Development of an in vitro mRNA decay system for Escherichia coli : Poly(A) polymerase I is necessary to trigger degradation

Abstract

Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from a strain carrying the wild-type gene for PAP I (pcnB ؉ ). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) tails are detected on the 3 end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I ؊ polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.In Escherichia coli, mRNA decay has been studied extensively in vivo with a variety of approaches. The identification of exo-and endoribonucleases and the use of mutant forms of these enzymes (1-5) has resulted in the recognition of decay intermediates in vivo (6-9) and hypotheses to explain the temporal process of decay for several messages (9-11). In addition, careful analysis of some messages has demonstrated physical characteristics of both the 5Ј-and 3Ј-untranslated regions that profoundly affect the accessibility of these mRNAs to ribonucleolytic attack (12-15). More recent evidence has indicated that factors other than ribonucleases and RNA structure may modulate message decay, particularly the role of translating ribosomes (16) and the activity of poly(A) polymerase (PAP) I (17, 18).Although our understanding of mRNA decay has progressed considerably, many questions remain regarding the overall biochemical mechanism. In other complex biological pathways, such as DNA replication and protein synthesis, in vitro studies have provided invaluable assistance for isolating and characterizing important system components. In the case of mRNA decay, however, no useful in vitro system has been described. To be of value, an in vitro reaction should accurately reflect the relative decay rates of short-and long-lived messages (19) and generate the same breakdown products observed in vivo. The use of labeled run-off mRNA transcripts in the presence of E. coli extracts has not met these criteria (unpublished results).As a first step in developing a reliable in vitro system, we present studies using mRNAs associated with polysomes as substrates for decay. This approach was adopted because in vivo decay has been shown to be affected by translation rates (16,20). In addition, we hypothesized that many of the factors necessary for mRNA decay might be associated with polyribosomes. Because of the complexity of such a system, we chose to focus initially on whether polyadenylylation affected mRNA decay rates in vitro.PAP activity was firs...

The Polyadenylation of RNA in Plants

“…This hypothesis is analogous to recent models in bacteria that postulate a role for polyadenylation in accelerated RNA turnover (e.g. Ingle and Kushner, 1996). Given the importance of posttranscriptional events, including mRNA turnover, in chloroplast gene expression (Mayfield et al, 1995), the potential link between RNA polyadenylation and turnover is very interesting.…”

The RNA binding protein Hfq interacts specifically with tRNAs

“…In eukaryotes, when processing defects occur, Trf4p, a component of the TRAMP complex, polyadenylates the misfolded tRNA to initiate degradation (Kadaba et al 2004;LaCava et al 2005;Vanacova et al 2005). Unlike cytoplasmic poly(A) tails, nuclear polyadenylation in eukaryotes targets the RNA for degradation in a process similar to bacterial RNA turnover, where mRNAs can be poly(A)-tailed to target them for degradeosome-mediated decay (Hajnsdorf et al 1995;O'Hara et al 1995;Ingle and Kushner 1996;Carpousis et al 1999).…”

Scarce adenylation in bacteriophage T4 mRNAs.

“…Polyadenylation at the 3' end of RNA facilitates degradation of mRNAs (O'Hara et al, 1995;Hajnsdorf et al, 1995;Ingle and Kushner, 1996;Mohanty and Kushner, 1999) as well as RNA fragments (Xu and Cohen, 1995;Hajnsdorf and Regnier, 1999) in E. coli . Interestingly, Sarker and his colleagues reported that bacteriophage T7 mRNA was polyadenylated (Johnson et al, 1998).…”