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

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 polyadenylated, we found that nonpolyadenylated [poly(A)-]mRNAs had a reduced translational capacity which was not due to an increase in their decay rates, but was attributable to a reduction in their efficiency of recruitment into polysomes. The defect in poly(A)- mRNAs affected a late step in translational initiation, was distinct from the phenotype associated with cap-deficient mRNAs, and resulted in a reduced ability to form 80S initiation complexes. Moreover, poly(A) added in trans inhibited translation from capped polyadenylated mRNAs but stimulated translation from capped poly(A)- mRNAs. We suggest that the presence of a 3' poly(A) tail may facilitate the binding of an initiation factor or ribosomal subunit at the mRNA 5' end.     

Translational regulation of the expression of ribosomal protein genes in Xenopus laevis

The mRNAs coding for ribosomal proteins (rp-mRNA) are subjected to translational control during Xenopus oogenesis and embryogenesis, and also during nutritional changes in Xenopus cultured cells. This regulation, which appears to respond to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA engaged on polysomes, each translated rp-mRNA molecule always remaining fully loaded with ribosomes. All rp-mRNAs analyzed up to now show this translational behavior, and also share some structural features in their untranslated portions. In particular they all have rather short 5' untranslated regions, similar to each other, and always start at the very 5' end with a stretch of several pyrimidines. Fusion to a reporter-coding sequence of the 5' untranslated region of r-protein S19 has shown that this is involved in the translational regulation.     

Translational discrimination of ribosomal protein mRNAs in the early Drosophila embryo

Most Drosophila mRNAs are actively translated in the early embryo, with the exception of the poorly translated ribosomal protein (r-protein) mRNAs. Two possible mechanisms for this translational discrimination were tested: (1) Translation of r-protein mRNAs is discriminated against by the limited activity of translational initiation factors in the early embryo and (2) translation of r-protein mRNAs is repressed by trans-acting factors that reversibly bind these mRNAs. Exogenously provided initiation factors promoted partial recruitment of r-protein mRNAs into polysomes, suggesting that modulation of initiation factor activity may play a role in the translational discrimination of r-protein mRNAs during embryogenesis. No evidence for involvement of reversibly binding trans-acting factors was obtained, although there are limitations in the interpretation of the latter experiments.     

Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.     

Translational regulation of human beta interferon mRNA: association of the 3'' AU-rich sequence with the poly(A) tail reduces translation efficiency in vitro.

The 3' AU-rich region of human beta-1 interferon (hu-IFN beta) mRNA was found to act as a translational inhibitory element. The translational regulation of this 3' AU-rich sequence and the effect of its association with the poly(A) tail were studied in cell-free rabbit reticulocyte lysate. A poly(A)-rich hu-IFN beta mRNA (110 A residues) served as an inefficient template for protein synthesis. However, translational efficiency was considerably improved when the poly(A) tract was shortened (11 A residues) or when the 3' AU-rich sequence was deleted, indicating that interaction between these two regions was responsible for the reduced translation of the poly(A)-rich hu-IFN beta mRNA. Differences in translational efficiency of the various hu-IFN beta mRNAs correlated well with their polysomal distribution. The poly(A)-rich hu-IFN beta mRNA failed to form large polysomes, while its counterpart bearing a short poly(A) tail was recruited more efficiently into large polysomes. The AU-rich sequence-binding activity was reduced when the RNA probe contained both the 3' AU-rich sequence and long poly(A) tail, supporting a physical association between these two regions. Further evidence for this interaction was achieved by RNase H protection assay. We suggest that the 3' AU-rich sequence may regulate the translation of hu-IFN beta mRNA by interacting with the poly(A) tail.     

Ribosomal protein, histone and calmodulin mRNAs are differently regulated at the translational level during oogenesis of Xenopus laevis

The localization of r-protein mRNA in subcellular compartments has been analysed. It was observed that the mRNA for a representative r-protein (L1) is diffuse in the cytoplasm, as shown by in situ hybridization experiments and that the distribution of rp-mRNA between polysomes and light mRNPs changes during oogenesis. In early oogenesis this mRNA is found mostly in subpolysomal fractions, whereas at the beginning of vitellogenesis (stage II) it becomes associated with polysomes where it remains in a constant amount at later stages. Histone and calmodulin mRNA, on the contrary, are mostly associated with non-polysomal fast-sedimenting particles throughout oogenesis. This suggests that the partition of different classes of mRNA between polysomes, light mRNP and heavy particles depends on their nature and might be determined by different requirements for these mRNAs during oogenesis.     

