 did not appear to markedly affect function in vivo. We do not understand why we have obtained different results upon mutation of the Q motif of Fal1 than others have found previously (44), but it is consistent with our overall results. Of the 13 RNA helicases tested in our combined analyses, only the Dbp10, Drs1, and Dbp6 Q motif mutations were lethal. 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.
Elements implicated in ATP binding: motifs I and II. Our results show that the mutations in motifs I and II, which are predicted to affect ATP binding and hydrolysis, were generally dominant negative when expressed in yeast. Moreover, of these, none of the motif I lysine mutants were able to support growth. Likewise, for many RNA helicases required for splicing, analogous mutations in splicing RNA helicases in motif I (Prp16 and Prp22) and motif II (Prp16, Prp22, and Prp43) are lethal and also cause dominant negative growth defects (19, 26, 38). Since a dominant negative effect is a very strong indication that these mutants have retained their ability to bind their RNA and/or RNP substrates, it is tempting to speculate that for most RNA helicases required for ribosome biogenesis, ATP binding is not essential for RNA-RNP substrate binding. Indeed, almost all of the dominant negative splicing factor mutants that have been characterized interact with the spliceosome and are often more stably bound than their wild-type counterparts (10, 31, 36, 39).
Substitutions in highly conserved residues in motif III are frequently tolerated. Previous results suggest that motif III has a role in coupling ATP hydrolysis with helicase activity but is not required for binding ATP. In the prototypical DEAD box protein eIF4-A and the DEAH box protein Prp22, substitution of the serine (SAT) in motif III abrogated RNA helicase activity but did not dramatically affect ATP hydrolysis (30, 40); in the case of Prp2, it also did not interfere with ATP hydrolysis (31). We showed that the serine-to-leucine substitutions in motif III only affected the DEAH box RNA helicases Dhr1 and Dhr2; the latter mutant also conferred a strong dominant negative growth defect when overexpressed (Table 1). Introduction of this mutation in the motif III of DEAH box LSU RNA helicases showed similar results, but the Mtr4 motif III mutant was the only one that conferred a strong dominant negative effect in vivo (1). Thus, it appears that an intact first amino acid in motif III (SAT or TAT for Dbp8) is not essential for function for many of the RNA helicases required for ribosome biogenesis.
The use of dominant negative RNA helicases as tools to study RNA helicase-RNP complex interactions in vivo. One of the goals of this project was to use the dominant negative mutant proteins to purify stalled or intermediate preribosomal complexes because dominant negative mutants are predicted to stably bind their RNA or RNP substrates. However, when we analyzed the association of TAP-tagged wild-type and dominant negative proteins with pre-rRNA precursors, both the wild-type and mutant proteins coimmunoprecipitated many diverse pre-rRNAs (data not shown). After testing different negative controls in the immunoprecipitations, we concluded that the observed interactions were probably related to overexpression of the helicases yielding nonspecific interactions. Expression of SSU RNA helicase mutants from a CEN-based low-copy-number plasmid (pGAD3) did not cause dominant negative growth defects (data not shown), indicating that very high levels of expression are absolutely essential to outcompete the wild-type proteins.
Collectively, our results with 23 mutations in the SSU RNA helicases, with 4 to 5 mutations per helicase, show that lethal mutations in RNA helicases involved in ribosome biogenesis predicted to block ATP binding and/or ATP hydrolysis are very often also dominant negative when overexpressed in yeast. This indicates that ATP binding and hydrolysis are essential for function but not always a prerequisite for RNA or RNP substrate binding. Moreover, mutation of residues that are conserved among all the tested RNA helicases did not have the same effect on every protein, strongly indicating that they do not all share the same biochemical properties. Thus, it is very likely that these RNA helicases have distinct functions in ribosome biogenesis.
|
ACKNOWLEDGMENTS |
|---|
This work was supported by Leslie H. Warner and Anna Fuller Postdoctoral Cancer Research fellowships (S.G.) and the National Institutes of Health (S.J.B.; GM52581).
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Blum, S.,
S. R. Schmid, A. Pause, P. Buser, P. Linder, N. Sonenberg,
and H. Trachsel. 1992. ATP hydrolysis by initiation
factor 4A is required for translation initiation in Saccharomyces
cerevisiae. Proc. Natl. Acad. Sci. USA
89:7664-7668.
