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.     

Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation

To study the mechanism and factors required to form the 3' ends of polyadenylated mRNAs, we have fractionated HeLa cell nuclear extracts carrying out the normally coupled cleavage and polyadenylation reactions. Each reaction is catalyzed by a distinct, separable activity. The partially purified cleavage enzyme (at least 360,000 MW) retained the specificity displayed in nuclear extracts, since substitutions in the AAUAAA signal sequence inhibited cleavage. In contrast, the fractionated poly(A) polymerase (300,000 MW) lost all specificity. When fractions containing the cleavage and polyadenylation activities were mixed, the efficiency and specificity of the polyadenylation reaction were restored. Interestingly, the cleavage activity by itself functioned well on only one of four precursor RNAs tested. However, when mixed with the poly(A) polymerase-containing fraction, the cleavage activity processed the four precursors with comparable efficiencies.     

Poly(A) polymerase (PAP) diversity in gene expression – Star-PAP vs canonical PAP

Almost all eukaryotic mRNAs acquire a poly(A) tail at the 3′-end by a concerted RNA processing event: cleavage and polyadenylation. The canonical PAP, PAPα, was considered the only nuclear PAP involved in general polyadenylation of mRNAs. A phosphoinositide-modulated nuclear PAP, Star-PAP, was then reported to regulate a select set of mRNAs in the cell. In addition, several non-canonical PAPs have been identified with diverse cellular functions. Further, canonical PAP itself exists in multiple isoforms thus illustrating the diversity of PAPs. In this review, we compare two nuclear PAPs, Star-PAP and PAPα with a general overview of PAP diversity in the cell. Emerging evidence suggests distinct niches of target pre-mRNAs for the two PAPs and that modulation of these PAPs regulates distinct cellular functions.     

Poly(A) polymerase and the regulation of cytoplasmic polyadenylation

Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Data presented here shows that complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. We discuss the hypothesis that CPSF is required for all polyadenylation reactions, but that the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzed polyadenylation and stimulated translation approximately 40-fold; stimulation was exclusively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation required PAPs catalytic core, but did not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA.     

Poly (A) polymerases in the nucleus and cytoplasm of frog oocytes: dynamic changes during oocyte maturation and early development.

Poly(A) can be added to mRNAs both in the nucleus and in the cytoplasm. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs provides a common mechanism of translational control. In this report, to begin to understand the regulation of polyadenylation activities during early development, we analyze poly (A) polymerases (PAPs) in oocytes and early embryos of the frog, Xenopus laevis. We have cloned and sequenced a PAP cDNA that corresponds to a maternal mRNA present in frog oocytes. This PAP is similar in size and sequence to mammalian nuclear PAPs. By immunoblotting using monoclonal antibodies raised against human PAP, we demonstrate that oocytes contain multiple forms of PAP that display different electrophoretic mobilities. The oocyte nucleus contains primarily the slower migrating forms of PAP, whereas the cytoplasm contains primarily the faster migrating species. The nuclear forms of PAP are phosphorylated, accounting for their retarded mobility. During oocyte maturation and early postfertilization development, preexisting PAPs undergo regulated phosphorylation and dephosphorylation events. Using the cloned PAP cDNA, we demonstrate that the complex changes in PAP forms seen during oocyte maturation may be due to modifications of a single polypeptide. These results demonstrate that the oocyte contains a cytoplasmic polymerase closely related to the nuclear enzyme and suggest models for how its activity may be regulated during early development.     

Characterization of a novel plant poly(A) polymerase

We have purified and characterized poly(A) polymerases (PAPs) from Pisum sativum, Brassica juncea, and Zea mays. Through chromatography on DEAE-Sepharose and heparin-Sepharose, these PAPs copurified as a single enzyme along with RNPs that could provide RNA substrates for the enzyme. More extensive purification by chromatography on MonoQ resulted in the resolution of the PAPs into as many as three fractions. One of these (PAP-I) contained a 43-kDa polypeptide immunologically related to the yeast PAP, and two others (PAP-II and PAP-III) contained RNAs that could serve as substrates for polyadenylation. These fractions by themselves possessed little PAP activity, but mixtures containing combinations of these displayed substantial activity. Similar PAP factors (PAP-I and PAP-III) were identified after fractionation of extracts prepared from Brassica juncea and Zea mays. The factors from one plant were completely interchangeable with those from different plants. We conclude that the poly(A) polymerases present in vegetative plant tissues consist of more than one component. In this respect, they are substantially different from other reported plant, mammalian, and yeast PAPs.     

