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.     

Yeast cells lacking 5''-->3'' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5'' cap structure.

Analysis of the slowed turnover rates of several specific mRNA species and the higher cellular levels of some of these mRNAs in Saccharomyces cerevisiae lacking 5'-->3' exoribonuclease 1 (xrn1 cells) has led to the finding that these yeast contain higher amounts of essentially full-length mRNAs that do not bind to oligo(dT)-cellulose. On the other hand, the length of mRNA poly(A) chains found after pulse-labeling of cells lacking the exoribonuclease, the cellular rate of synthesis of oligo(dT)-bound mRNA, and the initial rate of its deadenylation appeared quite similar to the same measurements in wild-type yeast cells. Examination of the 5' cap structure status of the poly(A)-deficient mRNAs by comparative analysis of the m7G content of poly(A)- and poly(A)+ RNA fractions of wild-type and xrn1 cells suggested that the xrn1 poly(A)- mRNA fraction is low in cap structure content. Further analysis of the 5' termini by measurements of the rate of 5'-->3' exoribonuclease 1 hydrolysis of specific full-length mRNA species showed that approximately 50% of the xrn1 poly(A)-deficient mRNA species lack the cap structure. Primer extension analysis of the 5' terminus of ribosomal protein 51A (RP51A) mRNA showed that about 30% of the poly(A)-deficient molecules of the xrn1 cells are slightly shorter at the 5' end. The finding of some accumulation of poly(A)-deficient mRNA species partially lacking the cap structure together with the reduction of the rate of mRNA turnover in cells lacking the enzyme suggest a possible role for 5'-->3' exoribonuclease 1 in the mRNA turnover process.     

Site-directed ribose methylation identifies 2'-OH groups in polyadenylation substrates critical for AAUAAA recognition and poly(A) addition

The importance of sugar contacts for the sequence-specific recognition that occurs during polyadenylation of mRNAs was investigated with chemically synthesized substrates containing 2'-O-CH3 groups at selected riboses. An RNA (5'-CUGCAAUAAACAAGU-UAA-3') with 2'-O-CH3 ribose at each nucleotide except for the AAUAAA sequence and 3'-terminal adenosine was efficiently polyadenylated in vitro. Methylation of single riboses within AAUAAA inhibited both poly(A) addition and binding of the specificity factor, but the magnitude of inhibition varied greatly at different nucleotides. Nucleotides that showed sensitivity to base substitutions did not necessarily show sensitivity to ribose methylation, and vice versa. The data indicate that the specificity factor interacts with AAUAAA through RNA-protein contacts involving essential recognition of both sugars and bases at different nucleotide positions.     

Poly(A)-tail-promoted translation in yeast: implications for translational control.

The cap structure and the poly(A) tail synergistically activate mRNA translation in vivo. Recent work using Saccharomyces cerevisiae spheroplasts and a yeast cell-free translation system revealed that the poly(A) tail can function as an independent promotor for ribosome recruitment, to internal initiation sites within an mRNA. This raises the question of how regulatory upstream open reading frames and translational repressor proteins binding to the 5'UTR can function, as well as how regulated polyadenylation can support faithful activation of protein synthesis. We investigated the function of the regulatory upstream open reading frame 4 from the yeast GCN 4 gene and the effect of IRP-1 binding to an iron-responsive element introduced into the 5' UTR of reporter mRNAs. Both manipulations effectively block cap-dependent translation, whereas ribosome recruitment promoted by the poly(A) tail under non-competitive conditions can efficiently bypass both blocks. We show that the synergistic use of both, the cap structure and the poly-A tail enforced by mRNA competition reinstates the full extent of translational control by both types of 5' UTR regulatory elements. With a view towards regulated polyadenylation, we studied the function of poly(A) tails of defined length on the translation of capped mRNAs. We find that poly(A) tail elongation increases translational efficiency, particularly under competitive conditions. Our results integrate recent findings on the function of the poly(A) tail into an understanding of translational control.     

cis-acting translational effects of the 5'' noncoding region of c-myc mRNA.

We have previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte lysate (A. Darveau, J. Pelletier, and N. Sonenberg, Proc. Natl. Acad. Sci. USA 82:2315-2319, 1985). We wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. Here we report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, we report that the cis-inhibitory effect is also exhibited in microinjected Xenopus oocytes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.     

Polyadenylation of maternal mRNA during oocyte maturation: poly(A) addition in vitro requires a regulated RNA binding activity and a poly(A) polymerase.

