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.     

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.     

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.     

Distinct roles of two Yth1p domains in 3'-end cleavage and polyadenylation of yeast pre-mRNAs

Yth1p is the yeast homologue of the 30 kDa subunit of mammalian cleavage and polyadenylation specificity factor (CPSF). The protein is part of the cleavage and polyadenylation factor CPF, which includes cleavage factor II (CF II) and polyadenylation factor I (PF I), and is required for both steps in pre-mRNA 3'-end processing. Yth1p is an RNA-binding protein that was previously shown to be essential for polyadenylation. Here, we demonstrate that Yth1p is also required for the cleavage reaction and that two protein domains have distinct roles in 3'-end processing. The C-terminal part is required in polyadenylation to tether Fip1p and poly(A) polymerase to the rest of CPF. A single point mutation in the highly conserved second zinc finger impairs both cleavage and polyadenylation, and affects the ability of Yth1p to interact with the pre-mRNA and other CPF subunits. Finally, we find that Yth1p binds to CYC1 pre-mRNA in the vicinity of the cleavage site. Our results indicate that Yth1p is important for the integrity of CPF and participates in the recognition of the cleavage site.     

Specific pre-cleavage and post-cleavage complexes involved in the formation of SV40 late mRNA 3'' termini in vitro.

Complexes form between processing factors present in a crude nuclear extract from HeLa cells and a simian virus 40 (SV40) late pre-mRNA which spans the polyadenylation [poly(A)] site. A specific 'pre-cleavage complex' forms on the pre-mRNA before cleavage. Formation of this complex requires the highly conserved sequence AAUAAA: it is prevented by mutations in AAUAAA, and by annealing DNA oligonucleotides to that sequence. After cleavage, the 5' half-molecule is found in a distinct 'post-cleavage complex'. In contrast, the 3' half-molecule is released. After cleavage and polyadenylation, polyadenylated RNA also is released. De novo formation of the post-cleavage complex requires AAUAAA and a nearby 3' terminus. Competition experiments suggest that a component which recognizes AAUAAA is required for formation of both pre- and post-cleavage complexes.     

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.     

The poly A polymerase Star-PAP controls 3'-end cleavage by promoting CPSF interaction and specificity toward the pre-mRNA

Star-PAP is a poly (A) polymerase (PAP) that is putatively required for 3'-end cleavage and polyadenylation of a select set of pre-messenger RNAs (mRNAs), including heme oxygenase (HO-1) mRNA. To investigate the underlying mechanism, the cleavage and polyadenylation of pre-mRNA was reconstituted with nuclear lysates. siRNA knockdown of Star-PAP abolished cleavage of HO-1, and this phenotype could be rescued by recombinant Star-PAP but not PAPα. Star-PAP directly associated with cleavage and polyadenylation specificity factor (CPSF) 160 and 73 subunits and also the targeted pre-mRNA. In vitro and in vivo Star-PAP was required for the stable association of CPSF complex to pre-mRNA and then CPSF 73 specifically cleaved the mRNA at the 3'-cleavage site. This mechanism is distinct from canonical PAPα, which is recruited to the cleavage complex by interacting with CPSF 160. The data support a model where Star-PAP binds to the RNA, recruits the CPSF complex to the 3'-end of pre-mRNA and then defines cleavage by CPSF 73 and subsequent polyadenylation of its target mRNAs.     

前体mRNA的选择性多聚腺苷酸化与人类疾病

真核细胞的前体mRNA必须经过复杂的加工过程才能成熟,包括5’端加帽、剪接和3’端加工,其中3’加工包括3’端的切割和多聚腺苷酸化.该过程由前体mRNA上的顺式作用元件以及多个蛋白质因子控制.组成哺乳动物前体mRNA3’端加工机器的核心蛋白质复合体有切割和多聚腺苷酸化特异性因子、切割刺激因子、切割因子Ⅰ和切割因子Ⅱ.其他因子包括poly(A)聚合酶、poly(A)结合蛋白、偶对蛋白(symplekin)等.哺乳动物基因通常含有多个ploy(A)位点,选择性多聚腺苷酸化不仅可产生具有不同长度3’UTR的mRNA异构体,还可能改变基因的CDS区.作为真核生物基因表达调控的关键机制,选择性多聚腺苷酸化在细胞生长、增殖和分化中起着重要作用.本文综述了哺乳动物前体mRNA的3’端加工过程,3’端加工机器的组成及功能,探讨了选择性多聚腺苷酸化在多种人类疾病中的作用机制,以期为读者带来一些新的见解.     

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.     

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.     

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.     

Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus.

