Components required for in vitro cleavage and polyadenylation of eukaryotic mRNA.

We have studied in vitro cleavage/polyadenylation of precursor RNA containing herpes simplex virus type 2 poly A site sequences and have analyzed four RNA/protein complexes which form during in vitro reactions. Two complexes, A and B, form extremely rapidly and are then progressively replaced by a third complex, C which is produced following cleavage and polyadenylation of precursor RNA. Substitution of ATP with cordycepin triphosphate prevents polyadenylation and the formation of complex C however a fourth complex, D results which contains cleaved RNA. A precursor RNA lacking GU-rich downstream sequences required for efficient cleavage/polyadenylation fails to form complex B and produces a markedly reduced amount of complex A. As these GU-rich sequences are required for efficient cleavage, this establishes a relationship between complex B formation and cleavage/polyadenylation of precursor RNA in vitro. The components required for in vitro RNA processing have been separated by fractionation of the nuclear extract on Q-Sepharose and Biorex 70 columns. A Q-Sepharose fraction forms complex B but does not process RNA. Addition of a Biorex 70 fraction restores cleavage activity at the poly A site but this fraction does not appear to contribute to complex formation. Moreover, in the absence of polyethylene glycol, precursor RNA is not cleaved and polyadenylated, however, complexes A and B readily form. Thus, while complex B is necessary for in vitro cleavage and polyadenylation, it may not contain all the components required for this processing.     

Characterization of the polyadenylation signal of influenza virus RNA.

It has been shown that a stretch of uridines (U's) near the 5' end of the virion RNA of influenza A virus is the polyadenylation site for viral mRNA synthesis. In addition, the RNA duplex made up the 3' and 5' terminal sequences adjacent to the U stretch is also involved in polyadenylation. We have further characterized the polyadenylation signal of influenza virus RNA with a ribonucleoprotein transfection system. We found that the optimal length of the U stretch is 5 to 7 uridine residues. We also showed that the upstream sequence at the 5' end is not involved in polyadenylation and that the optimal distance between the 5' end and the U stretch is 16 nucleotides. The combination of these features defines the polyadenylation site and differentiates this signal from other U stretches scattered throughout the genomes of influenza viruses.     

Maturation-specific polyadenylation and translational control: diversity of cytoplasmic polyadenylation elements, influence of poly(A) tail size, and formation of stable polyadenylation complexes.

Early embryonic development in Xenopus laevis is programmed in part by maternally derived mRNAs, many of which are translated at the completion of meiosis (oocyte maturation). Polysomal recruitment of at least one of these mRNAs, G10, is regulated by cytoplasmic poly(A) elongation which, in turn, is dependent upon the cytoplasmic polyadenylation element (CPE) UUUUUUAUAAAG and the hexanucleotide AAUAAA (L. L. McGrew, E. Dworkin-Rastl, M. B. Dworkin, and J. D. Richter, Genes Dev. 3:803-815, 1989). We have investigated whether sequences similar to the G10 RNA CPE that are present in other RNAs could also be responsible for maturation-specific polyadenylation. B4 RNA, which encodes a histone H1-like protein, requires a CPE of the sequence UUUUUAAU as well as the polyadenylation hexanucleotide. The 3' untranslated regions of Xenopus c-mos RNA and mouse HPRT RNA also contain U-rich CPEs since they confer maturation-specific polyadenylation when fused to Xenopus B-globin RNA. Polyadenylation of B4 RNA, which occurs very early during maturation, is limited to 150 residues, and it is this number that is required for polysomal recruitment. To investigate the possible diversity of factors and/or affinities that might control polyadenylation, egg extracts that faithfully adenylate exogenously added RNA were used in competition experiments. At least one factor is shared by B4 and G10 RNAs, although it has a much greater affinity for B4 RNA. Additional experiments demonstrate that an intact CPE and hexanucleotide are both required to compete for the polyadenylation apparatus. Gel mobility shift assays show that two polyadenylation complexes are formed on B4 RNA. Optimal complex formation requires an intact CPE and hexanucleotide but not ongoing adenylation. These data, plus additional RNA competition studies, suggest that stable complex formation is enhanced by an interaction of the trans-acting factors that bind the CPE and polyadenylation hexanucleotide.     

