Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal.

Abstract

An integral component of the splicing machinery, the Ul snRNP, is here implicated in the efficient polyadenylation of SV40 late mRNAs. This occurs as a result of an interaction between Ul snRNP-A protein and the upstream efficiency element (USE) of the polyadenylation signal. UV cross-linking and immunoprecipitation demonstrate that this interaction can occur while Ul snRNP-A protein is simultaneously bound to Ul RNA as part of the snRNP. The dual reactivity of Ul snRNP-A occurs because the protein has two RNA recognition motifs (RRMs). The target RNA of the first RRM (RRMl) has been shown previously to be the second stem-loop of Ul RNA. We have found that a target for the second RRM (RRM2) is within the AUUUGURA motifs of the USE of the SV40 late polyadenylation signal. RNA substrates containing the wild-type USE efficiently bind to Ul snRNP-A protein, whereas substrates fail to bind when motifs of the USE were replaced by linker sequences. The addition of an oligoribonucleotide containing a USE motif to an in vitro polyadenylation reaction inhibits polyadenylation of a substrate representing the SV40 late polyadenylation signal, whereas a mutant oligoribonucleotide, a nonspecific oligoribonucleotide, and an oligoribonucleotide containing the Ul RNA-binding site had much reduced or no inhibitory effects. In addition, antibodies to bacterially produced, purified Ul snRNP-A protein specifically inhibit in vitro polyadenylation of the SV40 late substrate. These data suggest that the Ul snRNP-A protein performs an important role in polyadenylation through interaction with the USE. Because this interaction can occur when Ul snRNP-A protein is part of the Ul snRNP, our data provide evidence to support a link between the processes of splicing and polyadenylation, as suggested by the exon definition model.

The snRNP-free U1A (SF-A) complex(es): Identification of the largest subunit as PSF, the polypyrimidine-tract binding protein-associated splicing factor

“…In agreement with the exon definition model and the possible involvement of snRNPs in last exon definition, we have shown that components of the U1 snRNP function in polyadenylation+ We have previously demonstrated that the U1snRNP-A protein (U1A) can bind to the SV40 late polyadenylation signal (Lutz & Alwine, 1994), and that purified U1A can interact with a component of the polyadenylation complex, the 160 kDa subunit of cleavage-polyadenylation specificity factor (CPSF) (Lutz et al+, 1996)+ Using purified components (CPSF, poly(A) polymerase, and precleaved RNA) we have shown that the addition of purified bacterially expressed U1A caused a concentration-dependent increase in both the overall level of polyadenylation and in poly(A) tail length (Lutz et al+, 1996)+ In agreement with this result we have shown that the purified recombinant U1A stabilized the interaction of CPSF with the AAUAAA-containing substrate RNA+ It is important to emphasize that in both the in vitro polyadenylation and binding analyses the U1A protein functioned in a form which was free of other U1snRNP components+ We refer to this form as snRNP-free U1A, or SF-A+ Our previous in vitro data suggests that specific concentrations of SF-A can mediate a general enhancement of polyadenylation (Lutz et al+, 1996; O'Connor et al+, 1997)+ Other experiments have indicated that U1A can also mediate a very specific negative feedback regulatory mechanism affecting the polyadenylation of its own mRNA+ This involves specific interactions with both the pre-mRNA encoding U1A and poly(A) polymerase (Gunderson et al+, 1994(Gunderson et al+, , 1997)+ These findings raise the question of how much U1A protein is present as SF-A in the cell+ It has been assumed that most, if not all, of the cellular U1A protein is tightly bound to U1 RNA as part of the snRNP+ This assumption was based upon the high affinity (K d ; 10 Ϫ9 ) of U1A for U1 RNA (Lutz-Freyermuth et al+, 1990;Jessen et al+, 1991), and upon the negative autoregulation of polyadenylation of U1A's own message (Boelens et al+, 1993)+ Recently we demonstrated that at least two classes of U1A can be detected in human cell nucleoplasm: U1A that is associated with the U1snRNP, and SF-A that is complexed with a distinct set of non-snRNP proteins, that is, the SF-A complex(es) (O'Connor et al+, 1997)+ Based upon the quantitation of cellular levels of U1A (Baserga & Steitz, 1993) we estimated that approximately 3% of the total U1A protein in the cell is part of the SF-A complex+ This corresponds to approximately 30,000 molecules+ Our previous data also indicated that SF-A has a physiologically relevant function: specifically, an antibody which is specific for SF-A and not snRNP-bound U1A can inhibit in vitro polyadenylation of an SV40 late polyadenylation substrate RNA+ As mentioned above SF-A has been shown to be in complex(es) with other non-snRNP proteins (O'Connor et al+, 1997)+ In this article, we demonstrate (1) that the largest protein in the SF-A complex(es), p105, is PSF, the polypyrimidine-tract binding protein-associated splicing factor (Patton et al+, 1993); (2) that PSF interacts with U1A both in vitro and in vivo; and (3) that SF-A has effects on both splicing and polyadenylation in a coupled splicing and polyadenylation reaction...…”

