Dbp3p, a putative RNA helicase in Saccharomyces cerevisiae, is required for efficient pre-rRNA processing predominantly at site A3.

In Saccharomyces cerevisiae, ribosomal biogenesis takes place primarily in the nucleolus, in which a single 35S precursor rRNA (pre-rRNA) is first transcribed and sequentially processed into 25S, 5.8S, and 18S mature rRNAs, leading to the formation of the 40S and 60S ribosomal subunits. Although many components involved in this process have been identified, our understanding of this important cellular process remains limited. Here we report that one of the evolutionarily conserved DEAD-box protein genes in yeast, DBP3, is required for optimal ribosomal biogenesis. DBP3 encodes a putative RNA helicase, Dbp3p, of 523 amino acids in length, which bears a highly charged amino terminus consisting of 10 tandem lysine-lysine-X repeats ([KKX] repeats). Disruption of DBP3 is not lethal but yields a slow-growth phenotype. This genetic depletion of Dbp3p results in a deficiency of 60S ribosomal subunits and a delayed synthesis of the mature 25S rRNA, which is caused by a prominent kinetic delay in pre-rRNA processing at site A3 and to a lesser extent at sites A2 and A0. These data suggest that Dbp3p may directly or indirectly facilitate RNase MRP cleavage at site A3. The direct involvement of Dbp3p in ribosomal biogenesis is supported by the finding that Dbp3p is localized predominantly in the nucleolus. In addition, we show that the [KKX] repeats are dispensable for Dbp3p's function in ribosomal biogenesis but are required for its proper localization. The [KKX] repeats thus represent a novel signaling motif for nuclear localization and/or retention.

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Dbp6p Is an Essential Putative ATP-Dependent RNA Helicase Required for 60S-Ribosomal-Subunit Assembly inSaccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.18.4.1855 ◽

1998 ◽

Vol 18 (4) ◽

pp. 1855-1865 ◽

Cited By ~ 58

Author(s):

Dieter Kressler ◽

Jesús de la Cruz ◽

Manuel Rojo ◽

Patrick Linder

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Subunit ◽

Rna Helicase ◽

Open Reading Frame ◽

Northern Hybridization ◽

Ribosomal Subunits ◽

Steady State Analysis ◽

Reading Frame ◽

Dead Box

A previously uncharacterizedSaccharomyces cerevisiaeopen reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore namedDBP6(DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.

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Correction for The DEAD-Box RNA Helicase Ded1p Affects and Accumulates in Saccharomyces cerevisiae P-Bodies

Molecular Biology of the Cell ◽

10.1091/mboc.23.14.2818 ◽

2012 ◽

Vol 23 (14) ◽

pp. 2818-2818 ◽

Cited By ~ 2

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Helicase ◽

P Bodies ◽

Dead Box ◽

The Dead

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Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

RNA ◽

10.1017/s1355838298981158 ◽

1998 ◽

Vol 4 (10) ◽

pp. 1268-1281 ◽

Cited By ~ 50

Author(s):

JESÚS DE LA CRUZ ◽

DIETER KRESSLER ◽

MANUEL ROJO ◽

DAVID TOLLERVEY ◽

PATRICK LINDER

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Helicase ◽

Ribosomal Subunits ◽

Late Step

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Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.7283 ◽

1997 ◽

Vol 17 (12) ◽

pp. 7283-7294 ◽

Cited By ~ 108

Author(s):

D Kressler ◽

J de la Cruz ◽

M Rojo ◽

P Linder

Keyword(s):

Saccharomyces Cerevisiae ◽

Translation Initiation ◽

18S Rrna ◽

Amino Acid Level ◽

Ribosomal Subunit ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Northern Hybridization ◽

Ribosomal Subunits ◽

Dead Box

A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation.

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The DEAD-Box RNA Helicase AtRH7/PRH75 Participates in Pre-rRNA Processing, Plant Development and Cold Tolerance in Arabidopsis

Plant and Cell Physiology ◽

10.1093/pcp/pcv188 ◽

2015 ◽

Vol 57 (1) ◽

pp. 174-191 ◽

Cited By ~ 21

Author(s):

Chun-Kai Huang ◽

Yu-Lien Shen ◽

Li-Fen Huang ◽

Shaw-Jye Wu ◽

Chin-Hui Yeh ◽

...

