Identification of a Human Cytoplasmic Poly(A) Nuclease Complex Stimulated by Poly(A)-binding Protein
Journal of Biological Chemistry
The poly(A) tail shortening in mRNA, called deadenylation, is the first rate-limiting step in eukaryotic mRNA turnover, and the polyadenylate-binding protein (PABP) appears to be involved in the regulation of this step. However, the precise role of PABP remains largely unknown in higher eukaryotes. Here we identified and characterized a human PABP-dependent poly(A) nuclease (hPAN) complex consisting of catalytic hPan2 and regulatory hPan3 subunits. hPan2 has intrinsically a 3 to 5
... to 5 exoribonuclease activity and requires Mg 2؉ for the enzyme activity. On the other hand, hPan3 interacts with PABP to simulate hPan2 nuclease activity. Interestingly, the hPAN nuclease complex has a higher substrate specificity to poly(A) RNA upon its association with PABP. Consistent with the roles of hPan2 and hPan3 in mRNA decay, the two subunits exhibit cytoplasmic co-localization. Thus, the human PAN complex is a poly(A)-specific exoribonuclease that is stimulated by PABP in the cytoplasm. Eukaryotic mRNAs have two major features, a 5Ј-terminal cap and a 3Ј-terminal poly(A) tail. Both of them play important roles in eukaryotic gene expression, especially in translation and mRNA decay processes. mRNAs are synergistically translated in the presence of both the 5Ј-cap and the 3Ј-poly(A) tail (1-4). During translation, the 5Ј-cap and the 3Ј-poly(A) tail are recognized by eIF4E and the poly(A)-binding protein (PABP), 1 respectively, and eIF4G mediates their association (5, 6). These result in the formation of a circularized mRNA (7) , which provides a structural basis for the hypothetical machinery of efficient translation; ribosomes after translation termination are recruited to the next cycle of translation initiation (8 -11). The removal of the poly(A) tail from mRNA leads to translation inhibition and is used as a strategy to silence certain maternal mRNAs during oocyte maturation and early embryonic development. On the other hand, both the 5Ј-cap and 3Ј-poly(A) tail are also involved in the regulation of mRNA decay (12). The removal of the poly(A) tail is the first rate-limiting step in the degradation of most mRNAs (13-15). This step is followed by the removal of the 5Ј-cap and exonucleolytic degradation of the mRNA body. Thus, deadenylation greatly affects gene expression in regard to abundance as well as the translation of mRNA. Many factors involved in mRNA decay have been identified. The 5Ј-cap is removed by decapping enzymes termed Dcps. Dcp1, Dcp2, and DcpS were identified in Saccharomyces cerevisiae and metazoans (16 -22). On the other hand, the 3Јpoly(A) tail is degraded by deadenylases, and the Ccr4/Pop2 and PAN nuclease (consisting of Pan2 and Pan3) were identified as the two major mRNA deadenylases in S. cerevisiae (23) (24) (25) (26) (27) . A strain lacking both Ccr4 and Pan2 exhibits no deadenylating activity (27) . Yeast PAN nuclease is characterized by the requirement of Pab1, the yeast PABP, for its deadenylating activity. In addition, two other deadenylases in metazoans were reported, namely the poly(A)-specific ribonuclease PARN (28, 29) and nocturnin (30). PARN is the most extensively investigated deadenylase in higher eukaryotes and is related to the enzyme activity involved in default deadenylation during Xenopus oocyte meiotic maturation (31). The most prominent feature of PARN is that its deadenylating activity is stimulated by the 5Ј-cap on mRNA (32-34). Nocturnin is a novel gene that is rhythmically expressed in the cytoplasm of retinal photoreceptor cells in a circadian clock-dependent manner (35) and is structurally related to the C-terminal deadenylase domain of Ccr4 (36). However, the orthologues of PARN and nocturnin are not present in S. cerevisiae. The roles of PABP in mRNA decay are enigmatic. Biochemical experiments showed that PABP can protect the poly(A) tail from degradation (37, 38) and that, in higher eukaryotes, PABP inhibits the deadenylating activity of PARN under physiological conditions (28, 31). On the other hand, genetic approaches indicate that poly(A) tail shortening rates of mRNA are significantly reduced in S. cerevisiae strains lacking the Pab1 (39 -41). In this study, we cloned the full-length human Pan2 (hPan2) and Pan3 (hPan3) cDNAs from HeLa cells. Biochemical analysis revealed that hPan2 is a Mg 2ϩ -dependent exoribonuclease and that hPan3 interacts with both hPan2 and PABP simultaneously. The intrinsic deadenylating activity of hPan2 is stimulated by PABP in the presence of hPan3, and the hPAN complex has a higher substrate specificity to poly(A) RNA when it associates with PABP. hPan2 and hPan3 exhibit cytoplasmic co-localization, consistent with their role in mRNA decay.