RNA processing in vitro produces mature 3'' ends of a variety of Saccharomyces cerevisiae mRNAs.

Ammonium sulfate fractionation of a Saccharomyces cerevisiae whole-cell extract yielded a preparation which carried out correct and efficient endonucleolytic cleavage and polyadenylation of yeast precursor mRNA substrates corresponding to a variety of yeast genes. These included CYC1 (iso-1-cytochrome c), HIS4 (histidine biosynthesis), GAL7 (galactose-1-phosphate uridyltransferase), H2B2 (histone H2B2), PRT2 (a protein of unknown function), and CBP1 (cytochrome b mRNA processing). The reaction processed these pre-mRNAs with varying efficiencies, with cleavage and polyadenylation exceeding 70% in some cases. In each case, the poly(A) tail corresponded to the addition of approximately 60 adenosine residues, which agrees with the usual length of poly(A) tails formed in vivo. Addition of cordycepin triphosphate or substitution of CTP for ATP in these reactions inhibited polyadenylation but not endonucleolytic cleavage and resulted in accumulation of the cleaved RNA product. Although this system readily generated yeast mRNA 3' ends, no processing occurred on a human alpha-globin pre-mRNA containing the highly conserved AAUAAA polyadenylation signal of higher eucaryotes. This sequence and adjacent signals used in mammalian systems are thus not sufficient to direct mRNA 3' end formation in yeast. Despite the lack of a highly conserved nucleotide sequence signal, the same purified fraction processed the 3' ends of a variety of unrelated yeast pre-mRNAs, suggesting that endonuclease cleavage and polyadenylation may produce the mature 3' ends of all mRNAs in S. cerevisiae.     

Conventional 3' end formation is not required for NMD substrate recognition in Saccharomyces cerevisiae

The recognition and rapid degradation of mRNAs with premature translation termination codons by the nonsense-mediated pathway of mRNA decay is an important RNA quality control system in eukaryotes. In mammals, the efficient recognition of these mRNAs is dependent upon exon junction complex proteins deposited on the RNA during pre-mRNA splicing. In yeast, splicing does not play a role in recognition of mRNAs that terminate translation prematurely, raising the possibility that proteins deposited during alternative pre-mRNA processing events such as 3' end formation might contribute to the distinction between normal and premature translation termination. We have utilized mRNAs with a 3' poly(A) tail generated by ribozyme cleavage to demonstrate that the normal process of 3' end cleavage and polyadenylation is not required for mRNA stability or the detection of a premature stop codon. Thus, in yeast, the distinction between normal and premature translation termination events is independent of both splicing and conventional 3' end formation.     

RNA polyadenylation and degradation in cyanobacteria are similar to the chloroplast but different from Escherichia coli

The mechanism of RNA degradation in Escherichia coli involves endonucleolytic cleavage, polyadenylation of the cleavage product by poly(A) polymerase, and exonucleolytic degradation by the exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase II. The poly(A) tails are homogenous, containing only adenosines in most of the growth conditions. In the chloroplast, however, the same enzyme, PNPase, polyadenylates and degrades the RNA molecule; there is no equivalent for the E. coli poly(A) polymerase enzyme. Because cyanobacteria is a prokaryote believed to be related to the evolutionary ancestor of the chloroplast, we asked whether the molecular mechanism of RNA polyadenylation in the Synechocystis PCC6803 cyanobacteria is similar to that in E. coli or the chloroplast. We found that RNA polyadenylation in Synechocystis is similar to that in the chloroplast but different from E. coli. No poly(A) polymerase enzyme exists, and polyadenylation is performed by PNPase, resulting in heterogeneous poly(A)-rich tails. These heterogeneous tails were found in the amino acid coding region, the 5' and 3' untranslated regions of mRNAs, as well as in rRNA and the single intron located at the tRNA(fmet). Furthermore, unlike E. coli, the inactivation of PNPase or RNase II genes caused lethality. Together, our results show that the RNA polyadenylation and degradation mechanisms in cyanobacteria and chloroplast are very similar to each other but different from E. coli.     

Emerging features of mRNA decay in bacteria

The problem of mRNA decay in E. coli has recently seen exciting progress, with the discoveries that key degradation enzymes are associated together in a high molecular weight degradosome and that polyadenylation promotes decay. Recent advances make it clear that mRNA decay in bacteria is far more interesting enzymatically than might have been predicted. In-depth study of specific mRNAs has revealed multiple pathways for degradation. Which pathway a given mRNA follows appears to depend in large part on the location of the initiating endonucleolytic cleavage within the mRNA. During the steps of mRNA decay, stable RNA structures pose formidable barriers to the 3' --> 5' exonucleases. However, polyadenylation is now emerging as a process that plays an important role in maintaining the momentum of exonucleolytic degradation by adding single-stranded extensions to the 3' ends of mRNAs and their decay intermediates, thereby facilitating further exonuclease digestion.     