RapA, Escherichia coli RNA polymerase SWI/SNF subunit-dependent polyadenylation of RNA

In this work, we describe RapA-dependent polyadenylation of model RNA substrates and endogenous, RNA polymerase-associated nucleic acid fragments. We demonstrate that the Escherichia coli RNA polymerase obtained through the classic purification procedure carries endogenous RNA oligonucleotides, which, in the presence of ATP, are polyriboadenylated in a RapA-dependent manner by an accessory poly(rA) polymerase. RNA polymerase isolated from poly(A) polymerase- (PAP-) and polynucleotide phosphorylase- (PNP-) deficient E. coli strain lacks accessory (rA)(n)-synthetic activity. Experiments with reconstituted RNA polymerase-PAP and RNA polymerase-PNP mixtures suggest that RapA enables the polyadenylation by PAP of RNA polymerase-associated RNA. Mutations disrupting RapA's ATP-hydrolytic function disrupt RapA-dependent polyadenylation, and the rapA(-)E. coli strain displays a measurable reduction in RNA polyadenylation. RapA-dependent polyadenylation can also be modulated by mutations in the section of RapA's SWI/SNF domain linked to interaction with single-stranded nucleic acid. We have developed enzymatic assays in which model, synthetic RNAs are polyriboadenylated in a RapA-dependent manner. Taken together, our results are consistent with RapA acting as an RNA polymerase-associated, ATP-dependent RNA translocase. Our work further links RapA to RNA remodeling and provides new mechanistic insights into the functional interaction between RNA polymerase and RapA.     

Translational control by cytoplasmic polyadenylation during Xenopus oocyte maturation: characterization of cis and trans elements and regulation by cyclin/MPF.

The expression of certain maternal mRNAs during oocyte maturation is regulated by cytoplasmic polyadenylation. To understand this process, we have focused on a maternal mRNA from Xenopus termed G10. This mRNA is stored in the cytoplasm of stage 6 oocytes until maturation when the process of poly(A) elongation stimulates its translation. Deletion analysis of the 3' untranslated region of G10 RNA has revealed that two sequence elements, UUUUUUAU and AAUAAA were both necessary and sufficient for polyadenylation and polysomal recruitment. In this communication, we have defined the U-rich region that is optimal for polyadenylation as UUUUUUAUAAAG, henceforth referred to as the cytoplasmic polyadenylation element (CPE). We have also identified unique sequence requirements in the 3' terminus of the RNA that can modulate polyadenylation even in the presence of wild-type cis elements. A time course of cytoplasmic polyadenylation in vivo shows that it is an early event of maturation and that it requires protein synthesis within the first 15 min of exposure to progesterone. MPF and cyclin can both induce polyadenylation but, at least with respect to MPF, cannot obviate the requirement for protein synthesis. To identify factors that may be responsible for maturation-specific polyadenylation, we employed extracts from oocytes and unfertilized eggs, the latter of which correctly polyadenylates exogenously added RNA. UV crosslinking demonstrated that an 82 kd protein binds to the U-rich CPE in egg, but not oocyte, extracts. The data suggest that progesterone, either in addition to or through MPF/cyclin, induces the synthesis of a factor during very early maturation that stimulates polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A simple procedure for isolation of eukaryotic mRNA polyadenylation factors.

