3' cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP

We have separated and purified three factors from HeLa cell nuclear extracts that together can accurately cleave and polyadenylate pre-mRNAs containing the adenovirus L3 polyadenylation site. One of the factors is a poly(A) polymerase with a molecular weight of approximately 50-60 kd. The second activity is a cleavage factor with a native molecular weight in the range of 70-120 kd. The third component is a factor (cleavage and polyadenylation factor, CPF) that is needed for the cleavage reaction and, in addition, confers specificity to the poly(A) polymerase activity; the native molecular weight of CPF is approximately 200 kd. Poly(A) polymerase together with CPF is sufficient to specifically polyadenylate pre-mRNA substrates that have been precleaved at the poly(A) addition site. In contrast, all three components are required for accurate cleavage and polyadenylation of pre-mRNA substrates. Further purification of CPF by buoyant density centrifugation, ion exchange, and affinity column chromatography or by gel filtration demonstrates that CPF activity resides in a ribonucleoprotein and copurifies with U11 snRNP.     

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.     

Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly.

To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively.     

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.     

Ara-ATP impairs 3''-end processing of pre-mRNAs by inhibiting both cleavage and polyadenylation.

Previous studies have demonstrated that Ara-ATP can inhibit poly(A) polymerase activity by competing with ATP. To elucidate the mechanism of action of this compound, its effect on the cleavage and polyadenylation of two specific substrates, SV40L and adenovirus L3 pre-mRNAs, was studied in HeLa nuclear extracts. Unlike cordycepin 5' triphosphate, Ara-ATP inhibited both cleavage and poly(A) addition. Addition of poly(A) polymerase fraction devoid of any other factors required for the processing reactions overcame the inhibitory effect on cleavage as well as polyadenylation of pre-mRNAs. These data suggest that Ara-ATP inhibits both cleavage and polyadenylation reactions by interacting with the ATP-binding site on poly(A) polymerase, the activity of which is essential for the cleavage reaction. Ara-ATP also blocked formation of the post-cleavage and polyadenylation-specific complexes, which further confirmed the inhibitory effect of the ATP analog on the two tightly coupled 3'-end processing reactions.     

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.     

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.     

The C proteins of heterogeneous nuclear ribonucleoprotein complexes interact with RNA sequences downstream of polyadenylation cleavage sites.

The heterogeneous nuclear ribonucleoprotein C1 and C2 proteins were preferentially cross-linked by treatment with UV light in nuclear extracts to RNAs containing six different polyadenylation signals. The domain required for the interaction was located downstream of the poly(A) cleavage site, since deletion of this segment from several polyadenylation substrate RNAs greatly reduced cross-linking efficiency. In addition, RNAs containing only downstream sequences were efficiently cross-linked to C proteins, while fully processed, polyadenylated RNAs were not. Analysis of mutated variants of the simian virus 40 late polyadenylation signal showed that uridylate-rich sequences located in the region between 30 and 55 nucleotides downstream of the cleavage site were required for efficient cross-linking of C proteins. This downstream domain of the simian virus 40 late poly(A) addition signal has been shown to influence the efficiency of the polyadenylation reaction. However, there was not a strict correlation between cross-linking of C proteins and the efficiency of polyadenylation.     

Polyadenylation of mRNA: minimal substrates and a requirement for the 2'' hydroxyl of the U in AAUAAA.