Expression of ribosomal-protein genes in Xenopus laevis development

Using probes to Xenopus laevis ribosomal-protein (r-protein) mRNAs, we have found that in the oocyte the accumulation of r-protein mRNAs proceeds to a maximum level, which is attained at the onset of vitellogenesis and remains stable thereafter. In the embryo, r-protein mRNA sequences are present at low levels in the cytoplasm during early cleavage (stages 2-5), become undetectable until gastrulation (stage 10) and accumulate progressively afterwards. Normalization of the amount of mRNA to cell number suggests an activation of r-protein genes around stage 10; however, a variation in mRNA turnover cannot be excluded. Newly synthesized ribosomal proteins cannot be found from early cleavage up to stage 26, with the exception of S3, L17 and L31, which are constantly made, and protein L5, which starts to be synthesized around stage 7. A complete set of ribosomal proteins is actively produced only in tailbud embryos (stages 28-32), several hours after the appearance of their mRNAs. Before stage 26 these mRNA sequences are found on subpolysomal fractions, whereas more than 50% of them are associated with polysomes at stage 31. Anucleolate mutants do not synthesize ribosomal proteins at the time when normal embryos do it very actively; nevertheless, they accumulate r-protein mRNAs.     

Sequence-specific adenylations and deadenylations accompany changes in the translation of maternal messenger RNA after fertilization of Spisula oocytes

A dramatic change in the pattern of protein synthesis occurs within ten minutes after fertilization of Spisula oocytes. This change is regulated entirely at the translational level. We have used DNA clones complementary to five translationally regulated messenger RNAs to follow shifts in mRNA utilization at fertilization and to characterize alterations in mRNA structure that accompany switches in translational activity in vivo. Four of the mRNAs studied are translationally inactive in the oocyte. After fertilization two of these mRNAs are completely recruited onto polysomes, and two are partially recruited. All four of these mRNAs have very short poly(A) tracts in the oocyte; after fertilization the poly(A) tails lengthen considerably. In contrast, a fifth mRNA, that encoding alpha-tubulin mRNA, is translated very efficiently in the oocyte and is rapidly lost from polysomes after fertilization. Essentially all alpha-tubulin mRNA in the oocyte is poly(A)+ and a large portion of this mRNA undergoes complete deadenylation after fertilization. These results reveal a striking relationship between changes in adenylation and translational activity in vivo. This correlation is not perfect, however. Evidence for and against a direct role for polyadenylation in regulating these translational changes is discussed. Changes in poly(A) tails are the only alterations in mRNA sizes that we have been able to detect. This indicates that, at least for the mRNAs studied here, translational activation is not due to extensive processing of larger translationally incompetent precursors. We have also isolated several complementary DNA clones to RNAs encoded by the mitochondrial genome. Surprisingly, the poly(A) tracts of at least two of the mitochondrial RNAs also lengthen in response to fertilization.     

Evidence for translational control of storage protein biosynthesis during embryogenesis ofAvena sativa L. (oat endosperm)

Oat polysomes direct the synthesisin vitro of a large number of products, the majority of which are the salt-soluble globulins (1,3,10,11,21). Total RNA or poly A⁺ RNA isolated from these polysomes directs the synthesis of the same number and types of products; however, the amount of globulins synthesized no longer represents the major products; rather, there is a decreased level of globulins and an increased amount of the other products synthesizedin vitro (6, 18). These results imply that the translational control can dictate final product levels. Reconstruction experiments using oat poly A⁺ mRNA and polysomal factors that are made free of endogenous RNA by nuclease digestion demonstrate that these factors do influence the translational specificity of oat globulin mRNA relative to other mRNAs. It is suggested that translational control is partially responsible for the levels of globulin in the mature grain.     

Ribosomal protein gene expression is cell type specific during development in Dictyostelium discoideum

Starvation for amino acids initiates the developmental cycle in the cellular slime mold, Dictyostelium discoideum. Upon starvation one of the earliest developmental events is the selective loss of the ribosomal protein mRNAs from polysomes. This loss depends upon sequences in the 5' non-translated leader of the ribosomal protein (r-protein) mRNAs. Here evidence is presented which indicates that those cells which will become prestalk cells express the ribosomal protein genes during development under starvation conditions. Cells which enter the prespore pathway shut off r-protein synthesis. The promoter and 5' non-translated leader sequences from two ribosomal protein genes, the rp-L11 and the rp-S9 genes, are fused to the Escherichia coli beta-galactosidase reporter gene. While beta-galactosidase enzyme activity is detected in situ in most growing cells, by 15 h of development beta-galactosidase enzyme activity is largely lost from the prespore cells although strong beta-galactosidase enzyme activity is present in the prestalk cells. These observations suggest the possibility that the ribosomal protein mRNAs are excluded from polysomes in a cell-type-specific manner.