2. Campodonico,
E., and B. Schwer. 2002. ATP-dependent remodeling of
the spliceosome: intragenic suppressors of release-defective mutants of
Saccharomyces cerevisiae Prp22. Genetics
160:407-415.
3. Caruthers, J. M., and D. B. McKay. 2002. Helicase structure and mechanism. Curr. Opin. Struct. Biol. 12:123-133.[CrossRef][Medline]
4. Colley,
A., J. D. Beggs, D. Tollervey, and D. L.
Lafontaine. 2000. Dhr1p, a putative DEAH-box RNA
helicase, is associated with the box C+D snoRNP U3. Mol.
Cell. Biol.
20:7238-7246.
5. Cordin, O., N. K. Tanner, M. Doere, P. Linder, and J. Banroques.2004 . The newly discovered Q motif of DEAD-box RNA helicases regulates RNA-binding and helicase activity. EMBO J. 23:2478-2487.[CrossRef][Medline]
6. Daugeron,
M. C., and P. Linder. 2001. Characterization
and mutational analysis of yeast Dbp8p, a putative RNA helicase
involved in ribosome biogenesis. Nucleic Acids Res.
29:1144-1155.
7. de la Cruz, J., D. Kressler, and P. Linder. 1999. Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families. Trends Biochem. Sci. 24:192-198.[CrossRef][Medline]
8. Dragon, F., J. E. Gallagher, P. A. Compagnone-Post, B. M. Mitchell, K. A. Porwancher, K. A. Wehner, S. Wormsley, R. E. Settlage, J. Shabanowitz, Y. Osheim, A. L. Beyer, D. F. Hunt, and S. J. Baserga. 2002. A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis.Nature 417:967-970.[CrossRef][Medline]
9. Dunbar, D. A., S. Wormsley, T. M. Agentis, and S. J. Baserga. 1997. Mpp10p, a U3 small nucleolar ribonucleoprotein component required for pre-18S rRNA processing in yeast. Mol. Cell. Biol. 17:5803-5812.[Abstract]
10. Edwalds-Gilbert, G., D. H. Kim, S. H. Kim, Y. H. Tseng, Y. Yu, and R. J. Lin. 2000. Dominant negative mutants of the yeast splicing factor Prp2 map to a putative cleft region in the helicase domain of DExD/H-box proteins.RNA 6:1106-1119.[Abstract]
11. Emery, B., J. de la Cruz, S. Rocak, O. Deloche, and P. Linder.2004 . Has1p, a member of the DEAD-box family, is required for 40S ribosomal subunit biogenesis in Saccharomyces cerevisiae. Mol. Microbiol. 52:141-158.[CrossRef][Medline]
12. Fatica, A., and D. Tollervey. 2002. Making ribosomes.Curr. Opin. Cell Biol. 14:313-318.[CrossRef][Medline]
13. Fromont-Racine, M., B. Senger, C. Saveanu, and F. Fasiolo. 2003. Ribosome assembly in eukaryotes. Gene 313:17-42.[CrossRef][Medline]
14. Gari, E., L. Piedrafita, M. Aldea, and E. Herrero. 1997. A set of vectors with a tetracycline-regulatable promoter system for modulating gene expression in Saccharomyces cerevisiae.Yeast 13:837-848.[CrossRef][Medline]
15. Gavin, A. C., M. Bosche, R. Krause, P. Grandi, M. Marzioch, A. Bauer, J. Schultz, J. M. Rick, A. M. Michon, C. M. Cruciat, M. Remor, C. Hofert, M. Schelder, M. Brajenovic, H. Ruffner, A. Merino, K. Klein, M. Hudak, D. Dickson, T. Rudi, V. Gnau, A. Bauch, S. Bastuck, B. Huhse, C. Leutwein, M. A. Heurtier, R. R. Copley, A. Edelmann, E. Querfurth, V. Rybin, G. Drewes, M. Raida, T. Bouwmeester, P. Bork, B. Seraphin, B. Kuster, G. Neubauer, and G. Superti-Furga.2002 . Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141-147.[CrossRef][Medline]
16. Grandi, P., V. Rybin, J. Bassler, E. Petfalski, D. Strauss, M. Marzioch, T. Schafer, B. Kuster, H. Tschochner, D. Tollervey, A. C. Gavin, and E. Hurt. 2002. 90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors. Mol. Cell 10:105-115.[CrossRef][Medline]
17. Granneman, S., and S. J. Baserga. 2004. Ribosome biogenesis: of knobs and RNA processing. Exp. Cell Res. 296:43-50.[CrossRef][Medline]
18. Herskowitz, I. 1987. Functional inactivation of genes by dominant negative mutations. Nature 329:219-222.[CrossRef][Medline]
19. Hotz,
H. R., and B. Schwer. 1998. Mutational
analysis of the yeast DEAH-box splicing factor Prp16.Genetics
149:807-815.