Specific maternal mRNAs receive poly(A) during early development as a means of translational regulation. In this report, we investigated the mechanism and control of poly(A) addition during frog oocyte maturation, in which oocytes advance from first to second meiosis becoming eggs. We analyzed polyadenylation in vitro in oocyte and egg extracts. In vivo, polyadenylation during maturation requires AAUAAA and a U-rich element. The same sequences are required for polyadenylation in egg extracts in vitro. The in vitro reaction requires at least two separable components: a poly(A) polymerase and an RNA binding activity with specificity for AAUAAA and the U-rich element. The poly(A) polymerase is similar to nuclear poly(A) polymerases in mammalian cells. Through a 2000-fold partial purification, the frog egg and mammalian enzymes were found to be very similar. More importantly, a purified calf thymus poly(A) polymerase acquired the sequence specificity seen during frog oocyte maturation when mixed with the frog egg RNA binding fraction, demonstrating the interchangeability of the two enzymes. To determine how polyadenylation is activated during maturation, we compared polymerase and RNA binding activities in oocyte and egg extracts. Although oocyte extracts were much less active in maturation-specific polyadenylation, they contained nearly as much poly(A) polymerase activity. In contrast, the RNA binding activity differed dramatically in oocyte and egg extracts: oocyte extracts contained less binding activity and the activity that was present exhibited an altered mobility in gel retardation assays. Finally, we demonstrate that components present in the RNA binding fraction are rate-limiting in the oocyte extract, suggesting that fraction contains the target that is activated by progesterone treatment. This target may be the RNA binding activity itself. We propose that in spite of the many biological differences between them, nuclear polyadenylation and cytoplasmic polyadenylation during early development may be catalyzed by similar, or even identical, components.     

A viral sequence in the 3''-untranslated region mimics a 5'' cap in facilitating translation of uncapped mRNA.

For recognition by the translational machinery, most eukaryotic cellular mRNAs have a 5' cap structure [e.g. m7G(5')ppp(5')N]. We describe a translation enhancer sequence (3'TE) located in the 3'-untranslated region (UTR) of the genome of the PAV barley yellow dwarf virus (BYDV-PAV) which stimulates translation from uncapped mRNA by 30- to 100-fold in vitro and in vivo to a level equal to that of efficient capped mRNAs. A four base duplication within the 3'TE destroyed the stimulatory activity. Efficient translation was recovered by addition of a 5' cap to this mRNA. Translation of both uncapped mRNA containing the 3'TE in cis and capped mRNA lacking any BYDV-PAV sequence was inhibited specifically by added 3'TE RNA in trans. This inhibition was reversed by adding initiation factor 4F (eIF4F), suggesting that the 3'TE, like the 5' cap, mediates eIF4F-dependent translation initiation. The BYDV-PAV 5'UTR was necessary for the 3'TE to function, except when the 3'TE itself was moved to the 5'UTR. Thus, the 3'TE is sufficient for recruiting the translation factors and ribosomes, while the viral 5'UTR may serve only for the long distance 3'-5' communication. Models are proposed to explain this novel mechanism of cap-independent translation initiation facilitated by the 3'UTR.     

Kinetic proofreading scanning models for eukaryotic translational initiation: the cap and poly(A) tail dependency of translation

Two simplified kinetic proofreading scanning (KPS) models were proposed to describe the 5' cap and 3' poly(A) tail dependency of eukaryotic translation initiation. In Model I, the initiation factor complex starts scanning and unwinding the secondary structure of the 5' untranslated region (UTR) from the 5' terminus of mRNA. In Model II, the initiation factor complex starts scanning from any binding site in the 5' UTR. In both models, following ATP hydrolysis, the initiation factor complex either dissociates from mRNA or continues to scan and unwind RNA secondary structure in the 5' UTR. This step repeats n times until the AUG codon is reached. These two models show very different cap and/or poly(A) tail dependency of translation initiation. The models predict that both cap and poly(A) tail dependencies of translation, and translatability of mRNAs are coupled with the structure of 5' UTR: the translation of mRNA with structured 5' UTR is strongly cap- and poly(A) tail-dependent; while translation of mRNA with unstructured 5' UTR is less cap- and poly(A) tail-dependent. We use these two models to explain: (1) the cap and poly(A) tail dependence of translation; (2) the effect of exogenous poly(A) on translation; (3) repression of host mRNA and translation of late adenovirus mRNA in the late phase of adenovirus infection; (4) repression of host mRNA and translation of Vaccinia virus mRNA in virus-infected cell; (5) heat shock repression of translation of normal mRNA and stimulation of translation of hsp mRNA; and (6) the synergistic effect of cap and poly(A) tail on stimulating translation. The kinetic proofreading scanning models provide a coherent interpretation of those phenomena.     

