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.

No abstract

TRAIL (TNF-related apoptosis-inducing ligand) death receptors DR4 and DR5 facilitate the selective elimination of malignant cells through the induction of apoptosis. From previous studies the regulation of the DR4 and DR5 cell-death pathways appeared similar; nevertheless in this study we screened a library of small interfering RNA (siRNA) for genes, which when silenced, differentially affect DR4-vs. DR5-mediated apoptosis. These experiments revealed that expression of the signal recognition particle (SRP) complex is essential for apoptosis mediated by DR4, but not DR5. Selective diminution of SRP subunits by RNA interference resulted in a dramatic decrease in cell surface DR4 receptors that correlated with inhibition of DR4-dependent cell death. Conversely, SRP silencing had little influence on cell surface DR5 levels or DR5-mediated apoptosis. Although loss of SRP function in bacteria, yeast and protozoan parasites causes lethality or severe growth defects, we observed no overt phenotypes in the human cancer cells studied-even in stable cell lines with diminished expression of SRP components. The lack of severe phenotype after SRP depletion allowed us to delineate, for the first time, a mechanism for the differential regulation of the TRAIL death receptors DR4 and DR5-implicating the SRP complex as an essential component of the DR4 cell-death pathway.

Poly(A)-specific ribonuclease (PARN) is the only mammalian exoribonuclease characterized thus far with high specificity for degrading the mRNA poly(A) tail. PARN belongs to the RNase D family of nucleases, a family characterized by the presence of four conserved acidic amino acid residues. Here, we show by site-directed mutagenesis that these residues of human PARN, i.e. 2؉ binding at both sites were affected in PARN polypeptides in which the conserved acidic amino acid residues were substituted to alanine. This suggests that these residues coordinate divalent metal ions. We conclude that the four conserved acidic amino acids are essential residues of the PARN active site and that the active site of PARN functionally and structurally resembles the active site for 3-exonuclease domain of Escherichia coli DNA polymerase I. Poly(A)-specific ribonuclease (PARN)1 is the only mammalian poly(A)-specific 3Ј-exoribonuclease identified and characterized thus far (1-5). It has been shown that PARN is an oligomeric, highly processive, and mRNA cap-interacting exonuclease (3, 6 -8). A reaction pathway for PARN degradation has been proposed, and key characteristics of this pathway are the release of 5Ј-AMP as the mononucleotide product and the requirement for a 3Ј located adenosine residue with a free 3Ј-hydroxyl group (5). PARN has been cloned from human and Xenopus laevis, and the PARN polypeptide is M r 74,000 in both cases (2, 9). However, the active mammalian enzyme is significantly larger due to its oligomeric structure and most likely consists of three identical subunits (3).The function of PARN in vivo is still unknown. However, compelling evidence suggests that PARN is a key nuclease involved in both mRNA decay (7) and mRNA poly(A) tail length control during X. laevis oocyte development (2, 9). The cap interacting and poly(A) degrading properties of PARN also provide evidence that PARN may play a role in controlling initiation of protein synthesis (3, 6 -8). Besides a possible role in controlling translation, the mRNA cap binding property of PARN has a direct role in stimulating PARN degradation efficiency (3, 6, 7) and in amplifying the processive mode of degradation (8). We have recently proposed a simple model for how PARN operates (8). In this model, oligomeric PARN interacts simultaneously with the mRNA 3Ј-end-located poly(A) tail and the 5Ј-end-located cap structure. It has been suggested, based on kinetic evidence, that the cap-binding site is separate from the active site of PARN (8).The amino acid sequence of PARN implies that PARN belongs to the RNase D family of nucleases (2,11,12), of which the 3Ј-exonuclease (or proofreading) domain of Escherichia coli DNA polymerase

No abstract