Multiple Exoribonucleases Catalyze Maturation of the 3′ Terminus of 16S Ribosomal RNA (rRNA)

Sulthana, Shaheen; Deutscher, Murray P.

doi:10.1074/jbc.c113.459172

Cited by 71 publications

(74 citation statements)

References 24 publications

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“…S11A). RNase II, RNase PH, PNPase, and RNase R are involved in 16S rRNA 3′ end maturation (29). However, we did not observe a strong effect of rph deletion in the i-tRNA 3GC background (SI Appendix, Fig.…”

Section: Significancementioning

confidence: 41%

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

Shetty

Varshney

2016

Proc. Natl. Acad. Sci. U.S.A.

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Ribosome biogenesis, a complex multistep process, results in correct folding of rRNAs, incorporation of >50 ribosomal proteins, and their maturation. Deficiencies in ribosome biogenesis may result in varied faults in translation of mRNAs causing cellular toxicities and ribosomopathies in higher organisms. How cells ensure quality control in ribosome biogenesis for the fidelity of its complex function remains unclear. Using Escherichia coli, we show that initiator tRNA (i-tRNA), specifically the evolutionarily conserved three consecutive GC base pairs in its anticodon stem, play a crucial role in ribosome maturation. Deficiencies in cellular contents of i-tRNA confer cold sensitivity and result in accumulation of ribosomes with immature 3′ and 5′ ends of the 16S rRNA. Overexpression of i-tRNA in various strains rescues biogenesis defects. Participation of i-tRNA in the first round of initiation complex formation licenses the final steps of ribosome maturation by signaling RNases to trim the terminal extensions of immature 16S rRNA.cold sensitivity | ribosome biogenesis | 3GC base pairs | RNase PH/RNAse R | 16S rRNA maturation R ibosomes are large and the most complex of the molecular machines in cells, using up to 40% of the cellular energy for their biogenesis (1). Ribosome biogenesis is a multistep process involving a coordinated network of RNA-RNA, RNA-protein, and protein-protein interactions. The process begins with the transcription of rRNA, its processing, nucleoside modifications, structural rearrangements, synthesis, and interaction of ribosomal proteins (r-proteins) (2). In cells, RNA processing enzymes, modification systems, and chaperone-like factors facilitate ribosome biogenesis. Any deficiencies in this process may produce ribosomes that fail to maintain the fidelity of protein synthesis and cause cellular toxicity/ death (3). Deficiencies in biogenesis factors impart cold sensitivity to Escherichia coli and affect bacterial drug resistance and virulence. In humans, genetic defects resulting in imperfect ribosome biogenesis are the cause of various ribosomopathies (4-7). Thus, a better understanding of ribosome biogenesis is a fundamental requirement to develop antimicrobials and various therapies (8-12).Of the two ribosomal subunits in E. coli, the small subunit contains a 1,542 nucleotide long rRNA (16S rRNA) along with 21 r-proteins, whereas the large subunit contains two rRNAs of 2,904 (23S rRNA) and 120 (5S rRNA) nucleotide lengths along with 33 r-proteins. The three rRNAs are synthesized as parts of a single transcript and the assembly of r-proteins begins cotranscriptionally. As the assembly advances, the precursor rRNA is cleaved by RNase III into immature units of 16S rRNA (as 17S), 23S rRNA (as 25S), and 5S rRNA (as 9S) rRNAs. The 17S rRNA retains unprocessed extensions of 110 and 33 nucleotides on the 5′ and 3′ ends, respectively. Trimming of the extensions by RNase G and other RNases matures 16S rRNA. Incorporation of r-proteins occurs in three stages. The primary r-proteins interact di...

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Section: Significancementioning

confidence: 41%

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

Shetty

Varshney

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Processing of the primary transcript of rrn operons to generate the mature 16S rRNA occurs in several steps, and 16S rRNA precursors accumulate in strains defective in specific nucleases, as well as in some ribosomal mutants (15,30,31). Analysis of the E. coli uS5 G27D mutant suggested a link between defective rRNA processing and translational fidelity (13,15).…”

Section: Resultsmentioning

confidence: 99%

The Loop 2 Region of Ribosomal Protein uS5 Influences Spectinomycin Sensitivity, Translational Fidelity, and Ribosome Biogenesis

Kamath

Gregory

O’Connor

2017

Antimicrob Agents Chemother

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Ribosomal protein uS5 is an essential component of the small ribosomal subunit that is involved in subunit assembly, maintenance of translational fidelity, and the ribosome's response to the antibiotic spectinomycin. While many of the characterized uS5 mutations that affect decoding map to its interface with uS4, more recent work has shown that residues distant from the uS4-uS5 interface can also affect the decoding process. We targeted one such interface-remote area, the loop 2 region (residues 20 to 31), for mutagenesis in Escherichia. coli and generated 21 unique mutants. A majority of the loop 2 alterations confer resistance to spectinomycin and affect the fidelity of translation. However, only a minority show altered rRNA processing or ribosome biogenesis defects.