20. Jankowsky,
E., C. H. Gross, S. Shuman, and A. M. Pyle.2001
. Active disruption of an RNA-protein interaction by a
DExH/D RNA helicase. Science
291:121-125.
21. Kressler, D., J. de la Cruz, M. Rojo, and P. Linder. 1997. Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 17:7283-7294.[Abstract]
22. Krogan, N. J., W. T. Peng, G. Cagney, M. D. Robinson, R. Haw, G. Zhong, X. Guo, X. Zhang, V. Canadien, D. P. Richards, B. K. Beattie, A. Lalev, W. Zhang, A. P. Davierwala, S. Mnaimneh, A. Starostine, A. P. Tikuisis, J. Grigull, N. Datta, J. E. Bray, T. R. Hughes, A. Emili, and J. F. Greenblatt. 2004. High-definition macromolecular composition of yeast RNA-processing complexes. Mol. Cell 13:225-239.[CrossRef][Medline]
23. Lee,
S. J., and S. J. Baserga. 1999.
Imp3p and Imp4p, two specific components of the U3 small nucleolar
ribonucleoprotein that are essential for pre-18S rRNA processing.Mol. Cell. Biol.
19:5441-5452.
24. Liang, W. Q., J. A. Clark, and M. J. Fournier. 1997. The rRNA-processing function of the yeast U14 small nucleolar RNA can be rescued by a conserved RNA helicase-like protein. Mol. Cell. Biol. 17:4124-4132.[Abstract]
25. Longtine, M. S., A. McKenzie, D. J. Demarini, N. G. Shah, A. Wach, A. Brachat, P. Philippsen, and J. R. Pringle. 1998. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14:953-961.[CrossRef][Medline]
26. Martin,
A., S. Schneider, and B. Schwer. 2002. Prp43 is an
essential RNA-dependent ATPase required for release of lariat-intron
from the spliceosome. J. Biol. Chem.
277:17743-17750.
27. O'Day,
C. L., F. Chavanikamannil, and J. Abelson.1996
. 18S rRNA processing requires the RNA helicase-like
protein Rrp3. Nucleic Acids Res.
24:3201-3207.
28. Oh,
J. Y., and J. Kim. 1999. ATP hydrolysis
activity of the DEAD box protein Rok1p is required for in vivo ROK1
function. Nucleic Acids Res.
27:2753-2759.
29. Pause,
A., N. Methot, and N. Sonenberg. 1993. The HRIGRXXR
region of the DEAD box RNA helicase eukaryotic translation initiation
factor 4A is required for RNA binding and ATP hydrolysis. Mol.
Cell. Biol.
13:6789-6798.
30. Pause, A., and N. Sonenberg. 1992. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 11:2643-2654.[Medline]
31. Plumpton, M., M. McGarvey, and J. D. Beggs. 1994. A dominant negative mutation in the conserved RNA helicase motif ‘SAT’ causes splicing factor PRP2 to stall in spliceosomes. EMBO J. 13:879-887.[Medline]
32. Raue, H. A. 2004. Pre-ribosomal RNA processing and assembly in Saccharomyces cerevisiae, p.199 -222. In M. O. Olson (ed.), The nucleolus. Kluwer Academic/Plenum Publishers, New York, N.Y.