Sensing of 5' monophosphate by Escherichia coli RNase G can significantly enhance association with RNA and stimulate the decay of functional mRNA transcripts in vivo

The RNase E/G family of endoribonucleases has a central role in RNA degradation and processing. Previous work has shown that their cleavage of substrates in vitro can be stimulated by the presence of a 5' monophosphate group. It has not however, established the importance of this activation for any natural RNA processing or decay pathway in vivo. Here we provide for Escherichia coli RNase G the first evidence that the sensing of a 5' monophosphate is required in vivo for the normal rapid decay of functional mRNAs; moreover, we show in vitro that, in contrast to a previous study, the presence of a 5' monophosphate can enhance the affinity of RNase G binding to RNA. The implications of these results along with our finding that the maturation of 16S rRNA is unaffected in cells containing an RNase G mutant impaired in 5' end sensing are discussed with regard to current models of RNA processing and decay and the molecular mechanism that underlies RNA cleavage by the RNase E/G family.     

Conditional defect in mRNA 3'' end processing caused by a mutation in the gene for poly(A) polymerase.

Maturation of most eukaryotic mRNA 3' ends requires endonucleolytic cleavage and polyadenylation of precursor mRNAs. To further understand the mechanism and function of mRNA 3' end processing, we identified a temperature-sensitive mutant of Saccharomyces cerevisiae defective for polyadenylation. Genetic analysis showed that the polyadenylation defect and the temperature sensitivity for growth result from a single mutation. Biochemical analysis of extracts from this mutant shows that the polyadenylation defect occurs at a step following normal site-specific cleavage of a pre-mRNA at its polyadenylation site. Molecular cloning and characterization of the wild-type allele of the mutated gene revealed that it (PAP1) encodes a previously characterized poly(A) polymerase with unknown RNA substrate specificity. Analysis of mRNA levels and structure in vivo indicate that shift of growing, mutant cells to the nonpermissive temperature results in the production of poly(A)-deficient mRNAs which appear to end at their normal cleavage sites. Interestingly, measurement of the rate of protein synthesis after the temperature shift shows that translation continues long after the apparent loss of polyadenylated mRNA. Our characterization of the pap1-1 defect implicates this gene as essential for mRNA 3' end formation in S. cerevisiae.     

Sok antisense RNA from plasmid R1 is functionally inactivated by RNase E and polyadenylated by poly(A) polymerase I

The hok/sok system of plasmid R1, which mediates plasmid stabilization by the killing of plasmid-free cells, codes for two RNA species, Sok antisense RNA and hok mRNA. Sok RNA, which is unstable, inhibits translation of the stable hok mRNA. The 64 nt Sok RNA folds into a single stem-loop domain with an 11 nt unstructured 5' domain. The initial recognition reaction between Sok RNA and hok mRNA takes place between the 5' domain and the complementary region in hok mRNA. In this communication we examine the metabolism of Sok antisense RNA. We find that RNase E cleaves the RNA 6 nt from its 5' end and that this cleavage initiates Sok RNA decay. The RNase E cleavage occurs in the part of Sok RNA that is responsible for the initial recognition of the target loop in hok mRNA and thus leads to functional inactivation of the antisense. The major RNase E cleavage product (denoted pSok_-6) is rapidly degraded by polynucleotide phosphorylase (PNPase). Thus, the RNase E cleavage tags pSok₋₆ for further rapid degradation by PNPase from its 3' end. We also show that Sok RNA is polyadenylated by poly(A) polymerase I (PAP I), and that the poly(A)-tailing is prerequisite for the rapid 3'-exonucleolytic degradation by PNPase.     

A small nuclear ribonucleoprotein associates with the AAUAAA polyadenylation signal in vitro

RNAs containing the polyadenylation sites for adenovirus L3 or E2a mRNA or for SV40 early or late mRNA are substrates for cleavage and poly(A) addition in an extract of HeLa cell nuclei. When polyadenylation reactions are probed with ribonuclease T1 and antibodies directed against either the Sm protein determinant or the trimethylguanosine cap structure at the 5' end of U RNAs in small nuclear ribonucleoproteins, RNA fragments containing the AAUAAA polyadenylation signal are immunoprecipitated. The RNA cleavage step that occurs prior to poly(A) addition is inhibited by micrococcal nuclease digestion of the nuclear extract. The immunoprecipitation of fragments containing the AAUAAA sequence can be altered, but not always abolished, by pretreatment with micrococcal nuclease. We discuss the involvement of small nuclear ribonucleoproteins in the cleavage and poly(A) addition reactions that form the 3' ends of most eukaryotic mRNAs.