Molecular Requirements for Duplex Recognition and Cleavage by Eukaryotic RNase III: Discovery of an RNA-dependent DNA Cleavage Activity of Yeast Rnt1p

Lamontagne, Bruno; Hannoush, Rami N.; Damha, Masad J.; Elela, Sherif Abou

doi:10.1016/j.jmb.2004.02.059

Cited by 23 publications

(30 citation statements)

References 53 publications

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“…Cleavage reactions were performed essentially as described previously (28) using either 0.2 pmol of purified Rnt1p (26) or total cell extracts (29). For the in vitro cleavage assay, 3 fmol of internally labeled RNA was incubated in the presence of 0.2 pmol of Rnt1p for 20 min at 30°C in 20 l of reaction buffer (30 mM Tris [pH 7.5], 5 mM spermidine, 10 mM MgCl 2 , 0.1 mM dithiothreitol, 0.1 mM EDTA [pH 7.5]).…”

Section: Methodsmentioning

confidence: 99%

Genome-Wide Prediction and Analysis of Yeast RNase III-Dependent snoRNA Processing Signals

Ghazal

Gervais-Bird

et al. 2005

Molecular and Cellular Biology

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In Saccharomyces cerevisiae, the maturation of both pre-rRNA and pre-small nucleolar RNAs (pre-snoRNAs) involves common factors, thereby providing a potential mechanism for the coregulation of snoRNA and rRNA synthesis. In this study, we examined the global impact of the double-stranded-RNA-specific RNase Rnt1p, which is required for pre-rRNA processing, on the maturation of all known snoRNAs. In silico searches for Rnt1p cleavage signals, and genome-wide analysis of the Rnt1p-dependent expression profile, identified seven new Rnt1p substrates. Interestingly, two of the newly identified Rnt1p-dependent snoRNAs, snR39 and snR59, are located in the introns of the ribosomal protein genes RPL7A and RPL7B. In vitro and in vivo experiments indicated that snR39 is normally processed from the lariat of RPL7A, suggesting that the expressions of RPL7A and snR39 are linked. In contrast, snR59 is produced by a direct cleavage of the RPL7B pre-mRNA, indicating that a single pre-mRNA transcript cannot be spliced to produce a mature RPL7B mRNA and processed by Rnt1p to produce a mature snR59 simultaneously. The results presented here reveal a new role of yeast RNase III in the processing of intron-encoded snoRNAs that permits independent regulation of the host mRNA and its associated snoRNA.Bacterial pre-rRNA processing is carried out by a defined set of nucleases (3-5, 43, 52). Key among this set is RNase III, initially isolated by its ability to bind and cleave duplex RNA (47, 48). RNase III generates the immediate precursors to the mature 16S and 23S rRNAs from the primary transcripts by cleaving within two extended RNA duplexes formed by longrange interactions that pair the termini of each rRNA (7, 63). These long-range interactions provide a simple method of coordinating the processing events at both ends of the transcript. In eukaryotes, pre-rRNA processing is more complex and requires many more small nucleolar RNAs (snoRNAs) and protein components with overlapping functions (13,15,16,41,46). For example, the removal of the 5Ј external-transcribed spacer requires 4 snoRNAs (U3, snR30, U14, and snR10) and about 64 snoRNAs are required for rRNA modifications (24, 57). snoRNAs are divided into two major subclasses: the first includes box C/D snoRNAs that function mostly as a guide for the methylation of rRNA (6, 20, 21, 55), while the second includes H/ACA snoRNAs that guide RNA pseudouridine formation (25,39,59). Most mammalian snoRNAs are encoded within intron sequences and are processed from either unspliced precursors or lariat species (18,19,64). In Saccharomyces cerevisiae, most snoRNAs are transcribed either as independent units or as a part of polycistronic transcript, while only 7 of the 66 known snoRNAs are located in the introns of mRNAs (14,44,53). Several polycistronic snoRNAs, and a few monocistronic ones, are processed by Rnt1p, the orthologue of the bacterial RNase III (30), which is also required for the processing of the pre-rRNA's 3Ј end (2,10,11,23,33). Following processing by Rnt1p, the RNAs are trimmed...