We have devised a simple chromatographic procedure which isolates five polyadenylation factors that are required for polyadenylation of eukaryotic mRNA. The factors were separated from each other by fractionation of HeLa cell nuclear extract in two consecutive chromatographic steps. RNA cleavage at the L3 polyadenylation site of human adenovirus 2 required at least four factors. Addition of adenosine residues required only two of these factors. The fractionation procedure separates two components that are both likely to be poly(A) polymerases. The candidate poly(A) polymerases were interchangeable and participated during both RNA cleavage and adenosine addition. They were discriminated from each other by chromatographic properties, heat sensitivity and divalent cation requirement. We have compared our data with published information and have been able to correlate the activities that we have isolated to previously identified polyadenylation factors. However, we have not been able to assign one of the candidate poly(A) polymerases to a previously identified poly(A) polymerase. This simple fractionation procedure can be used for generating an in vitro reconstituted system for polyadenylation within a short period of time.     

Characterization of the Distal Polyadenylation Site of the ?-Adducin (Add2) Pre-mRNA

Most genes have multiple polyadenylation sites (PAS), which are often selected in a tissue-specific manner, altering protein products and affecting mRNA stability, subcellular localization and/or translability. Here we studied the polyadenylation mechanisms associated to the beta-adducin gene (Add2). We have previously shown that the Add2 gene has a very tight regulation of alternative polyadenylation, using proximal PAS in erythroid tissues, and a distal one in brain. Using chimeric minigenes and cell transfections we identified the core elements responsible for polyadenylation at the distal PAS. Deletion of either the hexanucleotide motif (Hm) or the downstream element (DSE) resulted in reduction of mature mRNA levels and activation of cryptic PAS, suggesting an important role for the DSE in polyadenylation of the distal Add2 PAS. Point mutation of the UG repeats present in the DSE, located immediately after the cleavage site, resulted in a reduction of processed mRNA and in the activation of the same cryptic site. RNA-EMSA showed that this region is active in forming RNA-protein complexes. Competition experiments showed that RNA lacking the DSE was not able to compete the RNA-protein complexes, supporting the hypothesis of an essential important role for the DSE. Next, using a RNA-pull down approach we identified some of the proteins bound to the DSE. Among these proteins we found PTB, TDP-43, FBP1 and FBP2, nucleolin, RNA helicase A and vigilin. All these proteins have a role in RNA metabolism, but only PTB has a reported function in polyadenylation. Additional experiments are needed to determine the precise functional role of these proteins in Add2 polyadenylation.     

Hu proteins regulate polyadenylation by blocking sites containing U-rich sequences

A recent genome-wide bioinformatic analysis indicated that 54% of human genes undergo alternative polyadenylation. Although it is clear that differential selection of poly(A) sites can alter gene expression, resulting in significant biological consequences, the mechanisms that regulate polyadenylation are poorly understood. Here we report that the neuron-specific members of a family of RNA-binding proteins, Hu proteins, known to regulate mRNA stability and translation in the cytoplasm, play an important role in polyadenylation regulation. Hu proteins are homologs of the Drosophila embryonic lethal abnormal visual protein and contain three RNA recognition motifs. Using an in vitro polyadenylation assay with HeLa cell nuclear extract and recombinant Hu proteins, we have shown that Hu proteins selectively block both cleavage and poly(A) addition at sites containing U-rich sequences. Hu proteins have no effect on poly(A) sites that do not contain U-rich sequences or sites in which the U-rich sequences are mutated. All three RNA recognition motifs of Hu proteins are required for this activity. Overexpression of HuR in HeLa cells also blocks polyadenylation at a poly(A) signal that contains U-rich sequences. Hu proteins block the interaction between the polyadenylation cleavage stimulation factor 64-kDa subunit and RNA most likely through direct interaction with poly(A) cleavage stimulation factor 64-kDa subunit and cleavage and polyadenylation specificity factor 160-kDa subunit. These studies identify a novel group of mammalian polyadenylation regulators. Furthermore, they define a previously unknown nuclear function of Hu proteins.     

An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal.

