Multiple sequence elements and a maternal mRNA product control cdk2 RNA polyadenylation and translation during early Xenopus development.

Cytoplasmic poly(A) elongation is one mechanism that regulates translational recruitment of maternal mRNA in early development. In Xenopus laevis, poly(A) elongation is controlled by two cis elements in the 3' untranslated regions of responsive mRNAs: the hexanucleotide AAUAAA and a U-rich structure with the general sequence UUUUUAAU, which is referred to as the cytoplasmic polyadenylation element (CPE). B4 RNA, which contains these sequences, is polyadenylated during oocyte maturation and maintains a poly(A) tail in early embryos. However, cdk2 RNA, which also contains these sequences, is polyadenylated during maturation but deadenylated after fertilization. This suggests that cis-acting elements in cdk2 RNA signal the removal of the poly(A) tail at this time. By using poly(A) RNA-injected eggs, we showed that two elements which reside 5' of the CPE and 3' of the hexanucleotide act synergistically to promote embryonic deadenylation of this RNA. When an identical RNA lacking a poly(A) tail was injected, these sequences also prevented poly(A) addition. When fused to CAT RNA, the cdk2 3' untranslated region, which contains these elements, as well as the CPE and the hexanucleotide, promoted poly(A) addition and enhanced chloramphenicol acetyltransferase activity during maturation, as well as repression of these events after fertilization. Incubation of fertilized eggs with cycloheximide prevented the embryonic inhibition of cdk2 RNA polyadenylation but did not affect the robust polyadenylation of B4 RNA. This suggests that a maternal mRNA, whose translation occurs only after fertilization, is necessary for the cdk2 deadenylation or inhibition of RNA polyadenylation. This was further suggested when poly(A)+ RNA isolated from two-cell embryos was injected into oocytes that were then allowed to mature. Such oocytes became deficient for cdk2 RNA polyadenylation but remained proficient for B4 RNA polyadenylation. These data show that CPE function is developmentally regulated by multiple sequences and factors.     

Eukaryotic initiation factor 4G-poly(A) binding protein interaction is required for poly(A) tail-mediated stimulation of picornavirus internal ribosome entry segment-driven translation but not for X-mediated stimulation of hepatitis C virus translation

Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5' m(7)GpppN cap and the 3' poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRES-driven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3' end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3'-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.     

Plant mitochondrial polyadenylated mRNAs are degraded by a 3'- to 5'-exoribonuclease activity, which proceeds unimpeded by stable secondary structures

Recently, we and others have reported that mRNAs may be polyadenylated in plant mitochondria, and that polyadenylation accelerates the degradation rate of mRNAs. To further characterize the molecular mechanisms involved in plant mitochondrial mRNA degradation, we have analyzed the polyadenylation and degradation processes of potato atp9 mRNAs. The overall majority of polyadenylation sites of potato atp9 mRNAs is located at or in the vicinity of their mature 3'-extremities. We show that a 3'- to 5'-exoribonuclease activity is responsible for the preferential degradation of polyadenylated mRNAs as compared with non-polyadenylated mRNAs, and that 20-30 adenosine residues constitute the optimal poly(A) tail size for inducing degradation of RNA substrates in vitro. The addition of as few as seven non-adenosine nucleotides 3' to the poly(A) tail is sufficient to almost completely inhibit the in vitro degradation of the RNA substrate. Interestingly, the exoribonuclease activity proceeds unimpeded by stable secondary structures present in RNA substrates. From these results, we propose that in plant mitochondria, poly(A) tails added at the 3' ends of mRNAs promote an efficient 3'- to 5'- degradation process.     

In vitro cleavage of the simian virus 40 early polyadenylation site adjacent to a required downstream TG sequence.

Exogenous RNA containing the simian virus 40 early polyadenylation site was efficiently and accurately polyadenylated in in vitro nuclear extracts. Correct cleavage required ATP. In the absence of ATP, nonpoly(A)+ products accumulated which were 18 to 20 nucleotides longer than the RNA generated by correct cleavage; the longer RNA terminated adjacent to the downstream TG element required for polyadenylation. In the presence of ATP analogs, alternate cleavage was not observed; instead, correct cleavage without poly(A) addition occurred. ATP-independent cleavage of simian virus 40 early RNA had many of the same properties as correct cleavage including requirements for an intact AAUAAA element, a proximal 3' terminus, and extract small nuclear ribonucleoproteins. This similarity in reaction parameters suggested that ATP-independent cleavage is an activity of the normal polyadenylation machinery. The ATP-independent cleavage product, however, did not behave as an intermediate in polyadenylation. The alternate RNA did not preferentially chase into correctly cleaved material upon readdition of ATP; instead, poly(A) was added to the 3' terminus of the cleaved RNA during a chase. Purified ATP-independent cleavage RNA, however, was a substrate for correct cleavage when reintroduced into the nuclear extract. Thus, alternate cleavage of polyadenylation sites adjacent to a required downstream sequence element is directed by the polyadenylation machinery in the absence of ATP.     

Hierarchy of polyadenylation site usage by bovine papillomavirus in transformed mouse cells.

