Quality control of ribosomal RNA mediated by polynucleotide phosphorylase and RNase R

Abstract

Despite their overall accuracy, errors in macromolecular processes, such as rRNA synthesis and ribosome assembly, inevitably occur. However, whether these errors are remediated and how this might be accomplished is not known. In previous work, we showed that a double mutant strain lacking both polynucleotide phosphorylase (PNPase) and RNase R activities is inviable. In the course of examining the molecular basis for this phenotype, we found that shifting a temperature-sensitive mutant strain to 42°C led to cessation of growth and loss of cell viability. Northern analysis of RNA isolated from such cells after the temperature shift revealed that fragments of 16S and 23S rRNA accumulated to a high level, and that the amount of ribosomes and ribosomal subunits decreased due to defects in ribosome assembly. rRNA fragments were not detected at 31°C or when single mutant strains were grown at 42°C. Pulse-chase analysis showed that the rRNA fragments appeared within 5 min at 42°C, and that they accumulated before the loss of cell viability. The data are consistent with a model in which PNPase and RNase R mediate a previously unknown quality control process that normally removes defective rRNAs as soon as they are generated. In the absence of these RNases, rRNA fragments accumulate, leading to interference with ribosome maturation and ultimately to cell death.

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

“…This was necessary because double mutant strains lacking RNase II and PNPase (13) or RNase R and PNPase (14,15) are inviable, and cells lacking RNase PH and PNPase are slowed in growth (16). The mutant strain was grown at 37°C and transferred to 44°C for 1 h to inactivate PNPase such that the maturation of 16S RNA could be examined under conditions in which the activities of four exoribonucleases were lacking.…”

Direct observation of processive exoribonuclease motion using optical tweezers

“…B oth polynucleotide phosphorylase (PNPase) and RNase R are responsible for degrading a variety of transcripts in bacteria, such as mRNAs, defective rRNAs, and antisense regulatory RNAs (1)(2)(3)(4). Although strains of Escherichia coli remain viable with a loss of either enzyme, the elimination of both results in an accumulation of rRNA fragments and cell death (3).…”

“…Although strains of Escherichia coli remain viable with a loss of either enzyme, the elimination of both results in an accumulation of rRNA fragments and cell death (3). To degrade RNA, both ribonucleases require an unstructured binding site of at least 7-10 nt at the 3′ end of the substrate (5-7), a sequence that often is supplied by posttranscriptional polyadenylation (8)(9)(10).…”

Balance between Transcription and RNA Degradation Is Vital forSaccharomyces cerevisiaeMitochondria: Reduced Transcription Rescues the Phenotype of Deficient RNA Degradation

“…Perturbations in mitochondrial translation are known to affect the stability of mitochondrial DNA (Myers et al, 1985), which would explain the elevated frequency of mitochondrial petites observed in suv3⌬ strains (Stepien et al, 1992Golik et al, 1995). Defects in the quality control of ribosomal RNAs leading to defects in ribosome maturation were found to cause lethality in Escherichia coli strains devoid of the two major exoribonucleases, RNase R and polynucleotide phosphorylase (Cheng and Deutscher, 2003). Although the polynucleotide phosphorylase has been found in human mitochondria , it is conspicuously absent from the mitochondria of S. cerevisiae.…”