Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

Kiparisov, Sergey V.; Petrov, Alexey; Meškauskas, Arturas; Сергиев, Петр В.; Dontsova, Olga А.; Dinman, Jonathan D.

doi:10.1007/s00438-005-0020-9

Cited by 41 publications

(40 citation statements)

References 67 publications

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“…To choose among the numerous allelic variants of human 5S rRNAs reported to date (Kiparisov et al 2005), we performed RT-PCR and sequenced 5S rRNAs isolated from HeLa cells or from purified mitochondria. In both cases, the same 5S rRNA sequence was found to be the most abundant and was thus chosen for further experiments ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Second, 5S rRNA seems to be one of the most interesting RNA species from the point of view of its intracellular traffic, which implicates a network of possible itineraries for various subcellular localizations. Being an integral part of large ribosomal subunit of almost all living organisms, it appears to perform a very important function in the translation process, as recent evidence suggests Kiparisov et al 2005;Kouvela et al 2007). At the same time, it was shown that, at least in Xenopus laevis oocytes, to integrate a ribosome, a 5S rRNA molecule has to be first exported from the nucleus by the transcription factor IIIA (TFIIIA) (Pelham and Brown 1980;Guddat et al 1990) and reimported into the nucleolus with the ribosomal protein L5; the 5S rRNA-L5 complex is then involved in the central protuberance formation (Steitz et al 1988;Rudt and Pieler 1996).…”

Section: Introductionmentioning

confidence: 99%

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Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria

Smirnov¹,

Tarassov²,

Mager-Heckel³

et al. 2008

RNA

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RNA import into mitochondria is a widespread phenomenon. Studied in details for yeast, protists, and plants, it still awaits thorough investigation for human cells, in which the nuclear DNA-encoded 5S rRNA is imported. Only the general requirements for this pathway have been described, whereas specific protein factors needed for 5S rRNA delivery into mitochondria and its structural determinants of import remain unknown. In this study, a systematic analysis of the possible role of human 5S rRNA structural elements in import was performed. Our experiments in vitro and in vivo show that two distinct regions of the human 5S rRNA molecule are needed for its mitochondrial targeting. One of them is located in the proximal part of the helix I and contains a conserved uncompensated G:U pair. The second and most important one is associated with the loop E-helix IV region with several noncanonical structural features. Destruction or even destabilization of these sites leads to a significant decrease of the 5S rRNA import efficiency. On the contrary, the b-domain of the 5S rRNA was proven to be dispensable for import, and thus it can be deleted or substituted without affecting the 5S rRNA importability. This finding was used to demonstrate that the 5S rRNA can function as a vector for delivering heterologous RNA sequences into human mitochondria. 5S rRNA-based vectors containing a substitution of a part of the b-domain by a foreign RNA sequence were shown to be much more efficiently imported in vivo than the wild-type 5S rRNA.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria

Smirnov¹,

Tarassov²,

Mager-Heckel³

et al. 2008

RNA

View full text Add to dashboard Cite

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“…Purified ribosomes were treated with 1 mM puromycin and 1 mM GTP for 30 minutes at 37°C to remove aa-tRNAs and elongation factors. Treated ribosomes were then incubated with DMS as previously described (25). RNAs isolated from DMS-treated and untreated cells and ribosomes were annealed with 32 P-end-labeled primers (Table 2) by incubation at 70°C followed by slow cooling and holding for 5 min to 40°C followed by immediate transfer to ice.…”

Section: Methodsmentioning

confidence: 99%

“…Inset graphs indicate the fold changes in doubling times of the mutants in the presence of increasing drug concentrations normalized to that of wild-type cells under the same conditions. Sparsomycin lethal dose (g/ml) 25 …”

Section: Specific Repression Of ؊1 But Not ؉1 Prfmentioning

confidence: 99%

Specific Effects of Ribosome-Tethered Molecular Chaperones on Programmed −1 Ribosomal Frameshifting