The great majority of viral mRNAs in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV) have a common 3' end at the early polyadenylation site which is 23 nucleotides (nt) downstream of a canonical poly(A) consensus signal. Twenty percent of BPV mRNA from productively infected cells bypasses the early polyadenylation site and uses the late polyadenylation site approximately 3,000 nt downstream. To inactivate the BPV early polyadenylation site, the early poly(A) consensus signal was mutated from AAUAAA to UGUAAA. Surprisingly, this mutation did not result in significant read-through expression of downstream RNA. Rather, RNA mapping and cDNA cloning experiments demonstrate that virtually all of the mutant RNA is cleaved and polyadenylated at heterogeneous sites approximately 100 nt upstream of the wild-type early polyadenylation site. In addition, cells transformed by wild-type BPV harbor a small population of mRNAs with 3' ends located in this upstream region. These experiments demonstrate that inactivation of the major poly(A) signal induces preferential use of otherwise very minor upstream poly(A) sites. Mutational analysis suggests that polyadenylation at the minor sites is controlled, at least in part, by UAUAUA, an unusual variant of the poly(A) consensus signal approximately 25 nt upstream of the minor polyadenylation sites. These experiments indicate that inactivation of the major early polyadenylation signal is not sufficient to induce expression of the BPV late genes in transformed mouse cells.     

Accurate cleavage and polyadenylation of exogenous RNA substrate

Purified precursor RNA containing the L3 polyadenylation site of late adenovirus 2 mRNA is accurately cleaved and polyadenylated when incubated with nuclear extract from HeLa cells. The reaction is very efficient; 75% of the precursor is correctly processed. Cleavage is rapidly followed by polymerization of an initial poly(A) tract of approximately 130 nucleotides. Additional adenosine residues are added during further incubation. In the presence of the ATP analog alpha-beta-methylene-adenosine 5' triphosphate, the precursor RNA is cleaved but not polyadenylated, suggesting that processing is not coupled to the synthesis of the initial poly(A) tract. In the absence of free Mg2+, a small RNA of approximately 46 nucleotides is stabilized against degradation. Fingerprint analysis suggests this RNA is produced by endonucleolytic cleavage at the L3 site. Like the in vitro splicing reaction, the in vitro polyadenylation reaction is inhibited by adding antiserum against the small nuclear ribonucleoprotein particle containing U1 RNA.     

A small nuclear ribonucleoprotein associates with the AAUAAA polyadenylation signal in vitro

RNAs containing the polyadenylation sites for adenovirus L3 or E2a mRNA or for SV40 early or late mRNA are substrates for cleavage and poly(A) addition in an extract of HeLa cell nuclei. When polyadenylation reactions are probed with ribonuclease T1 and antibodies directed against either the Sm protein determinant or the trimethylguanosine cap structure at the 5' end of U RNAs in small nuclear ribonucleoproteins, RNA fragments containing the AAUAAA polyadenylation signal are immunoprecipitated. The RNA cleavage step that occurs prior to poly(A) addition is inhibited by micrococcal nuclease digestion of the nuclear extract. The immunoprecipitation of fragments containing the AAUAAA sequence can be altered, but not always abolished, by pretreatment with micrococcal nuclease. We discuss the involvement of small nuclear ribonucleoproteins in the cleavage and poly(A) addition reactions that form the 3' ends of most eukaryotic mRNAs.     

Localization and sequence analysis of poly(A) sites generating multiple dihydrofolate reductase mRNAs

The murine dihydrofolate reductase (DHFR) gene gives rise to multiple polyadenylated mRNAs displaying heterogeneity in the length of the 3' untranslated region. These species are present in the cytoplasm at levels that vary over 2 orders of magnitude, suggesting that certain poly(A) sites are preferred over others. Previous observations have shown that three out of the four major sites of polyadenylation do not display consensus hexanucleotide (AATAAA, ATTAAA) signals. We have further analyzed the sequences involved in directing multiple polyadenylation events on the DHFR gene by focusing our attention on the 4.1- and 5.6-kilobase mRNAs, the lowest abundance DHFR species observed on RNA blot analysis. Identification and sequence analysis of the poly(A) addition sites corresponding to these species revealed appropriately positioned consensus hexanucleotide signals; additional nearby poly(A) sites were also detected which apparently do not use consensus hexanucleotides to direct poly(A) addition to DHFR mRNAs of relatively lower abundance. We have also identified polyadenylation sites downstream of the 4.1- and 5.6-kilobase sites which display consensus hexanucleotide signals and correspond to messenger species too rare for detection by routine RNA blot analysis. Our data bring to 11 the number of known functional poly(A) addition sites associated with the DHFR gene.     

Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro.

We have partially purified a poly(A) polymerase (PAP) from HeLa cell nuclear extract which is involved in the 3'-end formation of polyadenylated mRNA. PAP had a molecular weight of approximately 50 to 60 kilodaltons. In the presence of manganese ions, PAP was able to polyadenylate RNA nonspecifically. However, in the presence of magnesium ions PAP required the addition of a cleavage and polyadenylation factor to specifically polyadenylate pre-mRNAs that contain an intact AAUAAA sequence and end at the poly(A) addition site (precleaved RNA substrates). The purified fraction containing PAP was also required in combination with a cleavage and polyadenylation factor and a cleavage factor for the correct cleavage at the poly(A) site of pre-mRNAs. Since the two activities of the PAP fractions, PAP and cleavage activity, could not be separated by extensive purification, we concluded that the two activities are contained in a single component, a PAP that is also required for the specific cleavage preceding the polyadenylation of pre-mRNA.