Conditional defect in mRNA 3'' end processing caused by a mutation in the gene for poly(A) polymerase.

Maturation of most eukaryotic mRNA 3' ends requires endonucleolytic cleavage and polyadenylation of precursor mRNAs. To further understand the mechanism and function of mRNA 3' end processing, we identified a temperature-sensitive mutant of Saccharomyces cerevisiae defective for polyadenylation. Genetic analysis showed that the polyadenylation defect and the temperature sensitivity for growth result from a single mutation. Biochemical analysis of extracts from this mutant shows that the polyadenylation defect occurs at a step following normal site-specific cleavage of a pre-mRNA at its polyadenylation site. Molecular cloning and characterization of the wild-type allele of the mutated gene revealed that it (PAP1) encodes a previously characterized poly(A) polymerase with unknown RNA substrate specificity. Analysis of mRNA levels and structure in vivo indicate that shift of growing, mutant cells to the nonpermissive temperature results in the production of poly(A)-deficient mRNAs which appear to end at their normal cleavage sites. Interestingly, measurement of the rate of protein synthesis after the temperature shift shows that translation continues long after the apparent loss of polyadenylated mRNA. Our characterization of the pap1-1 defect implicates this gene as essential for mRNA 3' end formation in S. cerevisiae.     

The autoregulatory translational control element of poly(A)-binding protein mRNA forms a heteromeric ribonucleoprotein complex

Repression of poly(A)-binding protein (PABP) mRNA translation involves the binding of PABP to the adenine-rich autoregulatory sequence (ARS) in the 5′-untranslated region of its own mRNA. In this report, we show that the ARS forms a complex in vitro with PABP, and two additional polypeptides of 63 and 105 kDa. The 63 and 105 kDa polypeptides were identified, as IMP1, an ortholog of chicken zip-code binding polypeptide, and UNR, a PABP binding polypeptide, respectively, by mass spectrometry of the ARS RNA affinity purified samples. Using a modified ribonucleoprotein (RNP) immunoprecipitation procedure we further show that indeed, both IMP1 and UNR bind to the ARS containing reporter RNA in vivo. Although both IMP1 and UNR could bind independently to the ARS RNA in vitro, their RNA-binding ability was stimulated by PABP. Mutational analyses of the ARS show that the presence of four of the six oligo(A) regions of the ARS was sufficient to repress translation and the length of the conserved pyrimidine spacers between the oligo(A) sequences was important for ARS function. The ability of mutant ARS RNAs to form the PABP, IMP1 and UNR containing RNP complex correlates well with the translational repressor activity of the ARS. There is also a direct relationship between the length of the poly(A) RNAs and their ability to form a trimeric complex with PABP, and to repress mRNA translation. UV crosslinking studies suggest that the ARS is less efficient than a poly(A) RNA of similar length, to bind to PABP. We show here that the ARS cannot efficiently form a trimeric complex with PABP; therefore, additional interactions with IMP1 and UNR to form a heteromeric RNP complex may be required for maximal repression of PABP mRNA translation under physiological conditions.     

Translation of maternal histone mRNAs in sea urchin embryos: a test of control by 5' cap methylation

Recent results have demonstrated the occurrence of mRNA cap methylation in the sea urchin embryo following fertilization. It has been suggested that this methylation event is responsible for the translational activation of maternal histone mRNAs in these embryos. We have used aphidicolin, an effective inhibitor of both DNA synthesis and cap methylation in cleavage stage sea urchin embryos, to examine the relationship between cap methylation and translation. At 5 micrograms/ml, a dose which rapidly abolishes DNA replication and blocks cleavage, we note no effect on recruitment or translation of maternal alpha-subtype histone mRNAs. This suggests that a postfertilization cap methylation event is not critical to the process of regulation of the translation of stored alpha-subtype histone mRNAs.     

Infrared CD of deoxy oligonucleotides. Conformational studies of 5''d(GCGC)3'', 5''d(CGCG)3'', 5''d(CCGG)3'', and 5''d(GGCC)3'' in low and high salt aqueous solution.

