Characterization of specific protein-RNA complexes associated with the coupling of polyadenylation and last-intron removal

Polyadenylation and splicing are highly coordinated on substrate RNAs capable of coupled polyadenylation and splicing. Individual elements of both splicing and polyadenylation signals are required for the in vitro coupling of the processing reactions. In order to understand more about the coupling mechanism, we examined specific protein-RNA complexes formed on RNA substrates, which undergo coupled splicing and polyadenylation. We hypothesized that formation of a coupling complex would be adversely affected by mutations of either splicing or polyadenylation elements known to be required for coupling. We defined three specific complexes (A(C)', A(C), and B(C)) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The A(C)' complex is formed by 30 s after mixing, the A(C) complex is formed between 1 and 2 min after mixing, and the B(C) complex is formed by 2 to 3 min after mixing. A(C)' is a precursor of A(C), and the A(C)' and/or A(C) complex is a precursor of B(C). Of the three complexes, B(C) appears to be a true coupling complex in that its formation was consistently diminished by mutations or experimental conditions known to disrupt coupling. The characteristics of the A(C)' complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the A(C)' complex is dependent on the polypyrimidine tract. The transition from A(C)' to A(C) appears to require an intact 3'-splice site. Formation of the B(C) complex requires both splicing elements and the polyadenylation signal. A unique polyadenylation-specific complex formed rapidly on substrates containing only the polyadenylation signal. This complex, like the A(C)' complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the B(C) complex. We also suggest a model in which the coupling mechanism may act as a dominant checkpoint in which aberrant definition of one exon overrides the normal processing at surrounding wild-type sites.     

Sedimentation analysis of polyadenylation-specific complexes.

Precursor RNA containing the adenovirus L3 polyadenylation site is assembled into a 50S complex upon incubation with HeLa nuclear extract at 30 degrees C. The cofactor and sequence requirements for 50S complex formation are similar to those of the in vitro polyadenylation reaction. Assembly of this complex requires ATP but is not dependent upon synthesis of a poly(A) tract. In addition, a 50S complex does not form on substrate RNA in which the AAUAAA hexanucleotide upstream of the poly(A) site has been mutated to AAGAAA or on RNA in which sequences between +5 and +48 nucleotides downstream of the site have been removed. These mutations also prevent in vitro processing of substrate RNA. Kinetic studies suggest that the 50S complex is an intermediate in the polyadenylation reaction. It forms at an early stage in the reaction and at later times contains both poly(A)+ RNA as well as unreacted precursor. U-type small nuclear ribonucleoprotein particles are components of the 50S complex, as shown by immunoprecipitation with antiserum specific to the trimethyl cap of these small nuclear RNAs.     

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.     

Spatial constraints on polyadenylation signal function

Efficient cleavage and polyadenylation of eukaryotic messenger RNAs require at least two signal elements: an AAUAAA or closely related sequence located 7-30 base pairs (bp) upstream of the site of processing, and a G/U- or U-rich sequence located 3' to the cleavage site. The herpes simplex virus type 1 thymidine kinase (tk) gene contains two copies of the AATAAA hexanucleotide and a GT-rich region. We have shown that the first AATAAA and the GT-rich region are essential for efficient processing, both in vivo and in vitro, whereas the second AATAAA does not appear to play any role in the formation of tk mRNA 3' ends. The failure of a signal containing only the second AATAAA and the GT-rich element to signal cleavage and polyadenylation suggested that these two elements might be too close together to constitute a functional polyadenylation signal. The experiments described in this report were directed at determining the effects on mRNA 3' end formation of alterations in spacing between signal elements. Wild-type tk contains 19 bp between these two elements. Constructs were made in which an AATAAA and the GT-rich region were separated by various distances ranging from 7 to 43 bp. The quantity and location of 3' ends of the tk mRNA produced by these constructs in Cos-1 cells were measured by S1 nuclease protection analysis. Signal efficiency was gradually reduced as the separation between the two signal elements was increased; with a separation of 43 bp, the signal functioned at approximately one-eighth the efficiency of the parental construction. Bringing the two signals closer together resulted in decreased signal efficiency; with a separation of 7 or 9 bp, no tk mRNA polyadenylated within the normal region was produced. Altering the sequences between these two elements without changing the distance had small effects on processing efficiency.     

