Functional defects in transfer RNAs lead to the accumulation of ribosomal RNA precursors

Slagter-Jäger, Jacoba G.; Puzis, Leopold; Gutgsell, Nancy S.; Belfort, Marlene; Jain, Chaitanya

doi:10.1261/rna.319407

Cited by 9 publications

(8 citation statements)

References 40 publications

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“…The cleaved RNA was fractionated on a 6% polyacrylamide-8 M urea sequencing gel, transferred to a positively charged nylon membrane (Nytran; Whatman Inc.), and probed with a radiolabeled oligonucleotide (5Ј-AAGGTT AAGCCTCACGGTTC-3Ј) complementary to the 3Ј cleavage product. Primer extension analysis to assess 5Ј-end maturation of 23S rRNA was carried out using a labeled specific oligonucleotide primer, as described previously (18).…”

Section: Methodsmentioning

confidence: 99%

Coordinated Regulation of 23S rRNA Maturation in Escherichia coli

Gutgsell

Jain

2010

J Bacteriol

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In Escherichia coli, rRNAs are transcribed as precursors and require processing at the 3 and 5 ends to generate mature RNA molecules. The largest of these RNAs, 23S rRNA, is matured at the 3 end by a set of exonucleases and at the 5 end by an unknown RNase. Whether the 3 and 5 maturation steps occur independently or are coupled has previously been unclear. By assessing the levels of precursors accumulating at the 3 and 5 ends, we provide evidence that these processes may be linked. Thus, each of several conditions that led to precursor accumulation at one end also did so at the other end. We also observed that each end undergoes maturation at similar rates, suggesting that the two processes could be coupled. Finally, we provide evidence that processing at the 3 end facilitates 5-end maturation. A model to explain the basis for the observed directionality of the reactions is proposed. This information will aid in the search for the enzyme responsible for final maturation of the 5 end of 23S rRNA.Many RNAs are transcribed as precursors and require processing for maturation and optimum functionality. This is especially true for rRNAs, and several model organisms have been studied extensively to define the steps involved in rRNA processing, including Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae (5,6,9,20). In E. coli, each of the three rRNAs is generated from a single rRNA operon transcript (6). The transcript is first cleaved at two sites by RNase III, generating precursors for 5S, 16S, and 23S rRNAs (16). For 5S rRNA, further processing steps involve endonucleolytic cleavage by RNase E at both the 5Ј and 3Ј ends, followed by final maturation of the 3Ј end by RNase T and by an unidentified enzyme at the 5Ј end (7, 11). 16S maturation proceeds via dual cleavages at the 5Ј end by RNase E and RNase G, whereas an unknown enzyme is responsible for 3Ј-end processing. Finally, 23S rRNA requires a set of 3Ј-to-5Ј exonucleases to carry out 3Ј-end maturation (12). However, the enzyme responsible for 5Ј-end maturation of 23S rRNA, presumably a novel endonuclease, has remained elusive despite nearly 3 decades of investigation (5). It has also been unclear whether processing of the two ends of this RNA represents coupled events or whether these events occur independently. Here we provide evidence that processing of the two ends constitutes linked events and that prior processing of the 3Ј end facilitates 5Ј-end maturation. MATERIALS AND METHODSStrains. MG1655* is a derivative of the sequenced strain MG1655 (2), which contains an engineered point mutation that converts a defective rph gene to wild type. Derivatives of MG1655* containing ⌬deaD, ⌬srmB, ⌬rph, ⌬rnb, or ⌬rnt alleles were constructed by transduction of deletion alleles that are interrupted by a kanamycin-resistant (Kan r ) marker (1). To construct MG1655* ⌬rph ⌬rnb ⌬rnt, the ⌬rph, ⌬rnb, and ⌬rnt alleles were introduced sequentially, and the Kan r marker was removed from the intermediate strains by recombination of frt sites that flank the kan gene...

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Section: Methodsmentioning

confidence: 99%

Coordinated Regulation of 23S rRNA Maturation in Escherichia coli

Gutgsell

Jain

2010

J Bacteriol

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“…Ribosomal profiles were generated by ultracentrifugation of cell extracts on a 14 to 32% sucrose gradient, as described previously (22). RNA was isolated using the hot phenol method and was analyzed by primer extension using a 23S rRNA-specific primer, as described previously (35).…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Growth Suppressors ofEscherichia coliStrains Lacking Phosphorolytic Ribonucleases