Cleavage-polyadenylation specificity factor (CPSF) is one of five separable factors known to be required for 3' cleavage and polyadenylation of mRNA precursors in vitro. Previous studies have shown that the cleavage and poly(A) addition reactions can be uncoupled in vitro and have suggested that CPSF may be the only factor essential for both of these subreactions. Here we report the purification of CPSF to near homogeneity from calf thymus and show that the purified factor contains three polypeptides of 165, 105, and 70 kDa. These polypeptides cosediment precisely with CPSF activity, which has a sedimentation coefficient of 11.5 S. Consistent with previous reports from our laboratory, purified CPSF does not contain a detectable RNA component, indicating that it is a multisubunit protein and not a small nuclear ribonucleoprotein. Extensively purified bovine CPSF can function with human poly(A) polymerase to bring about AAUAAA-dependent poly(A) addition or with human cleavage factors to catalyze accurate 3' cleavage of a pre-mRNA substrate. UV cross-linking and gel retention analyses demonstrate that highly purified CPSF interacts with one of these cleavage factors, the multisubunit cleavage-stimulation factor, to facilitate stable binding of both to an AAUAAA-containing pre-mRNA. Likewise, evidence is presented indicating that poly(A) polymerase and CPSF can interact directly.     

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.     

Conditional defect in mRNA 3'' end processing caused by a mutation in the gene for poly(A) polymerase.

Maturation of most eukaryotic mRNA 3' ends requires endonucleolytic cleavage and polyadenylation of precursor mRNAs. To further understand the mechanism and function of mRNA 3' end processing, we identified a temperature-sensitive mutant of Saccharomyces cerevisiae defective for polyadenylation. Genetic analysis showed that the polyadenylation defect and the temperature sensitivity for growth result from a single mutation. Biochemical analysis of extracts from this mutant shows that the polyadenylation defect occurs at a step following normal site-specific cleavage of a pre-mRNA at its polyadenylation site. Molecular cloning and characterization of the wild-type allele of the mutated gene revealed that it (PAP1) encodes a previously characterized poly(A) polymerase with unknown RNA substrate specificity. Analysis of mRNA levels and structure in vivo indicate that shift of growing, mutant cells to the nonpermissive temperature results in the production of poly(A)-deficient mRNAs which appear to end at their normal cleavage sites. Interestingly, measurement of the rate of protein synthesis after the temperature shift shows that translation continues long after the apparent loss of polyadenylated mRNA. Our characterization of the pap1-1 defect implicates this gene as essential for mRNA 3' end formation in S. cerevisiae.     

The murine IgM secretory poly(A) site contains dual upstream and downstream elements which affect polyadenylation.

Regulation of polyadenylation efficiency at the secretory poly(A) site plays an essential role in gene expression at the immunoglobulin (IgM) locus. At this poly(A) site the consensus AAUAAA hexanucleotide sequence is embedded in an extended AU-rich region and there are two downstream GU-rich regions which are suboptimally placed. As these sequences are involved in formation of the polyadenylation pre-initiation complex, we examined their function in vivo and in vitro . We show that the upstream AU-rich region can function in the absence of the consensus hexanucleotide sequence both in vivo and in vitro and that both GU-rich regions are necessary for full polyadenylation activity in vivo and for formation of polyadenylation-specific complexes in vitro . Sequence comparisons reveal that: (i) the dual structure is distinct for the IgM secretory poly(A) site compared with other immunoglobulin isotype secretory poly(A) sites; (ii) the presence of an AU-rich region close to the consensus hexanucleotide is evolutionarily conserved for IgM secretory poly(A) sites. We propose that the dual structure of the IgM secretory poly(A) site provides a flexibility to accommodate changes in polyadenylation complex components during regulation of polyadenylation efficiency.     

RNA processing in vitro produces mature 3'' ends of a variety of Saccharomyces cerevisiae mRNAs.

Ammonium sulfate fractionation of a Saccharomyces cerevisiae whole-cell extract yielded a preparation which carried out correct and efficient endonucleolytic cleavage and polyadenylation of yeast precursor mRNA substrates corresponding to a variety of yeast genes. These included CYC1 (iso-1-cytochrome c), HIS4 (histidine biosynthesis), GAL7 (galactose-1-phosphate uridyltransferase), H2B2 (histone H2B2), PRT2 (a protein of unknown function), and CBP1 (cytochrome b mRNA processing). The reaction processed these pre-mRNAs with varying efficiencies, with cleavage and polyadenylation exceeding 70% in some cases. In each case, the poly(A) tail corresponded to the addition of approximately 60 adenosine residues, which agrees with the usual length of poly(A) tails formed in vivo. Addition of cordycepin triphosphate or substitution of CTP for ATP in these reactions inhibited polyadenylation but not endonucleolytic cleavage and resulted in accumulation of the cleaved RNA product. Although this system readily generated yeast mRNA 3' ends, no processing occurred on a human alpha-globin pre-mRNA containing the highly conserved AAUAAA polyadenylation signal of higher eucaryotes. This sequence and adjacent signals used in mammalian systems are thus not sufficient to direct mRNA 3' end formation in yeast. Despite the lack of a highly conserved nucleotide sequence signal, the same purified fraction processed the 3' ends of a variety of unrelated yeast pre-mRNAs, suggesting that endonuclease cleavage and polyadenylation may produce the mature 3' ends of all mRNAs in S. cerevisiae.