We have identified an RNA-binding protein which interacts with the downstream element of the simian virus 40 late polyadenylation signal in a sequence-specific manner. A partially purified 50-kDa protein, which we have named DSEF-1, retains RNA-binding specificity as assayed by band shift and UV cross-linking analyses. RNA footprinting assays, using end-labeled RNA ladder fragments in conjunction with native gel electrophoresis, have identified the DSEF-1 binding site as 5'-GGGGGAGGUGUGGG-3'. This 14-base sequence serves as an efficient DSEF-1 binding site when placed within a GEM4 polylinker-derived RNA. Finally, the DSEF-1 binding site restored efficient in vitro 3' end processing to derivatives of the simian virus 40 late polyadenylation signal in which it substituted for the entire downstream region. DSEF-1, therefore, may be a sequence-specific binding factor which regulates the efficiency of polyadenylation site usage.     

细菌RNA聚腺苷酸化修饰及其调控机制与生理作用

聚腺苷酸化 (polyadenylation) 是指在底物RNA的3′-端加上一段聚腺苷酸残基的转录后修饰作用。1971年,第一次发现真核生物mRNA的3′-端存在多聚腺苷酸 (poly (A)) 尾,它保护mRNA免受核酸外切酶攻击,且对于转录终止、mRNA运输及翻译都起到重要作用,学者们一度将该现象认为是真核细胞mRNA的特征之一。时至今日,细菌RNA聚腺苷酸化现象的发现引起了学术界的高度重视,大量的研究结果不仅证明了该种修饰在细菌中普遍存在,而且发现其在细菌RNA的加工、降解及质量监控中扮演重要的角色;然而,与真核生物不同的是,在原核生物中该修饰倾向于使RNA去稳定化,即加速RNA的降解。本文综述了近年来细菌中RNA聚腺苷酸化修饰及其调控机制与生理作用的研究进展。     

Isolation and characterization of polyadenylation complexes assembled in vitro

We developed a two-step purification of mammalian polyadenylation complexes assembled in vitro. Biotinylated pre-mRNAs containing viral or immunoglobulin poly(A) sites were incubated with nuclear extracts prepared from mouse myeloma cells under conditions permissive for in vitro cleavage and polyadenylation and the mixture was fractionated by gel filtration; complexes containing biotinylated pre-mRNA and bound proteins were affinity purified on avidin-agarose resin. Western analysis of known components of the polyadenylation complex demonstrated copurification of polyadenylation factors with poly(A) site-containing RNA but not with control RNA substrates containing either no polyadenylation signals or a point mutation of the AAUAAA polyadenylation signal. Polyadenylation complexes that were assembled on exogenous RNA eluted from the Sephacryl column in fractions consistent with their size range extending from 2 to 4 x 10(6) Mr. Complexes endogenous to the extract were of approximately the same apparent size, but more heterogeneous in distribution. This method can be used to study polyadenylation/cleavage complexes that may form upon a number of different RNA sequences, an important step towards defining which factors might differentially associate with specific RNAs.     

Further analysis of cytoplasmic polyadenylation in Xenopus embryos and identification of embryonic cytoplasmic polyadenylation element-binding proteins.

