mRNA poly(A) tail, a 3' enhancer of translational initiation.

Abstract

To evaluate the hypothesis that the 3' poly(A) tract of mRNA plays a role in translational initiation, we constructed derivatives of pSP65 which direct the in vitro synthesis of mRNAs with different poly(A) tail lengths and compared, in reticulocyte extracts, the relative efficiencies with which such mRNAs were translated, degraded, recruited into polysomes, and assembled into messenger ribonucleoproteins or intermediates in the translational initiation pathway. Relative to mRNAs which were (14,19,39,48,50). These poly(A) "tails" are added post-transcriptionally in the nucleus, with an initial length, in mammalian cells, of approximately 200 to 250 adenylate residues (9). Following transport of mRNA to the cytoplasm, poly(A) tracts are gradually shortened so that, in steady state, poly(A) tail lengths are heterogeneous, ranging from 50 to 70 A's (2,8,10,38,59,80). Although it has been almost two decades since the discovery of these poly(A) tracts, their function(s) has yet to be clarified.Earlier results from our laboratory (37, 53, 54, 61, 79) led us to propose that poly(A) has a role in translation. More specifically, we proposed that an interaction of the cytoplasmic poly(A)-binding protein (PABP) with a critical minimum length of poly(A) facilitates the initiation of translation of polyadenylated [poly(A)+] but not nonpolyadenylated [poly(A)-] mRNAs. The results of several different experimental approaches have provided evidence which indirectly supports this hypothesis. These results include (i) the correlation of specific changes in mRNA poly(A) tail length with translational efficiency following fertilization in Spisula oocytes (72), during early development in Dictyostelium discoideum (61), oocyte maturation in Xenopus laevis (34,55), oocyte activation in mice (33,84,86), salivary gland development in Drosophila melanogaster (67), and in response to physiologic stimuli in the rat hypothalamus (11,69,89); (ii) the higher translational activity of poly(A)+ as opposed to poly(A)-mRNAs in vitro, as demonstrated in systems as diverse as cell extracts derived from rabbit reticulocytes (16, 73), Ehrlich ascites tumor cells (31), and wheat germ (73), as well as in microinjected Xenopus oocytes (15,21,34) and electroporated tobacco protoplasts (22); (iii) a correlation between the abundance and stability of PABPs and the rate of translational initiation in developing or heat-shocked Dictyostelium discoideum amoebae (53,

Replacement of the yeast TRP4 3??? untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail

“…Comparisons of mRNA levels, enzyme assays, and analyses of mRNA association with polyribosomes demonstrated that translation of the TRP4-ribozyme mRNA was reduced approximately 50-75%+ Impaired utilization of this mRNA may well reflect the important role of the poly(A) tail in translation initiation (Munroe & Jacobson, 1990;Tarun & Sachs, 1995;)+ Under most circumstances, it would be expected that a poly(A)-deficient mRNA would initiate poorly in yeast (Gallie, 1991;Proweller & Butler, 1994), an event consistent with the shift of approximately half of the ribozymecontaining mRNA to the nonpolysomal fractions of the cytoplasm (Fig+ 3)+ It is likely that the TRP4-ribozyme construct used here will prove valuable in further analyses of the mechanism of translation of poly(A) Ϫ mRNAs in yeast+ An additional translational regulatory phenomenon uncovered by these studies is suggested by the specific translational activity of the TRP4 ⌬39 UTR mRNA (Fig+ 2)+ Whereas the level of this mRNA is reduced more than threefold, relative to wild-type, PR-transferase activity is reduced less than half+ These results imply that the deletion has removed a TRP4 39 UTR sequence element normally capable of repressing translation of this mRNA+ The existence of such an element is not without precedent (Gray & Wickens, 1998)+ As summarized above, cells expressing the TRP4-ribozyme construct have decreased levels of Trp4p and are starved for tryptophan+ In light of the ability of yeast cells to alter their patterns of gene expression to ensure survival in different environments, it was not surprising that the TRP4-ribozyme construct elicited the activation of the general control system of amino acid biosynthesis+ The partial activation observed undoubtedly reflects the ability of cells harboring these constructs to synthesize modest amounts of tryptophan sufficient to support growth of trp4 cells+…”

The mechanism and regulation of deadenylation: Identification and characterization of Xenopus PARN

“…The role of poly(A) as a determinant of maternal mRNA translation during the meiotic maturation or subsequent fertilization of Xenopus oocytes is well established (Richter, 2000)+ This regulatory system discriminates between classes of mRNAs that are either polyadenylated or deadenylated during maturation+ One class of mRNAs, exemplified by G10, c-mos, and B4, contains the 39 UTR localized cis-sequences required for polyadenylation and subsequent translational activation (Dworkin & Dworkin, 1985;Fox et al+, 1989;McGrew et al+, 1989;Wormington, 1994)+ The cis sequences required for polyadenylation are the U-rich cytoplasmic polyadenylation element (CPE) and the ubiquitous nuclear polyadenylation element (AAUAAA) (McGrew & Richter, 1990;Paris & Richter, 1990)+ The deletion or mutational inactivation of either of these elements prevents both polyadenylation and translation (Fox et al+, 1989;McGrew et al+, 1989)+ In contrast, poly(A) removal is a default reaction deadenylating messages that lack a CPE such as those encoding ribosomal proteins and actin (Fox & Wickens, 1990;Varnum & Wormington, 1990)+ Deadenylated messages are dissociated from polysomes, thus preventing further translation+ Although the poly(A) tail is not necessarily sufficient for translatability (McGrew et al+, 1989), in no case has deadenylation been uncoupled from translational inactivation+ For example, the overexpression of poly(A)-binding protein (PABP) in Xenopus oocytes inhibits both maturation-specific deadenylation and translational silencing (Wormington et al+, 1996)+ The activity responsible for deadenylation in mature oocytes is initially nuclear associated, as poly(A) removal is a late maturation event that cannot be detected prior to nuclear envelope breakdown and is prevented if oocytes are enucleated prior to maturation (Varnum et al+, 1992)+ Importantly, Xenopus is the only system for which an in vivo function for deadenylation has been described (Fox & Wickens, 1990;Varnum & Wormington, 1990)+ The role of poly(A) in translation has been under intense scrutiny over the past few years (Sachs et al+, 1997;Sachs & Varani, 2000)+ The closed loop model of mRNA translation originally proposed by Munroe and Jacobson (1990) has been validated by subsequent biochemical and genetic evidence of interactions between the PABP and the cap-binding complex in yeast and mammalian systems (Tarun & Sachs, 1996;…”

An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

“…The stimulation of translation by the 59-m 7 G cap and 39-poly(A) tail in nucleasetreated RRL is additive and not synergistic (Munroe and Jacobson 1990). In contrast, cell-free translation extracts from HeLa cells show synergy between the 59-cap structure and 39-poly(A) for efficient translation (Svitkin et al 1996(Svitkin et al , 2001Bergamini et al 2000), but the efficiency of utilization of exogenous mRNAs is very low (Spirin 2004).…”