Assembly of a polyadenylation-specific 25S ribonucleoprotein complex in vitro.

Extracts from HeLa cell nuclei assemble RNAs containing the adenovirus type 2 L3 polyadenylation site into a number of rapidly sedimenting heterodisperse complexes. Briefly treating reaction mixtures prior to sedimentation with heparin reveals a core 25S assembly formed with substrate RNA but not an inactive RNA containing a U----C mutation in the AAUAAA hexanucleotide sequence. The requirements for assembly of this heparin-stable core complex parallel those for cleavage and polyadenylation in vitro, including a functional hexanucleotide, ATP, and a uridylate-rich tract downstream of the cleavage site. The AAUAAA and a downstream U-rich element are resistant in the assembly to attack by RNase H. The poly(A) site between the two protected elements is accessible, but is attacked more slowly than in naked RNA, suggesting that a specific factor or secondary structure is located nearby. The presence of a factor bound to the AAUAAA in the complex is independently demonstrated by immunoprecipitation of a specific T1 oligonucleotide containing the element from the 25S fraction. Precipitation of this fragment from reaction mixtures is blocked by the U----C mutation. However, neither ATP nor the downstream sequence element is required for binding of this factor in the nuclear extract, suggesting that recognition of the AAUAAA is an initial event in complex assembly.     

Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA.

Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.     

Purification and characterization of a mammalian polyadenylate polymerase involved in the 3' end processing of messenger RNA precursors

A polyadenylate polymerase involved in the polyadenylation of pre-mRNA has been purified 6,000-fold to apparent homogeneity from extracts of calf thymus. In the last purification step, anion exchange chromatography separates the enzyme into three major peaks that are indistinguishable by other physical or functional criteria. On denaturing polyacrylamide gels, the two predominant forms of poly(A) polymerase have molecular weights of 57,000 and 60,000. In solution, the enzyme is a monomer. It polymerizes exclusively ATP. The reaction is distributive and proceeds linearly without any lag phase. The requirement for a primer can be satisfied by any of a number of polyribonucleotides. A significantly higher activity in the presence of Mn2+ as opposed to Mg2+ is due to a hundredfold higher affinity for the primer terminus. In the presence of mg2+ and of a specificity factor partially purified from HeLa cells, the enzyme specifically polyadenylates an RNA that ends at the natural adenovirus L3 polyadenylation site. This reaction depends on the AAUAAA polyadenylation signal.     

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.     

Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro.

Recent in vivo studies have identified specific sequences between 56 and 93 nucleotides upstream of a polyadenylation [poly(A)] consensus sequence, AAUAAA, in human immunodeficiency virus type 1 (HIV-1) that affect the efficiency of 3'-end processing at this site (A. Valsamakis, S. Zeichner, S. Carswell, and J. C. Alwine, Proc. Natl. Acad. Sci. USA 88:2108-2112, 1991). We have used HeLa cell nuclear extracts and precursor RNAs bearing the HIV-1 poly(A) signal to study the role of upstream sequences in vitro. Precursor RNAs containing the HIV-1 AAUAAA and necessary upstream (U3 region) and downstream (U5 region) sequences directed accurate cleavage and polyadenylation in vitro. The in vitro requirement for upstream sequences was demonstrated by using deletion and linker substitution mutations. The data showed that sequences between 56 and 93 nucleotides upstream of AAUAAA, which were required for efficient polyadenylation in vivo, were also required for efficient cleavage and polyadenylation in vitro. This is the first demonstration of the function of upstream sequences in vitro. Previous in vivo studies suggested that efficient polyadenylation at the HIV-1 poly(A) signal requires a spacing of at least 250 nucleotides between the 5' cap site and the AAUAAA. Our in vitro analyses indicated that a precursor containing the defined upstream and downstream sequences was efficiently cleaved at the polyadenylation site when the distance between the 5' cap and the AAUAAA was reduced to at least 140 nucleotides, which is less than the distance predicted from in vivo studies. This cleavage was dependent on the presence of the upstream element.     