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“…The cggR probe was complementary to nt 37-66 of the cggR CDS. To control for RNA loading, membranes were stripped and probed either for 5S rRNA, as described (26), or for 16S rRNA, using an oligonucleotide complementary to nt 1405-1424 (27). Results were the average of two or more biological replicates.…”

Section: Methodsmentioning

confidence: 99%

Interaction of Bacillus subtilis Polynucleotide Phosphorylase and RNase Y

Salvo

Alabi²,

Liu

et al. 2016

Journal of Biological Chemistry

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Polynucleotide phosphorylase (PNPase), a 3-to-5 phosphorolytic exoribonuclease, is thought to be the primary enzyme responsible for turnover of Bacillus subtilis mRNA. The role of PNPase in B. subtilis mRNA decay has been analyzed previously by comparison of mRNA profiles in a wild-type strain versus a strain that is deleted for pnpA, the gene encoding PNPase. Bacterial mRNA turnover is an essential process that replenishes the ribonucleotide pool and plays a role in regulation of gene expression. We have been studying 3Ј-exonuclease activity in Bacillus subtilis, the Gram-positive model organism. Although at least four 3Ј-exonucleases are known to exist in B. subtilis (1), the phosphorolytic enzyme, polynucleotide phosphorylase (PNPase, 3 encoded by the pnpA gene), has long been thought to play the major role in mRNA turnover in this organism (2, 3). The B. subtilis pnpA gene was originally identified in a transposon insertion screen of competence-deficient mutants, and was also shown to be required for cold adaptation (4). A deletion strain lacking most of the pnpA coding sequence (the ⌬pnpA strain) was constructed, and additional phenotypes were observed: growth as non-motile chains, tetracycline sensitivity (5), and accumulation of mRNA decay intermediates (1, 6). More recently, reliance of global mRNA turnover on PNPase was examined in an RNA sequencing analysis of the B. subtilis transcriptome in wild-type and ⌬pnpA strains, and this demonstrated the broad impact of PNPase on mRNA turnover (7). Many of the above mentioned studies were performed before the discovery of RNase Y, a B. subtilis endoribonuclease that appears to function in initiation of decay similarly to the more well known RNase E of Escherichia coli (8,9). Endonucleolytic cleavage in the body of a message by RNase E in E. coli or RNase Y in B. subtilis generates mRNA fragments that are susceptible to further steps in mRNA turnover. In addition, just as RNase E of E. coli coordinates a complex of several proteins known as the "degradosome," so, too, there is evidence that RNase Y of B. subtilis is the organizing protein for a complex that includes, at least, PNPase, CshA (an RNA helicase), and the glycolytic enzymes, enolase and phosphofructokinase (8,10,11). A PNPase-RNase Y interaction was first indicated by bacterial-2-hybrid (B2H) assay and in vivo protein crosslinking experiments (8). This interaction has more recently been confirmed by surface plasmon resonance analysis, which showed a strong PNPase-RNase Y interaction with a K d of 5 nM (12). Structural mapping of regions of PNPase that interact with an RNase E peptide has been done for the E. coli enzyme (13). Structural studies have not been reported for the B. subtilis PNPaseRNase Y interaction.The existence of a PNPase-RNase Y complex raises the question of whether this interaction is relevant to the ability of PNPase to rapidly degrade upstream decay intermediates that are created by RNase Y cleavage. In addition, prior results of experiments in the strain that contained no PNPase...

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Multiple Exoribonucleases Catalyze Maturation of the 3′ Terminus of 16S Ribosomal RNA (rRNA)

Cited by 71 publications

References 24 publications

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

The Loop 2 Region of Ribosomal Protein uS5 Influences Spectinomycin Sensitivity, Translational Fidelity, and Ribosome Biogenesis

Interaction of Bacillus subtilis Polynucleotide Phosphorylase and RNase Y

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