Muldoon-Jacobs

Dinman

2006

Eukaryot Cell

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The ribosome-associated molecular chaperone complexes RAC (Ssz1p/Zuo1p) and Ssb1p/Ssb2p expose a link between protein folding and translation. Disruption of the conserved nascent peptide-associated complex results in cell growth and translation fidelity defects. To better understand the consequences of deletion of either RAC or Ssb1p/2p, experiments relating to cell growth and programmed ribosomal frameshifting (PRF) were assayed. Genetic analyses revealed that deletion of Ssb1p/Ssb2p or of Ssz1p/Zuo1p resulted in specific inhibition of ؊1 PRF and defects in Killer virus maintenance, while no effects were observed on ؉1 PRF. These factors may provide a new set of targets to exploit against viruses that use ؊1 PRF. Quantitative measurements of growth profiles of isogenic wild-type and mutant cells showed that translational inhibitors exacerbate underlying growth defects in these mutants. Previous studies have identified ؊1 PRF signals in yeast chromosomal genes and have demonstrated an inverse relationship between ؊1 PRF efficiency and mRNA stability. Analysis of published DNA microarray experiments reveals conditions under which Ssb1, Ssb2, Ssz1, and Zuo1 transcript levels are regulated independently of those of genes encoding ribosomal proteins. Thus, the findings presented here suggest that these trans-acting factors could be used by cells to posttranscriptionally regulate gene expression through ؊1 PRF.Programmed ribosomal frameshifting (PRF) is a posttranscriptional regulatory mechanism in which elongating ribosomes are directed to shift the reading frame in response to specific cis-acting signals in mRNAs. Many viruses, including human immunodeficiency virus type 1, use PRF to optimize the stoichiometric ratios between their structural and enzymatic proteins (5, 11). Changes in PRF efficiency alter those ratios, inhibiting virion morphogenesis (9). In particular, retroviruses appear to be very susceptible to such changes (4, 21, 33, 37). Thus, elucidation of the molecular mechanisms underlying PRF can aid in the rational design of antiviral therapeutics (reviewed in reference 8). Though first discovered in viruses, it is also becoming apparent that programmed Ϫ1 ribosomal frameshifting (Ϫ1 PRF) is also used to regulate expression of cellular genes (reviewed in reference 34). Given the widespread use of PRF, it is important to understand the molecular mechanisms underlying PRF and to identify cellular factors that might be used to regulate these processes.Ribosomes can be directed to shift by one base in either the 5Ј or 3Ј direction depending on the cis-acting signal. Programmed Ϫ1 ribosomal frameshifting is the result of a net shift of the translational reading frame by 1 base in the 5Ј direction. The shift is typically directed by a tripartite cis-acting mRNA element composed of a (from 5Ј to 3Ј) heptameric slippery site, a spacer, and a thermodynamically stable structure, typically an mRNA pseudoknot (reviewed in reference 5). It is generally accepted that Ϫ1 PRF requires a change in the forward kinetics...

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“…sap., H. sapiens. (C) The S. cerevisiae cytoplasmic 5S rRNA secondary structure (15,53) showing the position of the ⌿.…”

Section: Resultsmentioning

confidence: 99%

Different Mechanisms for Pseudouridine Formation in Yeast 5S and 5.8S rRNAs

Decatur

Schnare

2008

Molecular and Cellular Biology

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The selection of sites for pseudouridylation in eukaryotic cytoplasmic rRNA occurs by the base pairing of the rRNA with specific guide sequences within the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines (⌿s) in the cytoplasmic rRNA of Saccharomyces cerevisiae have been assigned to guide snoRNAs. Here, we examine the mechanism of ⌿ formation in 5S and 5.8S rRNA in which the unassigned ⌿s occur. We show that while the formation of the ⌿ in 5.8S rRNA is associated with snoRNP activity, the pseudouridylation of 5S rRNA is not. The position of the ⌿ in 5.8S rRNA is guided by snoRNA snR43 by using conserved sequence elements that also function to guide pseudouridylation elsewhere in the large-subunit rRNA; an internal stem-loop that is not part of typical yeast snoRNAs also is conserved in snR43. The multisubstrate synthase Pus7 catalyzes the formation of the ⌿ in 5S rRNA at a site that conforms to the 7-nucleotide consensus sequence present in other substrates of Pus7. The different mechanisms involved in 5S and 5.8S rRNA pseudouridylation, as well as the multiple specificities of the individual trans factors concerned, suggest possible roles in linking ribosome production to other processes, such as splicing and tRNA synthesis.RNA harbors a large array of structurally diverse, modified nucleosides (33, 93). Pseudouridine (5-ribosyluracil; ⌿) was the first discovered and is the most abundant (for a review, see reference 16). The isomerization of U to ⌿ involves the recognition of the specific U followed by the cleavage of the N-glycosidic bond, the rotation of the base, and the formation of a C-glycosidic bond (82). At the level of local RNA structure, ⌿ contributes to greater stability relative to that of U by providing an additional hydrogen-bonding capability that allows a water-mediated bridge to form between the base and the sugar-phosphate backbone as well as improved base stacking (for a review, see reference 16).In eukaryotes, ⌿ is found at specific sites in the rRNAs, tRNAs, and snRNAs. The cytoplasmic rRNA precursor (prerRNA) of eukaryotes is abundantly modified. As a result, in addition to about 54 ribose-methylated and 10 base-methylated nucleosides (61,89,108), 46 ⌿s are reported for the mature cytoplasmic rRNA of Saccharomyces cerevisiae (6,7,65,83,85). The pseudouridylation sites in eukaryotic, cytoplasmic rRNA are selected by the base pairing of the cytoplasmic rRNA with specific guide sequences present in the box H/ACA family of small nucleolar RNAs (snoRNAs) (for reviews, see references 10, 21, 54, and 114). These guide RNAs are associated with protein and function as ribonucleoprotein (RNP) particles, termed small nucleolar RNPs (snoRNPs).

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Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

Cited by 41 publications

References 67 publications

Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria

Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria

Specific Effects of Ribosome-Tethered Molecular Chaperones on Programmed −1 Ribosomal Frameshifting

Different Mechanisms for Pseudouridine Formation in Yeast 5S and 5.8S rRNAs

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