Keyword(s):

Cold Tolerance ◽

Plant Development ◽

Rna Helicase ◽

Processing Plant ◽

Rrna Processing ◽

Dead Box ◽

The Dead

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The DEAD-box RNA helicase, Dhh1, functions in mating by regulating Ste12 translation in Saccharomyces cerevisiae

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2007.12.169 ◽

2008 ◽

Vol 367 (3) ◽

pp. 680-686 ◽

Cited By ~ 16

Author(s):

Minhan Ka ◽

Young-Un Park ◽

Jinmi Kim

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Helicase ◽

Dead Box ◽

The Dead

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The rRNA-processing function of the yeast U14 small nucleolar RNA can be rescued by a conserved RNA helicase-like protein.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.4124 ◽

1997 ◽

Vol 17 (7) ◽

pp. 4124-4132 ◽

Cited By ~ 44

Author(s):

W Q Liang ◽

J A Clark ◽

M J Fournier

Keyword(s):

Mutational Analysis ◽

Rna Helicase ◽

Suppressor Gene ◽

Rrna Processing ◽

Base Pairing ◽

Small Nucleolar Rna ◽

Stem Loop ◽

Dead Box ◽

Extragenic Suppressor ◽

Nucleolar Rna

The phylogenetically conserved U14 small nucleolar RNA is required for processing of rRNA, and this function involves base pairing with conserved complementary sequences in 18S RNA. With a view to identifying other important U14 interactions, a stem-loop domain required for activity of Saccharomyces cerevisiae U14 RNAs (the Y domain) was first subjected to detailed mutational analysis. The mapping results showed that most nucleotides of the Y domain can be replaced without affecting function, except for loop nucleotides conserved among five different yeast species. Defective variants were then used to identify both intragenic and extragenic suppressor mutations. All of the intragenic mutations mapped within six nucleotides of the primary mutation, suggesting that suppression involves a change in conformation and that the loop element is involved in an essential intermolecular interaction rather than intramolecular base pairing. A high-copy extragenic suppressor gene, designated DBP4 (DEAD box protein 4), encodes an essential, putative RNA helicase of the DEAD-DEXH box family. Suppression by DBP4 (initially CA4 [T.-H. Chang, J. Arenas, and J. Abelson, Proc. Natl. Acad. Sci. USA 87:1571-1575, 1990]) restores the level of 18S rRNA and is specific for the Y domain but is not allele specific. DBP4 is predicted to function either in assembly of the U14 small nucleolar RNP or, more likely, in its interaction with other components of the rRNA processing apparatus. Mediating the interaction of U14 with precursor 18S RNA is an especially attractive possibility.

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Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7935 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7935-7941 ◽

Cited By ~ 157

Author(s):

M E Schmitt ◽

D A Clayton

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Yeast Cells ◽

Rrna Processing ◽

Rnase Mrp ◽

Gal1 Promoter ◽

5.8S Rrna ◽

Late Step ◽

Conditional Mutations ◽

A Site

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype.Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.

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Distinct RNA-unwinding mechanisms of DEAD-box and DEAH-box RNA helicase proteins in remodeling structured RNAs and RNPs

Biochemical Society Transactions ◽

10.1042/bst20170095 ◽

2017 ◽

Vol 45 (6) ◽

pp. 1313-1321 ◽

Cited By ~ 27

Author(s):

Benjamin Gilman ◽

Pilar Tijerina ◽

Rick Russell

Keyword(s):

Conformational Changes ◽

Protein Complexes ◽

Rna Helicase ◽

Conformational Transitions ◽

Messenger Rnas ◽

Base Pairs ◽

Dead Box ◽

Evolutionarily Conserved ◽

Partially Unfolded ◽

Rna Protein Complexes

Structured RNAs and RNA–protein complexes (RNPs) fold through complex pathways that are replete with misfolded traps, and many RNAs and RNPs undergo extensive conformational changes during their functional cycles. These folding steps and conformational transitions are frequently promoted by RNA chaperone proteins, notably by superfamily 2 (SF2) RNA helicase proteins. The two largest families of SF2 helicases, DEAD-box and DEAH-box proteins, share evolutionarily conserved helicase cores, but unwind RNA helices through distinct mechanisms. Recent studies have advanced our understanding of how their distinct mechanisms enable DEAD-box proteins to disrupt RNA base pairs on the surfaces of structured RNAs and RNPs, while some DEAH-box proteins are adept at disrupting base pairs in the interior of RNPs. Proteins from these families use these mechanisms to chaperone folding and promote rearrangements of structured RNAs and RNPs, including the spliceosome, and may use related mechanisms to maintain cellular messenger RNAs in unfolded or partially unfolded conformations.

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