Polyadenylation of stable RNA precursors in vivo

Abstract

Polyadenylation at the 3 terminus has long been considered a specific feature of mRNA and a few other unstable RNA species. Here we show that stable RNAs in Escherichia coli can be polyadenylated as well. RNA molecules with poly(A) tails are the major products that accumulate for essentially all stable RNA precursors when RNA maturation is slowed because of the absence of processing exoribonucleases; poly(A) tails vary from one to seven residues in length. The polyadenylation process depends on the presence of poly(A) polymerase I. A stochastic competition between the exoribonucleases and poly(A) polymerase is proposed to explain the accumulation of polyadenylated RNAs. These data indicate that polyadenylation is not unique to mRNA, and its widespread occurrence suggests that it serves a more general function in RNA metabolism.Cellular RNA molecules have long been divided into two groups. The first group, the stable RNAs, includes mainly rRNA, tRNA, and a variety of other small RNAs, and represents Ͼ95% of the total cellular RNA population. These RNAs have long lifetimes relative to the generation times of the cells in which they reside. The second group, unstable RNAs, consists primarily of mRNAs, which are a small fraction of the total RNA population and have half-lives that are usually much shorter than the generation time of the cell. The unstable RNAs generally contain a poly(A) tract at their 3Ј ends that contributes to their turnover; for mRNAs, poly(A) contributes to their role in translation (1-5). However, polyadenylation of stable RNAs has rarely been seen, and has not been considered important for stable RNA metabolism.In previous studies of the maturation of tRNA (6, 7), 5S RNA (8), and other small, stable RNAs (4.5S, 6S, M1, and tmRNA) (9) in Escherichia coli, it was shown that exoribonucleolytic trimming was a necessary step in the formation of the 3Ј termini of these various molecules. Thus, in the absence of the requisite 3Ј to 5Ј exoribonucleases, precursor products with extra nucleotides at their 3Ј ends accumulated. In some instances, however, these extra sequences were longer than expected. For example, 5S RNA is known to be released from long rRNA transcripts by RNase E endonucleolytic cleavages that leave three additional residues at each end of the processing intermediate (10-13). However, products were found to accumulate in some exoribonuclease-deficient strains that contained as many as 10 extra nucleotides at their 3Ј ends (8). Likewise, in a recent study of the maturation of M1 RNA, the catalytic subunit of RNase P, products with up to six additional 3Ј residues were observed (9), despite the fact that RNase E is thought to cleave this precursor at a position only one or two nucleotides downstream from the mature 3Ј terminus (14,15).In this paper we provide an explanation for these unexpected observations. To do so, we determined the nucleotide sequences at the 3Ј ends of the 5S and M1 RNA products that accumulate in the multiple exoribonuclease-deficient cells. This was acco...

Unpaired terminal nucleotides and 5′ monophosphorylation govern 3′ polyadenylation by Escherichia coli poly(A) polymerase I

“…Our in vitro findings imply that reduced poly(A) tail initiation by PAP I on small stable RNAs may account at least in part for their insensitivity to polyadenylation in vivo (22) and suggest a molecular basis for the differential polyadenylation observed for some cellular RNAs. Whereas 5S rRNA and certain other stable small RNAs have monophosphorylated 5Ј termini produced by endonucleolytic processing or exonucleolytic digestion, the length of unpaired terminal nucleotides on these molecules may be too short for PAP I to act efficiently.…”

“…1a) (6,19,20), undergo pcnBdependent (i.e., PAP I-dependent) polyadenylation in vivo, targeting these RNAs for rapid decay. However, mature tRNAs and ribosomal RNAs, which are highly stable within E. coli cells, normally lack detectable poly(A) tails (21)(22)(23).…”

Molecular Chaperones and Quality Control in Noncoding RNA Biogenesis

“…Although a requirement for polyadenylation in rRNA decay has not been reported, pre-23S rRNAs and 16S rRNAs containing short poly(A) tails have been observed in E. coli (Mohanty and Kushner 1999). Moreover, both overexpression of PAP and the absence of processing exoribonucleases result in accumulation of polyadenylated pre-23S rRNAs (Li et al 1998;Mohanty and Kushner 1999), suggesting that a balance normally exists between processing and polyadenylation of precursors. In E. coli, polyadenylation acts as a signal for both mRNA and noncoding RNA decay and assists 3t o 5´ exoribonucleases in initiating decay of structured RNAs by providing a single-stranded 3´ end (Deutscher 2006).…”

Effect of 3′ terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes

“…There is an accumulation of evidence showing that several cellular RNAs from evolutionarily distant species contain a single posttranscriptionally added adenylic acid residue on their 39 ends+ Our earlier studies showed that approximately 65% of the human SRP RNA, 7SK RNA, and U2 snRNA molecules contain a posttranscriptionally added adenylic acid residue on their 39 ends )+ A fraction of U2 and SRP RNAs from Xenopus oocytes and Saccharomyces cerevisiae also contain this posttranscriptional adenylation (Perumal et al+, 2000)+ In addition to these RNAs, there are many RNAs (e+g+, U6 snRNA and 5S rRNA) in which a small fraction of the RNAs contain 39 adenylic acid residues )+ Adenylation where one or two adenylic acid residues are posttranscriptionally added is known to occur in many RNAs, including some stable small RNAs of Escherichia coli (Attardi, 1985;Deutscher, 1990;Clayton, 1992;Li et al+, 1998)+ An enzymatic activity that posttranscriptionally adds a single adenylic acid residue to signal recognition particle (SRP) RNA has recently been identified and shown to be distinct from mRNA poly(A) polymerase (Sinha et al+, 1999)+ These data show that posttranscriptional adenylation, where a single adenylic acid residue is added to the 39 end of RNAs, is widespread in nature and conserved through evolution+…”