We have previously identified a HeLa cell 3 exonuclease specific for degrading poly(A) tails of mRNAs. Here we report on the purification and identification of a calf thymus 54-kDa polypeptide associated with a similar 3 exonuclease activity. The 54-kDa polypeptide was shown to be a fragment of the poly(A)-specific ribonuclease 74-kDa polypeptide. The native molecular mass of the nuclease activity was estimated to be 180 -220 kDa. Protein/protein cross-linking revealed an oligomeric structure, most likely consisting of three subunits. The purified nuclease activity released 5-AMP as the reaction product and degraded poly(A) in a highly processive fashion. The activity required monovalent cations and was dependent on divalent metal ions. The RNA substrate requirement was investigated, and it was found that the nuclease was highly poly(A)-specific and that only 3 end-located poly(A) was degraded by the activity. RNA substrates capped with m 7 G(5)ppp(5)G were more efficiently degraded than noncapped RNA substrates. Addition of free m 7 G(5)ppp(5)G cap analogue inhibited poly(A) degradation in vitro, suggesting a functional link between the RNA 5 end cap structure and poly(A) degradation at the 3 end of the RNA.

A novel universal neuropeptide display approach in the mass range of 300-5000 Da was developed to complement two-dimensional gel electrophoresis in the analysis of peptides and small proteins from brain tissue samples. For the analysis of neuropeptides we utilized on-line nanoscale capillary reversed phase liquid chromatography and electrospray ionization quadrupole-time of flight mass spectrometry. The method was employed for the analysis of a large number of peptides from three specific rat brain regions. Approximately 1500 peptides from each brain region were detected in the same analysis. Several of these peptides were sequenced using collision-induced dissociation and identified by database search tools. In addition, a method for comparing peptide elution profiles between samples was developed, to provide two- and three-dimensional computer graphics of the profiles and to pinpoint differences for statistical measurements. Among the characterized peptides were fragments from proteins such as hemoglobin, alpha-synuclein, stathmin, cyclophilin, actin, NADH dehydrogenase, cytochrome c oxidase and prosomatostatin, as well as the bioactive neuropeptides W-hemorphin-4, and LW-hemorphin-7. The present study showed that the combination of nanoscale reversed phase liquid chromatography and high-resolution tandem mass spectrometry provides a novel and powerful approach to investigate a large number of peptides and protein fragments in the brain.

We have identified a 3′ exonuclease in HeLa cell extracts which deadenylates mammalian mRNA and leaves the mRNA body intact after poly(A) removal. Only homopolymeric adenosine tails located at the 3′ end were efficiently removed by the exonuclease. The poly(A) removing activity did not require any specific sequences in the mRNA body either for poly(A) removal or for accumulation of the deadenylated mRNA. We conclude that the poly(A) removing activity is a 3′ exonuclease since (i) reaction intermediates gradually lose the poly(A) tail, (ii) degradation is prevented by the presence of a cordycepin residue at the 3′ end and (iii) RNAs having internally located poly(A) stretches are poor substrates for degradation. The possible involvement of the poly(A) removing enzyme in regulating mRNA translation and stability is discussed.

We have cloned human poly(A) polymerase (PAP) mRNA as cDNA in Escherichia coil. The primary structure of the mRNA was determined and compared to the bovine PAP mRNA sequence. The two sequences were 97% identical at the nucleotide level, which translated into 99% simlarity at the amino acid level. Polypeptides representing recombinant PAP were expressed in E. col, purified, and used as antigens to generate monoclonal antibodies. Western blot analysis using these monoclonal antibodies as probes revealed three PAPs, having estimated molecular masses of 90, 100, and 106 kDa in HeLa cell extracts. Fractionation of HeLa cells showed that the 90-kDa polypeptide was nuclear while the 100-and 106-kDa species were present in both nuclear and cytoplasmic fractions. The 106-kDa PAP was most likely a phosphorylated derivative of the 100-kDa species. PAP activity was recovered in vitro by using purified recombinant human PAP.Subsequent mutational analysis revealed that both the N-and C-terminal regions of PAP were important for activity and suggested that cleavage and polyadenylylation specificity factor (CPSF) interacted with the C-terminal region of PAP. Interestingly, tentative phosphorylation sites have been identified in this region, suggesting that phosphorylation/dephosphorylation may regulate the interaction between the two polyadenylylation factors PAP and CPSF.

The translation of specific maternal mRNAs is regulated during early development. For some mRNAs, an increase in translational activity is correlated with cytoplasmic extension of their poly(A) tails; for others, translational inactivation is correlated with removal of their poly(A) tails. Recent results in several systems suggest that events at the 3 end of the mRNA can affect the state of the 5 cap structure, m 7 G(5)ppp(5)G. We focus here on the potential role of cap modifications on translation during early development and on the question of whether any such modifications are dependent on cytoplasmic poly(A) addition or removal. To do so, we injected synthetic RNAs into Xenopus oocytes and examined their cap structures and translational activities during meiotic maturation. We draw four main conclusions. First, the activity of a cytoplasmic guanine-7-methyltransferase increases during oocyte maturation and stimulates translation of an injected mRNA bearing a nonmethylated GpppG cap. The importance of the cap for translation in oocytes is corroborated by the sensitivity of protein synthesis to cap analogs and by the inefficient translation of mRNAs bearing nonphysiologically capped 5 termini. Second, deadenylation during oocyte maturation does not cause decapping, in contrast to deadenylation-triggered decapping in Saccharomyces cerevisiae. Third, the poly(A) tail and the N-7 methyl group of the cap stimulate translation synergistically during oocyte maturation. Fourth, cap ribose methylation of certain mRNAs is very inefficient and is not required for their translational recruitment by poly(A). These results demonstrate that polyadenylation can cause translational recruitment independent of ribose methylation. We propose that polyadenylation enhances translation through at least two mechanisms that are distinguished by their dependence on ribose modification.