33. Rehrauer,
W. M., and S. C. Kowalczykowski.1993
. Alteration of the nucleoside triphosphate (NTP)
catalytic domain within Escherichia coli recA protein
attenuates NTP hydrolysis but not joint molecule formation.J. Biol. Chem.
268:1292-1297.
34. Rigaut, G., A. Shevchenko, B. Rutz, M. Wilm, M. Mann, and B. Séraphin. 1999. A generic protein purification method for protein complex characterization and proteome exploration.Nat. Biotechnol. 17:1030-1032.[CrossRef][Medline]
35. Schmid,
S. R., and P. Linder. 1991. Translation
initiation factor 4A from Saccharomyces cerevisiae: analysis
of residues conserved in the D-E-A-D family of RNA helicases.Mol. Cell. Biol.
11:3463-3471.
36. Schneider,
S., H. R. Hotz, and B. Schwer. 2002.
Characterization of dominant-negative mutants of the DEAH-box
splicing factors Prp22 and Prp16. J. Biol.
Chem.
277:15452-15458.
37. Schneider,
S., and B. Schwer. 2001. Functional domains of the
yeast splicing factor Prp22p. J. Biol. Chem.
276:21184-21191.
38. Schwer, B., and C. H. Gross. 1998. Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing.EMBO J. 17:2086-2094.[CrossRef][Medline]
39. Schwer,
B., and C. Guthrie. 1992. A dominant negative mutation
in a spliceosomal ATPase affects ATP hydrolysis but not
binding to the spliceosome. Mol. Cell. Biol.
12:3540-3547.
40. Schwer, B., and T. Meszaros. 2000. RNA helicase dynamics in pre-mRNA splicing. EMBO J. 19:6582-6591.[CrossRef][Medline]
41. Schwer, B. 2001. A new twist on RNA helicases: DExH/D box proteins as RNPases. Nat. Struct. Biol. 8:113-116.[CrossRef][Medline]
42. Sung, P., D. Higgins, L. Prakash, and S. Prakash. 1988. Mutation of lysine-48 to arginine in the yeast RAD3 protein abolishes its ATPase and DNA helicase activities but not the ability to bind ATP. EMBO J. 7:3263-3269.[Medline]
43. Sung,
P., and S. A. Stratton. 1996. Yeast Rad51
recombinase mediates polar DNA strand exchange in the absence of ATP
hydrolysis. J. Biol. Chem.
271:27983-27986.
44. Tanner, K. N., O. Cordin, J. Banroques, M. Doere, and P. Linder.2003 . The Q motif: a newly identified motif in DEAD box helicases may regulate ATP binding and hydrolysis. Mol. Cell 11:127-138.[CrossRef][Medline]
45. Tanner, N. K., and P. Linder. 2001. DExD/H box RNA helicases: from generic motors to specific dissociation functions.Mol. Cell 8:251-262.[CrossRef][Medline]
46. Tschochner, H., and E. Hurt. 2003. Pre-ribosomes on the road from the nucleolus to the cytoplasm. Trends Cell Biol. 13:255-263.[CrossRef][Medline]
47. Venema, J., C. Bousquet-Antonelli, J. P. Gelugne, M. Caizergues-Ferrer, and D. Tollervey. 1997. Rok1p is a putative RNA helicase required for rRNA processing. Mol. Cell. Biol. 17:3398-3407.[Abstract]
48. Vos,
H. R., R. Bax, A. W. Faber, J. C. Vos,
and H. A. Raue. 2004. U3 snoRNP and Rrp5p
associate independently with Saccharomyces cerevisiae 35S
pre-rRNA, but Rrp5p is essential for association of Rok1p.Nucleic Acids Res.
32:5827-5833.
49. Wehner, K. A., and S. J. Baserga. 2002. The sigma 70-like motif: a eukaryotic RNA binding domain unique to a superfamily of proteins required for ribosome biogenesis. Mol. Cell 9:329-339.[CrossRef][Medline]
50. Zhang,
M., and M. R. Green. 2001. Identification
and characterization of yUAP/Sub2p, a yeast homolog of the essential
human pre-mRNA splicing factor hUAP56. Genes Dev.
15:30-35.
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
