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 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.     

Uptake and utilization of mRNA by myogenic cells in culture

Primary chick myoblast cultures demonstrate the ability to take up exogenously supplied polyadenylated RNA and express the encoded information in a specific manner. This expression is shown to exhibit tissue specificity. Analysis of creatine kinase activity monitored at various times of incubation in the presence of either polyadenylated or nonpolyadenylated RNA indicates that only the poly(A)+ mRNA is capable of being actively translated. Radioactively labled poly(A)+ mRNA is taken up by the cell cultures in a time-dependent manner and subsequently shown to be associated with polysomes. This association with polysomes does not occur in the presence of puromycin and is unaffected by actinomycin D. Thus, nonspecific interaction with polysomes and induction of new RNA synthesis are ruled out and the association of the exogenously supplied poly(A)+ mRNA with polysomes is indicative of its translation in the recipient cells. When heterologous mRNA (globin) is supplied to the myoblasts, it is also taken up and properly translated. In addition, exogenously supplied myosin heavy chain mRNA is found associated with polysomes consisting of 4-10 ribosomes in myoblast cell cultures while in myotubes it is associated with very large polysomes, thus reflecting the different translational efficiencies that this message exhibits at two very different stages of myogenesis. The results indicate that muscle cell cultures can serve as an in vitro system to study translational controls and their roles in development.     

Regulation of in vitro translation by double-stranded RNA in mammalian cell mRNA preparations.

Polyadenylated mRNA has been purified from a variety of human and mouse cell sources. These preparations are actively translated in the wheat germ cell-free system but have only poor ability to stimulate the nuclease-treated reticulocyte lysate. The translation of endogenous and exogenous globin mRNA is strongly inhibited by the poly(A)+ RNA preparations in reticulocyte lysates. Both polysomal and non-polysomal RNA have similar effects but poly(A)+ RNA is almost 2000-fold more inhibitory than poly(A)-RNA on a weight basis. The inhibition is abolished in the presence a high concentration of poly(I).poly(C). Analysis of endogenous eIF-2 in the lysate reveals that the subunit becomes extensively phosphorylated in the presence of the inhibitory poly(A)+ RNA. Prolonged incubation of lysate with poly(A)+ RNA also causes some nucleolytic degradation of polysomal globin mRNA. These characteristics suggest that some eukaryotic cell mRNAs contain regions of double-stranded structure which are sufficiently extensive to activate translational control mechanisms in the reticulocyte lysate.     

Changes in polyadenylation of lactate dehydrogenase-X mRNA during spermatogenesis in mice

The expression of the mRNA for mouse testicular lactate dehydrogenase (LDH-X) was examined by RNA:cDNA hybridization in situ in the testis and by Northern analyses of meiotic and postmeiotic spermatogenic cell populations. Silver grains accumulated in cells inside the second layer from the periphery of the seminiferous tubule, confirming previous findings that LDH-X mRNA first appears in the spermatocyte and continues to accumulate until the late spermatid stage. Northern analyses showed that meiotic and postmeiotic cells contained 1.2 and 1.3 kb classes of hybridizing mRNA, respectively. RNase H digestion of oligo (dT)-hybridized RNA and poly(U)-Sepharose column chromatography with differential elution by formamide revealed that the difference in size of the two classes of mRNAs was due to the poly(A) tail length of the LDH-X mRNA. When the distribution of the LDH-X mRNA was examined across polysome gradients, both mRNAs were partially associated with polysomes. These results suggest that the changes in the polyadenylation of LDH-X mRNA were associated with the meiotic division during spermatogenesis in the mouse. They raise the possibility that the stable accumulation of the LDH-X mRNAs in the postmeiotic cells is enhanced by poly(A) tails of increased length.     

Roles of a Trypanosoma brucei 5'->3' exoribonuclease homolog in mRNA degradation

The genome of the kinetoplastid parasite Trypanosoma brucei encodes four homologs of the Saccharomyces cerevisiae 5'-->3' exoribonucleases Xrn1p and Xrn2p/Rat1p, XRNA, XRNB, XRNC, and XRND. In S. cerevisiae, Xrn1p is a cytosolic enzyme involved in degradation of mRNA, whereas Xrn2p is involved in RNA processing in the nucleus. Trypanosome XRND was found in the nucleus, XRNB and XRNC were found in the cytoplasm, and XRNA appeared to be in both compartments. XRND and XRNA were essential for parasite growth. Depletion of XRNA increased the abundances of highly unstable developmentally regulated mRNAs, perhaps by delaying a deadenylation-independent decay pathway. Degradation of more stable or unregulated mRNAs was not affected by XRNA depletion although a slight decrease in average poly(A) tail length was observed. We conclude that in trypanosomes 5'-->3' exonuclease activity is important in degradation of highly unstable, regulated mRNAs, but that for other mRNAs another step is more important in determining the decay rate.     

Accumulation of polyadenylated mRNA during liver regeneration.

Cytoplasmic and polysomal polyadenylated mRNA [poly(A)+-mRNA] increased by 120% prior to the onset of DNA synthesis during the regeneration of rat liver following partial hepatectomy. Despite this large change in cytoplasmic mRNA and an approximately 50% increase in total nuclear RNA, the amount of polyadenylated nuclear RNA increased by only 15--20% during this time. Neither the average size of nuclear or of cytoplasmic polyadenylated mRNA nor the length of their poly(adenylic acid) [poly(A)] tracts changed during liver regeneration. Polysomal poly-(A)+-mRNA increased proportionately more and at a faster rate than rRNA during the first day following partial hepatectomy. Normal livers contained a substantial proportion of cytoplasmic poly(A)+-mRNA not associated with polysomes but this proportion was not altered in 3-h regenerating liver. Thus, in regenerating liver, most preexisting cytoplasmic mRNA does not appear to be recruited into polysomes prior to the substantial increase in the amount of cytoplasmic poly(A)+-mRNA.     

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.     

Contribution of polyadenylate sequences to the translational efficiency of globin messenger RNAs.

mRNAs from reticulocyte polysomes were fractionated by chromatography on poly(U)-Sepharose and thermal elution. The molar ratio of alpha- to beta-globin mRNA was found to be 2:1 and 1:1 respectively in short- and long-poly(A) size classes. Translational analyses indicated that the globin mRNAs containing long poly(A) tracts (with a mean length of about 70 nucleotides) directed protein synthesis with higher rates than did mRNA containing short poly(A) tracts (15-35 nucleotides). Experiments performed with sub-saturating mRNA concentrations showed that the digestion with RNAase H induced a decrease in the translational capacity of both globin mRNAs and an increase in the alpha- to beta-globin synthesis ratio. No correlation was observed between the size of the poly(A) tail in mRNA and the optimal K+ requirement for translation.