Infrared (vibrational) circular dichroism (VCD) spectra have been obtained for the self-complementary tetranucleotides, 5'd(CGCG)3', 5'd(GCGC)3', 5'd(CCGG)3', and 5'd(GGCC)3'. In buffered aqueous solution at low salt concentration, these tetramers exhibit spectra associated with right-handed polymers, although the spectra differ significantly for the four species. In high salt solution, a B-->Z transition occurs in 5'd(CGCG)3', while the other tetranucleotides appear only slightly altered. Temperature dependent studies of these oligonucleotides reveal a greater amount of thermal stability for the tetramers in low salt than for the high salt solutions. VCD intensities computed via the exciton formalism are compared with observed results.     

Nonenzymic adenosine 5'-diphosphate ribosylation of poly(adenosine diphosphate ribose)

Poly(adenosine 5'-diphosphate ribose) [poly(ADP-ribose]) is spontaneously ADP-ribosylated when it is incubated with nicotinamide adenine dinucleotide, especially in 0.5 M NaCl and at an alkaline pH. The ADP-ribose residues are monomeric and are attached to the middle of polymer chains. The linkage is similar to, and may be identical with, that of the branch points that are created in cells. RNA is also spontaneously ADP-ribosylated, but not DNA.     

Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates.

We demonstrate here that both procaspase-3 (32 kDa) and PARP are calpain substrates. In calcium-channel opener maitotoxin-treated cells, a 30 kDa caspase-3 fragment is produced in a time and concentration-dependent manner. Formation of this fragment is prevented by calpain inhibitors but not by the pancaspase inhibitor, carbobenzoxy-Asp-CH(2)OC(O)-2,6-dichlorobenzene (Z-D-DCB) nor the selective proteasome inhibitor lactacystin. In maitotoxin-treated cells, PARP (113 kDa) is also cleaved into a 40 kDa immunoreactive fragment, in a calpain-inhibitor-sensitive manner. Both procaspase-3 and PARP are also cleaved in vitro by purified micro-calpain to a 30 kDa fragment and a 40 kDa fragment, respectively. Finally, we show that staurosporine-mediated caspase-3 activation is interrupted by maitotoxin pretreatment.     

Interaction between a poly(A)-specific ribonuclease and the 5' cap influences mRNA deadenylation rates in vitro

We have used an in vitro system that reproduces in vivo aspects of mRNA turnover to elucidate mechanisms of deadenylation. DAN, the major enzyme responsible for poly(A) tail shortening in vitro, specifically interacts with the 5' cap structure of RNA substrates, and this interaction is greatly stimulated by a poly(A) tail. Several observations suggest that cap-DAN interactions are functionally important for the networking between regulated mRNA stability and translation. First, uncapped RNA substrates are inefficiently deadenylated. Second, a stem-loop structure in the 5' UTR dramatically reduces deadenylation by interfering with cap-DAN interactions. Third, the addition of cap binding protein eIF4E inhibits deadenylation in vitro. These data provide insights into the early steps of substrate recognition that target an mRNA for degradation.     

Cytoplasmic polyadenylation element (CPE)- and CPE-binding protein (CPEB)-independent mechanisms regulate early class maternal mRNA translational activation in Xenopus oocytes

Meiotic cell cycle progression during vertebrate oocyte maturation requires the correct temporal translation of maternal mRNAs encoding key regulatory proteins. The mechanism by which specific mRNAs are temporally activated is unknown, although both cytoplasmic polyadenylation elements (CPE) within the 3'-untranslated region (3'-UTR) of mRNAs and the CPE-binding protein (CPEB) have been implicated. We report that in progesterone-stimulated Xenopus oocytes, the early cytoplasmic polyadenylation and translational activation of multiple maternal mRNAs occur in a CPE- and CPEB-independent manner. We demonstrate that polyadenylation response elements, originally identified in the 3'-UTR of the mRNA encoding the Mos proto-oncogene, direct CPE- and CPEB-independent polyadenylation of an early class of Xenopus maternal mRNAs. Our findings refute the hypothesis that CPE sequences alone account for the range of temporal inductions of maternal mRNAs observed during Xenopus oocyte maturation. Rather, our data indicate that the sequential action of distinct 3'-UTR-directed translational control mechanisms coordinates the complex temporal patterns and extent of protein synthesis during vertebrate meiotic cell cycle progression.