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.     

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.     

Mitochondrial poly(A) polymerase and polyadenylation

Polyadenylation of mitochondrial RNAs in higher eukaryotic organisms have diverse effects on their function and metabolism. Polyadenylation completes the UAA stop codon of a majority of mitochondrial mRNAs in mammals, regulates the translation of the mRNAs, and has diverse effects on their stability. In contrast, polyadenylation of most mitochondrial mRNAs in plants leads to their degradation, consistent with the bacterial origin of this organelle. PAPD1 (mtPAP, TUTase1), a noncanonical poly(A) polymerase (ncPAP), is responsible for producing the poly(A) tails in mammalian mitochondria. The crystal structure of human PAPD1 was reported recently, offering molecular insights into its catalysis. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.     

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.     

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.     

Purification of the cleavage and polyadenylation factor involved in the 3'-processing of messenger RNA precursors

Polyadenylation of messenger RNA precursors requires the nucleotide sequence AAUAAA and two factors: poly(A) polymerase and a specificity factor termed cleavage and polyadenylation factor (CPF). We have purified CPF from calf thymus and from HeLa cells to near homogeneity. Four polypeptides with molecular masses of 160, 100, 73, and 30 kDa cofractionate with CPF activity. Glycerol gradient centrifugation and gel filtration indicate that these four proteins form one large complex with a sedimentation constant of 12 S, a Stokes radius near 100 A, and a native molecular mass near 500 kDa. Purified CPF binds specifically to an RNA that contains the AAUAAA sequence. Mutation of the AAUAAA sequence inhibits CPF binding as well as polyadenylation. Purified CPF contains only trace amounts of RNA and does not react with antibodies against common epitopes of small nuclear ribonucleoprotein particles. Thus, contrary to previous indications, CPF does not appear to be a small nuclear ribonucleoprotein particle.     

RNA structure is a critical determinant of poly(A) site recognition by cleavage and polyadenylation specificity factor.

Sequence conservation among mammalian poly(A) sites is limited to the sequence AAUAAA, coupled with an amorphous downstream U- or GU-rich region. Since these sequences may also occur within the coding region of mRNAs, additional information must be required to define authentic poly(A) sites. Several poly(A) sites have been shown to contain sequences outside the core elements that enhance the efficiency of 3' processing in vivo and in vitro. The human immunodeficiency virus type 1, equine infectious anemia virus, and adenovirus L1 3' processing enhancers have been shown to promote the binding of cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of AAUAAA, to the pre-mRNA, thereby facilitating the assembly of a stable 3' processing complex. We have used in vitro selection to examine the mechanism by which the human immunodeficiency virus type 1 3' processing enhancer promotes the interaction of CPSF with the AAUAAA hexamer. Surprisingly, RNAs selected for efficient polyadenylation were related by structure rather than sequence. Therefore, in the absence of extensive sequence conservation, our results strongly suggest that RNA structure is a critical determinant of poly(A) site recognition by CPSF and may play a key role in poly(A) site definition.     

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.     

Cloning and characterization of Arabidopsis homologues of the animal CstF complex that regulates 3' mRNA cleavage and polyadenylation

The 3' cleavage and polyadenylation of mRNAs has been studied in detail in animals and yeast, but not in plants. Aimed at elucidating the regulation of mRNA 3' end formation in plants, three Arabidopsis cDNAs encoding homologues of the animal proteins CstF-64, CstF-77 and CstF-50 that form the cleavage stimulating factor of the polyadenylation machinery have been cloned. It is shown experimentally that the N-terminal domain of the Arabidopsis CstF-64 homologue binds the mRNA 3' non-coding region in an analogous manner to the animal protein. It is also shown that the Arabidopsis CstF-64 and CstF-77 homologues strongly interact with each other in a similar way to their animal counterparts. These results imply that these Arabidopsis homologues belong to the polyadenylation machinery of nuclear mRNAs.     

Cleavage and polyadenylation of messenger RNA precursors in vitro occurs within large and specific 3' processing complexes.

We have investigated the assembly of complexes associated with in vitro cleavage and polyadenylation of synthetic pre-mRNAs by native gel electrophoresis. Incubation of SP6-generated pre-mRNA containing the adenovirus L3 polyadenylation site in HeLa cell nuclear extract results in the rapid assembly of specific complexes. Formation of these complexes precedes the appearance of cleaved intermediates and polyadenylated products and is dependent on an intact polyadenylation signal within the pre-mRNA. The specific complexes do not form on RNAs with point mutations in the AAUAAA sequence upstream of the L3 polyadenylation site. Furthermore, such mutant RNAs cannot compete for factors involved in the assembly of specific complexes on wild-type pre-mRNA. Upon complex formation a 67-nucleotide region of the L3 pre-mRNA is protected from RNase T1 digestion. This region contains both the upstream AAUAAA signal and the GU-rich downstream sequences. Cleavage and polyadenylation occur within the specific complexes and the processed RNA is subsequently released. We propose that the assembly of specific complexes represents an essential step during pre-mRNA 3' end formation in vitro.     

Interaction of poly(A) polymerase with the 25-kDa subunit of cleavage factor I.

Mammalian poly(A) polymerase (PAP), a key enzyme in the pre-mRNA 3'-end processing reaction, carries the catalytic domain in the N-terminal region, an RNA binding domain, two nuclear localization signals, and a serine/threonine-rich regulatory domain in the C-terminal region. Using LexA-based yeast two-hybrid screening, we identified a cDNA encoding the 25-kDa subunit of cleavage factor I (CFI-25) as a protein that interacts with the C-terminal region of mouse PAP. The glutathione S-transferase pull-down assay and the immunoprecipitation experiment revealed that PAP directly interacts with CFI-25 and that the C-terminal 69 residues of PAP and the N-terminal 60 residues of CFI-25 are sufficient for the interaction between CFI-25 and PAP. Since CFI is known to function in the assembly of the pre-mRNA 3'-processing complex, this interaction may play an important role in the assembly of the processing complex and/or in the regulation of PAP activity within the complex.     

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.