mRNA-specific polyadenylation can be assayed in vitro by using synthetic RNAs that end at or near the natural cleavage site. This reaction requires the highly conserved sequence AAUAAA. At least two distinct nuclear components, an AAUAAA specificity factor and poly(A) polymerase, are required to catalyze the reaction. In this study, we identified structural features of the RNA substrate that are critical for mRNA-specific polyadenylation. We found that a substrate that contained only 11 nucleotides, of which the first six were AAUAAA, underwent AAUAAA-specific polyadenylation. This is the shortest substrate we have used that supports polyadenylation: removal of a single nucleotide from either end of this RNA abolished the reaction. Although AAUAAA appeared to be the only strict sequence requirement for polyadenylation, the number of nucleotides between AAUAAA and the 3' end was critical. Substrates with seven or fewer nucleotides beyond AAUAAA received poly(A) with decreased efficiency yet still bound efficiently to specificity factor. We infer that on these shortened substrates, poly(A) polymerase cannot simultaneously contact the specificity factor bound to AAUAAA and the 3' end of the RNA. By incorporating 2'-deoxyuridine into the U of AAUAAA, we demonstrated that the 2' hydroxyl of the U in AAUAAA was required for the binding of specificity factor to the substrate and hence for poly(A) addition. This finding may indicate that at least one of the factors involved in the interaction with AAUAAA is a protein.     

A novel plant in vitro assay system for pre-mRNA cleavage during 3'-end formation

Messenger RNA (mRNA) maturation in eukaryotic cells requires the formation of the 3' end, which includes two tightly coupled steps: the committing cleavage reaction that requires both correct cis-element signals and cleavage complex formation, and the polyadenylation step that adds a polyadenosine [poly(A)] tract to the newly generated 3' end. An in vitro biochemical assay plays a critical role in studying this process. The lack of such an assay system in plants hampered the study of plant mRNA 3'-end formation for the last two decades. To address this, we have now established and characterized a plant in vitro cleavage assay system, in which nuclear protein extracts from Arabidopsis (Arabidopsis thaliana) suspension cell cultures can accurately cleave different pre-mRNAs at expected in vivo authenticated poly(A) sites. The specific activity is dependent on appropriate cis-elements on the substrate RNA. When complemented by yeast (Saccharomyces cerevisiae) poly(A) polymerase, about 150-nucleotide poly(A) tracts were added specifically to the newly cleaved 3' ends in a cooperative manner. The reconstituted polyadenylation reaction is indicative that authentic cleavage products were generated. Our results not only provide a novel plant pre-mRNA cleavage assay system, but also suggest a cross-kingdom functional complementation of yeast poly(A) polymerase in a plant system.     

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.     

hnRNP F influences binding of a 64-kilodalton subunit of cleavage stimulation factor to mRNA precursors in mouse B cells

Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued for trans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular proteins, the ratio of hnRNP F to H or H' was found to be higher in memory B cells than in plasma cells. In memory B cells the activity of CstF-64 binding to pre-mRNA, but not its amount, was reduced. Second, examination of the complexes formed on input pre-mRNA in nuclear extracts revealed large assemblages containing hnRNP H, H', and F but deficient in CstF-64 in memory B-cell extracts but not in plasma cells. Formation of these large complexes is dependent on the region downstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs compete for a similar region. Third, using a recombinant protein we showed that hnRNP F could bind to the region downstream of a poly(A) site, block CstF-64 association with RNA, and inhibit the cleavage reaction. Fourth, overexpression of recombinant hnRNP F in plasma cells resulted in a decrease in the endogenous Ig heavy-chain mRNA secretory form-to-membrane ratio. These results demonstrate that mammalian hnRNP F can act as a negative regulator in the pre-mRNA cleavage reaction and that increased expression of F in memory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.     

Analysis of adenovirus type 2 L1 RNA 3''-end formation in vivo and in vitro.

Downstream sequence requirements for efficient cleavage and polyadenylation at the adenovirus type 2 L1 poly(A) site were determined in vivo in 293 cells and in vitro by using RNA precursors in HeLa cell nuclear extracts. The two cleavage sites used were found to differ in sensitivity to 3'-end deletion in vivo and in vitro.     

