Attachment of the RNA degradosome to the bacterial inner cytoplasmic membrane prevents wasteful degradation of rRNA in ribosome assembly intermediates.

RNA processing and degradation shape the transcriptome by generating stable molecules that are necessary for translation (rRNA and tRNA) and by facilitating the turnover of mRNA, which is necessary for the posttranscriptional control of gene expression. In bacteria and the plant chloroplast, RNA degradosomes are multienzyme complexes that process and degrade RNA. In many bacterial species, the endoribonuclease RNase E is the central component of the RNA degradosome. RNase E-based RNA degradosomes are inner membrane proteins in a large family of gram-negative bacteria (ß- and γ-Proteobacteria). Until now, the reason for membrane localization was not understood. Here, we show that a mutant strain of Escherichia coli, in which the RNA degradosome is localized to the interior of the cell, has high levels of 20S and 40S particles that are defective intermediates in ribosome assembly. These particles have aberrant protein composition and contain rRNA precursors that have been cleaved by RNase E. After RNase E cleavage, rRNA fragments are degraded to nucleotides by exoribonucleases. In vitro, rRNA in intact ribosomes is resistant to RNase E cleavage, whereas protein-free rRNA is readily degraded. We conclude that RNA degradosomes in the nucleoid of the mutant strain interfere with cotranscriptional ribosome assembly. We propose that membrane-attached RNA degradosomes in wild-type cells control the quality of ribosome assembly after intermediates are released from the nucleoid. That is, the compact structure of mature ribosomes protects rRNA against cleavage by RNase E. Turnover of a proportion of intermediates in ribosome assembly explains slow growth of the mutant strain. Competition between mRNA and rRNA degradation could be the cause of slower mRNA degradation in the mutant strain. We conclude that attachment of the RNA degradosome to the bacterial inner cytoplasmic membrane prevents wasteful degradation of rRNA precursors, thus explaining the reason for conservation of membrane-attached RNA degradosomes throughout the ß- and γ-Proteobacteria.

Detachment of the RNA degradosome from the inner membrane of Escherichia coli results in a global slowdown of mRNA degradation, proteolysis of RNase E and increased turnover of ribosome-free transcripts.

The reason for RNase E attachment to the inner membrane is largely unknown. To understand the cell biology of RNA degradation, we have characterized a strain expressing RNase E lacking the membrane attachment site (cytoplasmic RNase E). Genome-wide data show a global slowdown in mRNA degradation. There is no correlation between mRNA stabilization and the function or cellular location of encoded proteins. The activity of cRNase E is comparable to the wild-type enzyme in vitro, but the mutant protein is unstable in vivo. Autoregulation of cRNase E synthesis compensates for protein instability. cRNase E associates with other proteins to assemble a cytoplasmic RNA degradosome. CsrB/C sRNAs, whose stability is regulated by membrane-associated CsrD, are stabilized. Membrane attachment of RNase E is thus necessary for CsrB/C turnover. In contrast to mRNA stability, ribosome-free transcripts are sensitive to inactivation by cRNase E. Our results show that effects on RNA degradation are not due to the differences in the activity or level of cRNase E, or failure to assemble the RNA degradosome. We propose that membrane attachment is necessary for RNase E stability, functional interactions with membrane-associated regulatory factors and protection of ribosome-free transcripts from premature interactions with RNase E in the nucleoid.

Analysis of tetra- and hepta-nucleotides motifs promoting -1 ribosomal frameshifting in Escherichia coli.

Programmed ribosomal -1 frameshifting is a non-standard decoding process occurring when ribosomes encounter a signal embedded in the mRNA of certain eukaryotic and prokaryotic genes. This signal has a mandatory component, the frameshift motif: it is either a Z_ZZN tetramer or a X_XXZ_ZZN heptamer (where ZZZ and XXX are three identical nucleotides) allowing cognate or near-cognate repairing to the -1 frame of the A site or A and P sites tRNAs. Depending on the signal, the frameshifting frequency can vary over a wide range, from less than 1% to more than 50%. The present study combines experimental and bioinformatics approaches to carry out (i) a systematic analysis of the frameshift propensity of all possible motifs (16 Z_ZZN tetramers and 64 X_XXZ_ZZN heptamers) in Escherichia coli and (ii) the identification of genes potentially using this mode of expression amongst 36 Enterobacteriaceae genomes. While motif efficiency varies widely, a major distinctive rule of bacterial -1 frameshifting is that the most efficient motifs are those allowing cognate re-pairing of the A site tRNA from ZZN to ZZZ. The outcome of the genomic search is a set of 69 gene clusters, 59 of which constitute new candidates for functional utilization of -1 frameshifting.