Early development in Xenopus laevis is programmed in part by maternally inherited mRNAs that are synthesized and stored in the growing oocyte. During oocyte maturation, several of these messages are translationally activated by poly(A) elongation, which in turn is regulated by two cis elements in the 3' untranslated region, the hexanucleotide AAUAAA and a cytoplasmic polyadenylation element (CPE) consisting of UUUUUAU or similar sequence. In the early embryo, a different set of maternal mRNAs is translationally activated. We have shown previously that one of these, C12, requires a CPE consisting of at least 12 uridine residues, in addition to the hexanucleotide, for its cytoplasmic polyadenylation and subsequent translation (R. Simon, J.-P. Tassan, and J.D. Richter, Genes Dev. 6:2580-2591, 1992). To assess whether this embryonic CPE functions in other maternal mRNAs, we have chosen Cl1 RNA, which is known to be polyadenylated during early embryogenesis (J. Paris, B. Osborne, A. Couturier, R. LeGuellec, and M. Philippe, Gene 72:169-176, 1988). Wild-type as well as mutated versions of Cl1 RNA were injected into fertilized eggs and were analyzed for cytoplasmic polyadenylation at times up to the gastrula stage. This RNA also required a poly(U) CPE for cytoplasmic polyadenylation in embryos, but in this case the CPE consisted of 18 uridine residues. In addition, the timing and extent of cytoplasmic poly(A) elongation during early embryogenesis were dependent upon the distance between the CPE and the hexanucleotide. Further, as was the case with Cl2 RNA, Cl1 RNA contains a large masking element that prevents premature cytoplasmic polyadenylation during oocyte maturation. To examine the factors that may be involved in the cytoplasmic polyadenylation of both C12 and C11 RNAs, we performed UV cross-linking experiments in egg extracts. Two proteins with sizes of ~36 and ~45 kDa interacted specifically with the CPEs of both RNAs, although they bound preferentially to the C12 CPE. The role that these proteins might play in cytoplasmic polyadenylation is discussed.     

Functional analysis of point mutations in the AAUAAA motif of the SV40 late polyadenylation signal.

We have constructed 14 independent point mutations in the conserved AAUAAA element of the SV40 late polyadenylation signal in order to study the recognition and function of alternative polyadenylation signals. A variant RNA containing an AUUAAA was polyadenylated at 20% the level of wild-type substrate RNA, while all other derivatives tested were not functional in vitro. The AUUAAA variant RNA formed specific complexes in native polyacrylamide gels and crosslinked to the AAUAAA-specific 64kd polypeptide, but at a lower efficiency than wild-type substrate RNA.     

Polyadenylation of RNAs Associated with a Nucleus-localized Phosphorolytic Nuclease

The exosome is a protein complex consisting of a variety of 3′→5′ exoribonucleases that functions both in the processing of rRNA precursors and in the degradation of mRNA. A prokaryotic counterpart of the exosome known as the degradosome exists in bacteria and chloroplasts. Interestingly, RNA polyadenylation has been implicated in degradosome functioning, giving rise to the possibility of a similar role in exosome function. Using phosphorolytic breakdown of RNA as an assay, we have purified an exosome-like activity from pea nuclear extracts. This activity copurifies with at least one Arabidopsis exosome subunit homologue. Recombinant Arabidopsis poly(A) polymerase and purified chloroplast poly(A) polymerase can polyadenylate RNAs that copurify with the exosome-like activity, even though the quantity of this co-purifying RNA is well below the affinity of the PAPs for free RNA. These results suggest a role for polyadenylation in exosome function, perhaps analogous to the role that polyadenylation plays in facilitating RNA breakdown by the bacterial degradosome.     

A host factor that binds near the termini of hepatitis B virus pregenomic RNA.

The terminal regions of hepatitis B virus (HBV) pregenomic RNA (pgRNA) harbors sites governing many essential functions in the viral life cycle, including polyadenylation, translation, RNA encapsidation, and DNA synthesis. We have examined the binding of host proteins to a 170-nucleotide region from the 5' end of HBV pgRNA; a large portion of this region is duplicated at the 3' end of this terminally redundant RNA. By UV cross-linking labeled RNA to HepG2 cell extracts, we have identified a 65-kDa factor (p65) of nuclear origin which can specifically bind to this region. Two discrete binding sites were identified within this region; in vitro cross-competition experiments suggest that the same factor binds to both elements. One binding site (termed UBS) overlaps a portion of the highly conserved stem-loop structure (epsilon), while the other site (termed DBS) maps 35 nucleotides downstream of the hexanucleotide polyadenylation sequence. Both binding sites are highly pyrimidine rich and map to regions previously found to be important in the regulation of viral polyadenylation. However, functional analysis of mutant binding sites in vivo indicates that p65 is not involved in the polyadenylation of HBV pgRNA. Potential roles for the factor in viral replication in vivo are discussed.