Nuclear location of ribonuclease H and increased level of magnesium-dependent ribonuclease H in rat liver on thioacetamide treatment

Rat liver nuclei were isolated in aqueous solutions of low ionic strength or anhydrous glycerol. The presence of ribonuclease H (RNase H) [EC 3.1.4.34] activity in the cytoplasm is due to extraction of the nuclear enzyme by buffer and inorganic salts. Two forms of RNase H were separated from rat liver nuclei by affinity chromatography using a DNA-cellulose column. When the RNase H in the wash solution of nuclei with 0.3 M sucrose and in nuclear solution extracted with 0.15 M NaCl were fractionated on a single-stranded DNA-cellulose column, two peaks corresponding to Mn2+- and Mg2+-dependent RNases H were eluted at 0.1 M and 0.2 M NaCl, respectively, and a peak having both RNase H activities was recovered in the wash-through fraction from the column. Among the enzymes from these two fractions in the nuclei, the activity of the Mg2+-dependent RNase H which binds to DNA-cellulose increased several-fold within 24 h of a single injection of thioacetamide. The activities of Mg2+-dependent RNase H extracted with higher-salt solution from the nuclei and recovered in the flow-through fraction from the DNA-cellulose column and the Mn2+-dependent RNase H activities were relatively unaffected by an injection of thioacetamide.     

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.     

Novel function of the human presqualene diphosphate phosphatase as a type II phosphatidate phosphatase in phosphatidylcholine and triacylglyceride biosynthesis pathways

Phosphatidate phosphatases, PAPs, are key enzymes in lipid biosynthesis and signaling. Type I PAP enzymes participate in de-novo phospholipid biosynthesis, whereas type II PAP enzymes have an established role in lipid signaling. To identify novel human type II PAPs potentially involved in de-novo phospholipid synthesis we used bioinformatics to screen for enzymes with an active site exposed to the cytosolic side of membranes. Two related enzymes, a novel lipid phosphatase related protein (LPRP-A) and a presqualene diphosphate phosphatase (PA-PSP) met this criterion. PA-PSP and LPRP-A have differential tissue and subcellular distribution, and novel yet differential roles in lipid metabolism. Specifically, PA-PSP, but not LPRP-A, was a potent Mg(2+)-independent, NEM-insensitive type II PAP. Subcellular fractionation detection indicated that both proteins were associated with membranes, while immunofluorescent deconvolution imaging revealed that these membranes were exclusively from the nuclear envelope and the endoplasmic reticulum. PA-PSP overexpression, but not LPRP-A, accelerated the synthesis of phosphatidylcholine and caused accumulation of triacylglycerol with concomitant decrease in the rate of phosphatidylinositol synthesis. Coexpression of human CTP:phosphocholine cytidylyltransferase-alpha with PA-PSP enhanced the effect of PA-PSP on phosphatidylcholine levels, yet attenuated its effect on triacylglycerol. Taken together, our studies provide the first evidence that the eukaryotic, ER-resident PA-PSP is a bifunctional enzyme with specific type II PAP activity, and regulates, in addition to type I PAPs, the de-novo biosynthesis of phospholipids and triacylglycerols.     

Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor.