Jain

2009

J Bacteriol

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RNases are involved in critical aspects of RNA metabolism in all organisms. Two classes of RNases that digest RNA from an end (exo-RNases) are known: RNases that use water as a nucleophile to catalyze RNA degradation (hydrolytic RNases) and RNases that use inorganic phosphate (phosphorolytic RNases). It has been shown previously that the absence of the two known Escherichia coli phosphorolytic RNases, polynucleotide phosphorylase and RNase PH, leads to marked growth and ribosome assembly defects. To investigate the basis for these defects, a screen for growth suppressors was performed. The majority of suppressor mutations were found to lie within nsrR, which encodes a nitric oxide (NO)-sensitive transcriptional repressor. Further analysis showed that the suppressors function not by inactivating nsrR but by causing overexpression of a downstream gene that encodes a hydrolytic RNase, RNase R. Additional studies revealed that overexpression of another hydrolytic RNase, RNase II, similarly suppressed the growth defects. These results suggest that the requirement for phosphorolytic RNases for robust cellular growth and efficient ribosome assembly can be bypassed by increased expression of hydrolytic RNases.Many aspects of RNA metabolism are performed by RNases, a class of enzymes defined by their ability to cleave phosphodiester bonds in RNA (13, 30). Most organisms contain multiple RNases, and the crucial cellular functions of these enzymes include a role in RNA processing and maturation, turnover of unstable RNAs, and degradation of defective RNAs (14,15). Genetic analysis has shown that several of these enzymes are essential for cell viability, whereas in other cases the absence of certain combinations of RNases can cause synthetic defects (15, 29).For many years, Escherichia coli has been an important model organism for studying RNase function. E. coli is known to harbor about 20 RNases, a number that is likely to increase since the RNases responsible for several processes are still unknown (15). The RNases can be classified into two groups: endo-RNases and exo-RNases. Endo-RNases cleave RNA internally and generate RNA fragments. Exo-RNases cleave one terminal phosphodiester bond at a time, either from the 5Ј or the 3Ј end, releasing mononucleotides as a product.Currently, E. coli has eight known exo-RNases, each of which possesses 3Ј-to-5Ј activity (38). Six of these enzymes, RNase II, RNase D, RNase T, RNase R, RNase BN, and oligoribonuclease, are hydrolytic RNases that use water as a nucleophile, generating nucleotide monophosphates as a product. Two exo-RNases, polynucleotide phosphorylase (PNPase) and RNase PH, use inorganic phosphate as a nucleophile and release nucleotide diphosphates during RNA digestion. These enzymes are referred to as phosphorolytic RNases. PNPase, which was identified over 50 years ago, is implicated in the digestion of mRNA fragments generated during mRNA turnover and of structured RNAs with the assistance of helicases to open RNA duplexes (18,32). In addition, PNPase mutants exhibit...

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“…For RNA isolation, cells were grown under the conditions specified and RNA was extracted from using the hot-phenol method (Aiba et al 1981). Primer extension reactions to assay 59-end maturation of 23S rRNA were carried out using a radioactively labeled oligonucleotide primer that anneals 35-55 nucleotides from the 59 end of mature 23S rRNA, as described previously (Slagter-Jäger et al 2007). The primer extension products were separated on a 6% polyacrylamide-8 M urea sequencing gel, dried, and visualized using a Molecular Dynamics Storm 840 PhosphorImager.…”

Section: Ribosome and Rna Analysismentioning

confidence: 99%

Functional and molecular analysis of Escherichia coli strains lacking multiple DEAD-box helicases

Jagessar¹,

Jain²

2010

RNA

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DEAD-box RNA helicases are enzymes that unwind RNA duplexes and are found in virtually all organisms. Most organisms harbor multiple DEAD-box helicases, suggesting that these factors participate in distinct aspects of RNA metabolism. To define the individual and collective contribution of the five DEAD-box helicases in the bacterium Escherichia coli (E. coli ), nonpolar deletion mutants lacking single or multiple DEAD-box genes were constructed. An analysis of the single-deletion strains indicated that the absence of either the DeaD or SrmB RNA helicase causes growth and/or ribosomal defects under typical laboratory growth conditions. The analysis of strains lacking multiple DEAD-box genes showed cumulative growth defects at low temperatures. A strain deleted for all five DEAD-box genes was also constructed for these studies, representing the first time all DEAD-box genes have been removed in any organism. Additional investigations revealed that the growth and ribosomal defects of such a DEAD-box deficient strain can be sharply attenuated under alternative conditions, indicating that the defects caused by a lack of DEAD-box genes are modulated by growth context.

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Functional defects in transfer RNAs lead to the accumulation of ribosomal RNA precursors

Cited by 9 publications

References 40 publications

Coordinated Regulation of 23S rRNA Maturation in Escherichia coli

Coordinated Regulation of 23S rRNA Maturation in Escherichia coli

Identification and Characterization of Growth Suppressors ofEscherichia coliStrains Lacking Phosphorolytic Ribonucleases

Functional and molecular analysis of Escherichia coli strains lacking multiple DEAD-box helicases

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