Roles of polyadenylation and nucleolytic cleavage in the filamentous phage mRNA processing and decay pathways in Escherichia coli

Abstract

To define basic features of mRNA processing and decay in Escherichia coli, we have examined a set of mRNAs encoded by the filamentous phage f1 that have structures typical of bacterial mRNAs. They bear a stable hairpin stem-loop on the 3' end left from rho-independent termination and are known to undergo processing by RNase E. A small percentage of the f1 mRNAs were found to bear poly(A) tails that were attached to heterogeneous positions near the common 3' end. In a poly(A) polymerase-deficient host, the later-appearing processed mRNAs were stabilized, and a novel small RNA accumulated. This approximately 125-nt RNA proved to arise via RNase E cleavage from the 3'-terminal region of the mRNAs bearing the terminator. Normally ribosomes translating gene VIII appear to protect this cleavage site from RNase E, so that release of the fragment from the mRNAs occurs very slowly. The data presented define additional steps in the f1 mRNA processing and decay pathways and clarify how features of the pathways are used in establishing and maintaining the persistent filamentous phage infection. Although the primary mode of decay is endonucleolytic cleavage generating a characteristic 5' --> 3' wave of products, polyadenylation is involved in part in degradation of the processed mRNAs and is required for turnover of the 125-nt mRNA fragment. The results place polyadenylation at a later rather than an initiating step of decay. They also provide a clear illustration of how stably structured RNA 3' ends act as barriers to 3' --> 5' exonucleolytic mRNA decay.

Defects in RNA polyadenylation impair both lysogenization by and lytic development of Shiga toxin-converting bacteriophages

“…Polyadenylation of different mRNAs of bacteriophage T7 was reported, but it appeared that this modification occurred after nucleolytic cleavage of the primary transcripts (Johnson et al, 1998). Similar results were described after analysis of mRNAs encoded by filamentous bacteriophage f1, where polyadenylation has been suggested to occur at the later stages of transcript degradation (Goodrich & Steege, 1999). Contrary to reports on T7-and f1-derived transcripts, no significant polyadenylation of mRNAs of phage T4 could be found (Yonesaki, 2002).…”

The catalytic domain of RNase E shows inherent 3′ to 5′ directionality in cleavage site selection

“…Attack by RNase E may be further modulated or circumvented by the presence of RNA binding proteins or ribosomes at particular sites of substrates, or by the concurrent actions of other ribonucleases, and such factors potentially may account for differences in the directionality of RNase E-mediated decay reported for different substrates in vivo (17,46,55). Additionally, full-length primary transcripts, which contain 5Ј-triphosphorylated termini, are cleaved poorly by RNase E (18,20), and consequently may lack the 5Ј end-binding activity that we have shown here for 5Ј-monophosphorylated termini.…”

“…It has been proposed that RNase E enters substrates at the 5Ј end and then scans toward the 3Ј end for cleavage sites (8,9,17,20). The ability of 2Ј-O-methyl substitutions to inhibit cleavage at ordinarily susceptible internucleotide bonds, together with evidence that noncleavable recognition sequences may retain RNase E, suggested an approach to test this model directly.…”

“…RNase E cleaves preferentially at specific sites in single-strand RNA segments rich in AϩU nucleotides (13)(14)(15)(16)). An inherent mode of action involving entry of RNase E at the 5Ј end of substrates followed by a 5Ј to 3Ј wave of cleavages has been inferred from the greater in vivo stability of fragments at the 3Ј ends of certain RNA substrates (17)(18)(19)(20) together with evidence that (i) the enzyme prefers a free 5Ј end for endonucleolytic cleavage (18,20), (ii) RNase E degradation efficiency is affected by 5Ј-phosphorylation in vivo (10) and in vitro (19,20), and (iii) 5Ј regions of secondary structure can impede cleavages in vivo (21). However, RNA degradation in vivo also can occur in the opposite direction (e.g., ref.…”

Global analysis of Escherichia coli RNA degradosome function using DNA microarrays

“…Investigations of the decay of individual transcripts (16)(17)(18)(19) and global investigations of mRNA abundance in rne mutants (20) have indicated a broadly important role for this enzyme. However, the RNase E region that provides a scaffold for degradosome formation is not essential for cell survival and growth (17,21,22), and truncated RNase E proteins lacking this domain are active in vivo as well as in vitro (8,23).…”