Evolutionary and functional insights into the Ski2-like helicase family in Archaea: a comparison of Thermococcales ASH-Ski2 and Hel308 activities.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The Ski2-like proteins are primordial helicases that play an active role in eukaryotic RNA homeostasis pathways, with multiple homologs having specialized functions. The significance of the expansion and diversity of Ski2-like proteins in Archaea, the third domain of life, has not yet been established. Here, by studying the phylogenetic diversity of Ski2-like helicases among archaeal genomes and the enzymatic activities of those in Thermococcales, we provide further evidence of the function of this protein family in archaeal metabolism of nucleic acids. We show that, in the course of evolution, ASH-Ski2 and Hel308-Ski2, the two main groups of Ski2-like proteins, have diverged in their biological functions. Whereas Hel308 has been shown to mainly act on DNA, we show that ASH-Ski2, previously described to be associated with the 5'-3' aRNase J exonuclease, acts on RNA by supporting an efficient annealing activity, but also an RNA unwinding with a 3'-5' polarity. To gain insights into the function of Ski2, we also analyse the transcriptome of Thermococcus barophilus ΔASH-Ski2 mutant strain and provide evidence of the importance of ASH-Ski2 in cellular metabolism pathways related to translation.

The interplay of mRNA stimulatory signals required for AUU-mediated initiation and programmed -1 ribosomal frameshifting in decoding of transposable element IS911.

The IS911 bacterial transposable element uses -1 programmed translational frameshifting to generate the protein required for its mobility: translation initiated in one gene (orfA) shifts to the -1 frame and continues in a second overlapping gene (orfB), thus generating the OrfAB transposase. The A-AAA-AAG frameshift site of IS911 is flanked by two stimulatory elements, an upstream Shine-Dalgarno sequence and a downstream stem-loop. We show here that, while they can act independently, these stimulators have a synergistic effect when combined. Mutagenic analyses revealed features of the complex stem-loop that make it a low-efficiency stimulator. They also revealed the dual role of the upstream Shine-Dalgarno sequence as (i) a stimulator of frameshifting, by itself more potent than the stem-loop, and (ii) a mandatory determinant of initiation of OrfB protein synthesis on an AUU codon directly preceding the A6G motif. Both roles rely on transient base pairing of the Shine-Dalgarno sequence with the 3' end of 16S rRNA. Because of its effect on frameshifting, the Shine-Dalgarno sequence is an important determinant of the level of transposase in IS911-containing cells, and hence of the frequency of transposition.

Genetic variability of the frameshift region in IS911 transposable elements from Escherichia coli clinical isolates.

The IS911 bacterial transposable element has been analyzed for its mechanism of transposition and for the way it controls the expression of its genes by programmed -1 translational frameshifting. In the present study the prevalence of IS911 has been determined in the Enterobacteriaceae family and in other Gram-negative bacilli. Three variants, found in Escherichia coli clinical isolates and having mutations in the region implicated in frameshifting, were functionally characterized. All three were altered in their frameshifting and transposition abilities, suggesting that the frameshift region of IS911 may constitute a target for mutations reducing the transposition frequency of this mobile element in natural populations of E. coli.

Apical loop-internal loop RNA pseudoknots: a new type of stimulator of -1 translational frameshifting in bacteria.

Nearly all members of a widespread family of bacterial transposable elements related to insertion sequence 3 (IS3), therefore called the IS3 family, very likely use programmed -1 ribosomal frameshifting to produce their transposase, a protein required for mobility. Comparative analysis of the potential frameshift signals in this family suggested that most of the insertion sequences from the IS51 group contain in their mRNA an elaborate pseudoknot that could act as a recoding stimulator. It results from a specific intramolecular interaction between an apical loop and an internal loop from two stem-loop structures. Directed mutagenesis, chemical probing, and gel mobility assays of the frameshift region of one element from the IS51 group, IS3411, provided clear evidences of the existence of the predicted structure. Modeling was used to generate a three-dimensional molecular representation of the apical loop-internal loop complex. We could demonstrate that mutations affecting the stability of the structure reduce both frameshifting and transposition, thus establishing the biological importance of this new type of RNA structure for the control of transposition level.