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Section: Methodsmentioning

confidence: 99%

Genome-Wide Prediction and Analysis of Yeast RNase III-Dependent snoRNA Processing Signals

Ghazal

Gervais-Bird

et al. 2005

Molecular and Cellular Biology

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“…To test this possibility, we monitored the impact on the expression of the telomerase subunits of a point mutation in RNT1 (D247R), which leads to an impaired Rnt1p RNA-cleavage activity without affecting other functions (45,46). Total RNA was extracted from wild type, rnt1⌬, or rnt1-D247R cells and analyzed using Northern blots.…”

Section: Figure 3 Rnt1p Catalytic Activity Is Required For the Normamentioning

confidence: 99%

RNase III-dependent Regulation of Yeast Telomerase

Larose

Laterreur

Ghazal

et al. 2007

Journal of Biological Chemistry

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In bakers' yeast, in vivo telomerase activity requires a ribonucleoprotein (RNP) complex with at least four associated proteins (Est2p, Est1p, Est3p, and Cdc13p) and one RNA species (Tlc1). The function of telomerase in maintaining chromosome ends, called telomeres, is tightly regulated and linked to the cell cycle. However, the mechanisms that regulate the expression of individual components of telomerase are poorly understood. Here we report that yeast RNase III (Rnt1p), a double-stranded RNA-specific endoribonuclease, regulates the expression of telomerase subunits and is required for maintaining normal telomere length. Deletion or inactivation of RNT1 induced the expression of Est1, Est2, Est3, and Tlc1 RNAs and increased telomerase activity, leading to elongation of telomeric repeat tracts. In silico analysis of the different RNAs coding for the telomerase subunits revealed a canonical Rnt1p cleavage site near the 3 end of Est1 mRNA. This predicted structure was cleaved by Rnt1p and its disruption abolished cleavage in vitro. Mutation of the Rnt1p cleavage signal in vivo impaired the cell cycle-dependent degradation of Est1 mRNA without affecting its steady-state level. These results reveal a new mechanism that influences telomeres length by controlling the expression of the telomerase subunits.The ends of eukaryotic chromosomes are capped with special structures made of tandem DNA repeats and associated proteins, called telomeres. These structures protect chromosomes from end-to-end fusion, recombination, and nucleolytic degradation (1, 2). However, because the conventional DNA replication machinery cannot fully duplicate the ends of linear chromosomes, telomeric DNA will shorten with each round of replication, leading to a replicative senescence (3). To solve this end replication problem, most eukaryotes use the activity of an enzyme called telomerase to ensure the maintenance of telomeric DNA (4). Telomerase is a ribonucleoprotein reverse transcriptase that can extend the 3Ј end of chromosomes using its RNA subunit as a template for the addition of telomeric repeats.In vitro, telomerase activity requires a reverse transcriptase (Tert, Est2p in yeast) and an RNA template (Terc, Tlc1 in yeast)

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“…18,36 In order to understand how A1-substrates are selected for cleavage, we compared the A1-substrates pattern of Rnt1p-dependent protection from hydroxyl radicals generated by Fe (II)-EDTA 32 to that exhibited by G2-substrates. As shown in Figure 4(a), R48 tet-NGNN exhibited a strong protection in the third and fourth nucleotide of the loop and the first four nucleotides of the stem near the loop 3′ end.…”

Section: Rnt1p Binds To G2 and A1-loops Using Different Sets Of Nuclementioning

confidence: 99%

Characterization of the Reactivity Determinants of a Novel Hairpin Substrate of Yeast RNase III

Ghazal

Elela

2006

Journal of Molecular Biology

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Molecular Requirements for Duplex Recognition and Cleavage by Eukaryotic RNase III: Discovery of an RNA-dependent DNA Cleavage Activity of Yeast Rnt1p

Cited by 23 publications

References 53 publications

Genome-Wide Prediction and Analysis of Yeast RNase III-Dependent snoRNA Processing Signals

Genome-Wide Prediction and Analysis of Yeast RNase III-Dependent snoRNA Processing Signals

RNase III-dependent Regulation of Yeast Telomerase

Characterization of the Reactivity Determinants of a Novel Hairpin Substrate of Yeast RNase III

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