U1, U2, and U4/U6 small nuclear ribonucleoproteins are required for in vitro splicing but not polyadenylation

The requirement for individual U RNAs in splicing and polyadenylation was investigated using oligonucleotide-directed cleavage of snRNAs in in vitro processing extracts. Cleavage of U1, U2, or U4 RNA inhibited splicing but not polyadenylation of short precursor RNAs. Thus each snRNA and the snRNP in which it is assembled participates in the splicing reaction. Splicing activity was recovered when extracts containing cleaved U RNAs were mixed in pairwise combinations, indicating that U1, U2, and U4/U6 snRNPs independently interact with the assembling spliceosome. The involvement of multiple snRNPs in the splicing of simple precursor RNAs suggests that the spliceosome is a large complex assembly consisting of multiple snRNPs whose activity is dependent on the structural integrity of the individual U RNAs.     

Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3'' processing signal AAUAAA.

Cleavage and polyadenylation factor (CPF) is required for the cleavage as well as for the subsequent polyadenylation reaction during 3' processing of messenger RNA precursors. Here, we have investigated the interaction of CPF and poly(A) polymerase with short RNA substrates. CPF activates poly(A) polymerase to elongate RNA primers carrying the canonical hexamer recognition signal AAUAAA. CPF specifically binds to such RNA as shown by gel mobility shift assays and competition experiments. Upon binding of CPF, two polypeptides of 35 kDa and 160 kDa can be covalently crosslinked to the RNA by irradiation with UV light. These polypeptides may correspond to the smallest and the largest subunit contained in purified CPF fractions. In addition, chemical modification-exclusion experiments demonstrate that CPF interacts directly with the AAUAAA recognition signal in the RNA. The entire hexamer signal is involved in binding of CPF since modification of any of its bases interferes with complex formation.     

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.     

Analysis of RNA cleavage at the adenovirus-2 L3 polyadenylation site.

Processing at the L3 polyadenylation site of human adenovirus-2 involves endonucleolytic cleavage generating the 3' terminal sequence -UAOH to which adenosine residues are added. This dinucleotide is 19 nucleotides downstream of the AAUAAA polyadenylation signal. The ATP analog cordycepin triphosphate (3' dATP) inhibits poly(A) synthesis, but precursor RNA is processed to give a product terminating in -UAAH. Addition of only one adenosine analog demonstrates that the initial poly(A) tract is synthesized by polymerization of single residues rather than by ligation of preformed poly(A). Cleavage is not coupled to polyadenylation since incubation with an ATP analog containing a non-hydrolyzable alpha--beta bond generates a product with a 3' terminus coincident with the -UAOH) addition site. Addition of this accurately processed RNA to a nuclear extract results in efficient polyadenylation, suggesting that downstream sequences are not required for synthesis of the poly(A) tract. Finally, processing at the L3 poly(A) site may involve both endonucleolytic and exonucleolytic activities.     

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.     

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.     

The RNA helicase Mtr4p modulates polyadenylation in the TRAMP complex

Many steps in nuclear RNA processing, surveillance, and degradation require TRAMP, a complex containing the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for decay or trimming by the nuclear exosome. It has been unclear how polyadenylation by TRAMP differs from polyadenylation by conventional poly(A) polymerase, which produces poly(A) tails that stabilize RNAs. Using reconstituted S. cerevisiae TRAMP, we show that TRAMP inherently suppresses poly(A) addition after only 3-4 adenosines. This poly(A) tail length restriction is controlled by Mtr4p. The helicase detects the number of 3'-terminal adenosines and, over several adenylation steps, elicits precisely tuned adjustments of ATP affinities and rate constants for adenylation and TRAMP dissociation. Our data establish Mtr4p as a critical regulator of polyadenylation by TRAMP and reveal that an RNA helicase can control the activity of another enzyme in a highly complex fashion and in response to features in RNA.     

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.     

Assembly of a processive messenger RNA polyadenylation complex.

Polyadenylation of mRNA precursors by poly(A) polymerase depends on two specificity factors and their recognition sequences. These are cleavage and polyadenylation specificity factor (CPSF), recognizing the polyadenylation signal AAUAAA, and poly(A) binding protein II (PAB II), interacting with the growing poly(A) tail. Their effects are independent of ATP and an RNA 5'-cap. Analysis of RNA-protein interactions by non-denaturing gel electrophoresis shows that CPSF, PAB II and poly(A) polymerase form a quaternary complex with the substrate RNA that transiently stabilizes the binding of poly(A) polymerase to the RNA 3'-end. Only the complex formed from all three proteins is competent for the processive synthesis of a full-length poly(A) tail.