The Cleavage and Polyadenylation Specificity Factor in Xenopus laevis Oocytes Is a Cytoplasmic Factor Involved in Regulated Polyadenylation

During early development, specific mRNAs receive poly(A) in the cytoplasm. This cytoplasmic polyadenylation reaction correlates with, and in some cases causes, translational stimulation. Previously, it was suggested that a factor similar to the multisubunit nuclear cleavage and polyadenylation specificity factor (CPSF) played a role in cytoplasmic polyadenylation. A cDNA encoding a cytoplasmic form of the 100-kDa subunit of Xenopus laevis CPSF has now been isolated. The protein product is 91% identical at the amino acid sequence level to nuclear CPSF isolated from Bos taurus thymus. This report provides three lines of evidence that implicate the X. laevis homologue of the 100-kDa subunit of CPSF in the cytoplasmic polyadenylation reaction. First, the protein is predominantly localized to the cytoplasm of X. laevis oocytes. Second, the 100-kDa subunit of X. laevis CPSF forms a specific complex with RNAs that contain both a cytoplasmic polyadenylation element (CPE) and the polyadenylation element AAUAAA. Third, immunodepletion of the 100-kDa subunit of X. laevis CPSF reduces CPE-specific polyadenylation in vitro. Further support for a cytoplasmic form of CPSF comes from evidence that a putative homologue of the 30-kDa subunit of nuclear CPSF is also localized to the cytoplasm of X. laevis oocytes. Overexpression of influenza virus NS1 protein, which inhibits nuclear polyadenylation through an interaction with the 30-kDa subunit of nuclear CPSF, prevents cytoplasmic polyadenylation, suggesting that the cytoplasmic X. laevis form of the 30-kDa subunit of CPSF is involved in this reaction. Together, these results indicate that a distinct, cytoplasmic form of CPSF is an integral component of the cytoplasmic polyadenylation machinery.     

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.     

Requirements for accurate and efficient mRNA 3'' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro.

Using a pre-RNA containing the simian virus 40 early introns and poly(A) addition site, we investigated several possible requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation in a HeLa cell nuclear extract. Splicing and 3' end formation occurred under the same conditions but did not appear to be coupled in any way in vitro. Like splicing, 3' end cleavage and polyadenylation each required Mg2+, although spermidine could substitute in the cleavage reaction. Additionally, cleavage of this pre-RNA, but not others, was totally blocked by EDTA, indicating that structural features of pre-RNA may affect the ionic requirements of 3' end formation. The ATP analog 3' dATP inhibited both cleavage and polyadenylation even in the presence of ATP, possibly reflecting the coupled nature of these activities. A 5' cap structure appears not to be required for mRNA 3' end processing in vitro because neither the presence or absence of a 5' cap on the pre-RNA nor the addition of cap analogs to reaction mixtures had any effect on the efficiency of 3' end processing. Micrococcal nuclease pretreatment of the nuclear extract inhibited cleavage and polyadenylation. However, restoration of activity was achieved by addition of purified Escherichia coli RNA, suggesting that the inhibition caused by such a nuclease treatment was due to a general requirement for mass of RNA rather than to destruction of a particular nucleic acid-containing component such as a small nuclear ribonucleoprotein.     

A multisubunit 3'' end processing factor from yeast containing poly(A) polymerase and homologues of the subunits of mammalian cleavage and polyadenylation specificity factor.

Polyadenylation is the second step in 3' end formation of most eukaryotic mRNAs. In Saccharomyces cerevisiae, this step requires three trans-acting factors: poly(A) polymerase (Pap1p), cleavage factor I (CF I) and polyadenylation factor I (PF I). Here, we describe the purification and subunit composition of a multiprotein complex containing Pap1p and PF I activities. PF I-Pap1p was purified to homogeneity by complementation of extracts mutant in the Fip1p subunit of PF I. In addition to Fip1p and Pap1p, the factor comprises homologues of all four subunits of mammalian cleavage and polyadenylation specificity factor (CPSF), as well as Ptalp, which previously has been implicated in pre-tRNA processing, and several as yet uncharacterized proteins. As expected for a PF I subunit, pta1-1 mutant extracts are deficient for polyadenylation in vitro. PF I also appears to be functionally related to CPSF, as it polyadenylates a substrate RNA more efficiently than Pap1p alone. Possibly, the observed interaction of the complex with RNA tethers Pap1p to its substrate.     

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.