To determine the role of poly(A) polymerase in 3'-end processing of mRNA, the effect of purified poly(A) polymerase antibodies on endonucleolytic cleavage and polyadenylation was studied in HeLa nuclear extracts, using adenovirus L3 pre-mRNA as the substrate. Both Mg2+- and Mn2+-dependent reactions catalyzing addition of 200 to 250 and 400 to 800 adenylic acid residues, respectively, were inhibited by the antibodies, which suggested that the two reactions were catalyzed by the same enzyme. Anti-poly(A) polymerase antibodies also inhibited the cleavage reaction when the reaction was coupled or chemically uncoupled with polyadenylation. These antibodies also prevented formation of specific complexes between the RNA substrate and components of nuclear extracts during cleavage or polyadenylation, with the concurrent appearance of another, antibody-specific complex. These studies demonstrate that (i) previously characterized poly(A) polymerase is the enzyme responsible for addition of the poly(A) tract at the correct cleavage site and probably for the elongation of poly(A) chains and (ii) the coupling of these two 3'-end processing reactions appears to result from the potential requirement of poly(A) polymerase for the cleavage reaction. The results suggest that the specific endonuclease is associated with poly(A) polymerase in a functional complex.     

Potential role of poly(A) polymerase in the assembly of polyadenylation-specific RNP complexes.

To elucidate the mechanism by which poly(A) polymerase functions in the 3'-end processing of pre-mRNAs, polyadenylation-specific RNP complexes were isolated by sedimentation in sucrose density gradients and the fractions were analyzed for the presence of the enzyme. At early stages of the reaction, the RNP complexes were resolved into distinct peaks which sedimented at approximately 18S and 25S. When reactions were carried out under conditions which support cleavage or polyadenylation, the pre-mRNA was specifically assembled into the larger 25S RNP complexes. Polyclonal antibodies raised against the enzyme purified from a rat hepatoma, which have been shown to inhibit cleavage and polyadenylation (Terns, M., and Jacob, S. T., Mol. Cell. Biol. 9:1435-1444, 1989) also prevented assembly of the 25S polyadenylation-specific RNP complexes. Furthermore, formation of these complexes required the presence of a chromatographic fraction containing poly(A) polymerase. UV cross-linking analysis indicated that the purified enzyme could be readily cross-linked to pre-mRNA but in an apparent sequence-independent manner. Reconstitution studies with the fractionated components showed that formation of the 25S RNP complex required the poly(A) polymerase fraction. Although the enzyme has not been directly localized to the specific complexes, the data presented in this report supports the role of poly(A) polymerase as an essential component of polyadenylation-specific complexes which functions both as a structural and enzymatic constituent.     

The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro.

The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This sequence is required for accurate and efficient cleavage and polyadenylation of pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late pre-mRNAs are cleaved and polyadenylated in vitro in a HeLa cell nuclear extract, and that cleavage in vitro is abolished by each of four different single-base changes in AAUAAA. In this same extract, precleaved RNAs (RNAs with 3' termini at the polyadenylation site) are efficiently polyadenylated. This in vitro polyadenylation reaction also requires the AAUAAA sequence.     

Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase

In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity.     

CstF-64 and 3′-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα

Almost all eukaryotic mRNAs have a poly (A) tail at the 3′-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3′-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3′-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3′-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation.     

PAP- and GLD-2-type poly(A) polymerases are required sequentially in cytoplasmic polyadenylation and oogenesis in Drosophila

Cytoplasmic polyadenylation has an essential role in activating maternal mRNA translation during early development. In vertebrates, the reaction requires CPEB, an RNA-binding protein and the poly(A) polymerase GLD-2. GLD-2-type poly(A) polymerases form a family clearly distinguishable from canonical poly(A) polymerases (PAPs). In Drosophila, canonical PAP is involved in cytoplasmic polyadenylation with Orb, the Drosophila CPEB, during mid-oogenesis. We show that the female germline GLD-2 is encoded by wispy. Wispy acts as a poly(A) polymerase in a tethering assay and in vivo for cytoplasmic polyadenylation of specific mRNA targets during late oogenesis and early embryogenesis. wispy function is required at the final stage of oogenesis for metaphase of meiosis I arrest and for progression beyond this stage. By contrast, canonical PAP acts with Orb for the earliest steps of oogenesis. Both Wispy and PAP interact with Orb genetically and physically in an ovarian complex. We conclude that two distinct poly(A) polymerases have a role in cytoplasmic polyadenylation in the female germline, each of them being specifically required for different steps of oogenesis.