The poly(A)‐binding protein facilitates in vitro translation of poly(A)‐rich mRNA

Abstract

To investigate the role of the 73-kDa poly(A)-binding protein in protein synthesis, the effect of the addition of homo-polyribonucleotides on the translation of polyadenylated and non-adenylated mRNA was studied in the rabbit reticulocyte lysate. Poly(A) was found to be the most effective polynucleotide in inhibiting duck-globin mRNA translation, whereas it had no effect on the translation of polyribosomal duck-globin mRNP, or on the endogenous synthesis of the rabbit reticulocyte lysate. The translation of poly(A)-free mRNA was not affected by the addition of poly(A).Furthermore, we found that the inhibiting effect of poly(A) can be reversed by addition of purified poly(A)-binding protein. It is thus likely that the 73-kDa poly(A)-binding protein is an essential factor necessary for poly(A)-rich mRNA translation.The presence of a stretch of adenylate residues at the 3' end of eukaryotic mRNA has been known for over a decade, but its precise role still remains undefined [l -41. Only a few classes of somatic-cell mRNA do not possess a poly(A) tail long enough to be retained on an oligo(dT) column, as those coding for the histones and some viral mRNAs such as reovirus, TMV and TYMV [5 -71. Investigation of the cytoplasmic role of poly(A) has concentrated in the past on mRNA stability and efficiency of translation.A role of the poly(A) sequence in maintaining message stability was suggested by microinjection experiments showing, in the case of rabbit-globin mRNA and human-histone mRNA, a stringent relationship between the presence of a poly(A) tail and mRNA stability in the frog oocyte and HeLa cells [6,[8][9][10]. This relationship is however apparently not true for all types of mRNA. For instance interferon [ l l ] and mengovirus mRNA [12], whether in their natural polyadenylated or enzymatically deadenylated form, have the same half-life in oocytes. Furthermore, all ten reovirus mRNAs have been shown to be stable for several days following microinjection [7]. A study of the endogenous Xenopus leavis oocyte mRNA coding for the histones has shown that as much as 25-50% is non-adenylated and yet is clearly stable during the long period of oogenesis [13, 141. As another possibility, a function of the poIy(A) tail (and the protein associating with it) in mRNA translation was suggested [15, 161. The efficiency of translation of enzymatically deadenylated mRNA has been compared to that of natural poly(A)-rich mRNA in a variety of in vitro proteinsynthesizing systems. A significant difference between the two forms of mRNA, suggesting a facilitating effect of the poly(A) tail on translation, was only observed when systems with a high rate of re-initiation, such as the reticulocyte lysate, were employed. On the other hand, in the wheat germ or Krebs In eukaryotic cells, the mRNA recovered from polyribosomes dissociated with EDTA or puromycin in the form of mRNP is found tightly associated with a major protein of about 73 kDa, as well as with several other minor proteins [18, 191. This 73-kDa protein appears to be ubiqui...

RNA pseudoknot domain of tobacco mosaic virus can functionally substitute for a poly(A) tail in plant and animal cells.

“…The TMV 3'-UTR and a poly(A) tail do not necessarily operate through the same mechanism, however. The inhibitory effect of exogenous poly(A) on translation of polyadenylated mRNA (Jacobson and Favreau 1983;Grossi de SA et al 1988) suggests that free poly(A) successfully competes with the polyadenylated mRNA for the poly(A}-binding protein, a factor required for efficient expression. In contrast, exogenous poly(A) has no effect on the expression of TMV mRNA (Grossi de SA et al 1988), suggesting that the pseudoknot domain and tRNA-like structure do not require poly(A) binding protein for efficient function.…”

The Major Core Protein of Messenger Ribonucleoprotein Particles (p50) Promotes Initiation of Protein Biosynthesis in Vitro

“…p50 is not the only mRNP protein implicated in the initiation phase of protein synthesis. Another major mRNP protein, p70 (or poly(A)-binding protein), appears to be involved in protein synthesis initiation, too (37)(38)(39)(40). Whether poly(A)-binding protein and p50 interact directly on mRNAs has not yet been determined.…”

Multiple functions for the poly(A)-binding protein in mRNA decapping and deadenylation in yeast.

“…It is the major cytoplasmic poly(A)-binding protein and is highly conserved among eukaryotic organisms (Sachs et al 1986;Grange et al 1987;Zelus et al 1989;Belostotsky and Meagher 1993). Additionally, several lines of evidence argue that Pablp plays a role in stimulating translation initiation (Grossi de Sa et al 1988;Sachs and Davis 1989), which suggests that this protein can influence events at the 5' end of an mRNA while bound to the 3' poly(A) tail.…”