“…We next wanted to confirm that PSF was a component of the SF-A complex(es)+ Previously we have shown that the SF-A complex(es) in human 293T cell nucleoplasmic extracts migrates in fractions 7-11 of a 5-30% sucrose gradient+ In contrast, the U1snRNP is found in fractions 17-23 (O'Connor et al+, 1997)+ We prepared similar sucrose gradients using human 293T cell nucleoplasm, separated the resulting fractions by SDS-PAGE, and transferred the gel to nitrocellulose+ The resulting Western blot was probed with a rabbit polyclonal anti-PSF antibody obtained from J+ Patton (Patton et al+, 1993)+ Figure 3 shows that the p105 band migrating in fractions 9-11 is specifically detected by the anti-PSF antibody+ In addition, no other PSF is detected in the gradient; the presence of a PSF signal in fraction 25 is the result of "spill over" from the HeLa lane next to it+ To ascertain the position of U1A in the gradient, the blot was then probed (without stripping) with a rabbit polyclonal antibody that recognizes U1A (Lutz & Alwine, 1994)+ U1A was found in fractions 9-11, where the SF-A complex(es) migrate, as well as in fractions 17-23, where the U1snRNP migrates+ These data confirm that PSF migrates in the portion of the gradient where the SF-A complex(es) are found and suggests that PSF is part of the SF-A complex(es)+ FIGURE 2. Peptide sequencing reveals the identity of p105 as the splicing factor PSF+ The amino acid sequence of PSF is shown at the top+ The two peptides sequenced from p105 are shown below+ Both peptides are shown in bold type within the sequence of PSF+ FIGURE 3.…”

“…SF-A complex(es) was purified for peptide sequencing using a large scale immunoprecipitation protocol+ One milliliter of monoclonal antibody 12E12 ascites fluid was chemically coupled to 2 mL of prewashed Gamma Bind-Plus Sepharose (Pharmacia, capacity ϭ 17 mg/mL) using dimethylpimelimidate as previously described (Harlow & Lane, 1988)+ Nucleoplasm was prepared as described (O'Connor et al+, 1997) from ten 150-mm plates of human 293T cells, and was incubated with the coupled antibody for 2 h at 4 8C+ The bound antigens were eluted in 0+1 M glycine pH 3+ Eluted fractions were analyzed by SDS-PAGE (12% acrylamide 68:1)+ The proteins were visualized by silver staining the gels+ The fractions containing the most abundant amounts of SF-A complex(es) were pooled and preparatively fractionated by SDS-PAGE (12% acrylamide 68:1) and transferred electrophoretically to polyvinylidene difluoride (PVDF) membrane+ The p105 band was visualized by staining with Amido Black (0+1% Amido Black in 10% acetic acid), excised, and subjected to peptide sequencing+ p105 from the PVDF membrane was digested with Achromobacter protease I in 50 mM Tris-HCl (pH 9+0) containing 0+05% Tween 20 for 24 h at 30 8C+ The peptide fragments were extracted with 50% acetonitrile containing 0+064% TFA+ The digest was separated by HPLC (Hewlett Packard 1090) using a Vydac C18 column (1+0 ϫ 250 mm, 10 micron)+ The peptides were sequenced on an automated protein sequencer (ABI Procise 494)+ Sucrose-gradient fractionation followed by Western blotting Sucrose gradients of 5-30% (w/v) were prepared using human 293T cell nucleoplasm as described previously (O'Connor et al+, 1997)+ Briefly, gradients were centrifuged in a Beckman SW28 rotor at 25,000 RPM for 42 h at 4 8C+ Thirty 1-mL fractions were taken using a BioComp model 150 gradient fractionator+ Thirty microliters of every other fraction were separated by SDS-PAGE (12+5% acrylamide 29:1) and were electrophoretically transferred to nitrocellulose+ The membrane was subsequently blocked in PBS containing 5% nonfat dry milk+ Primary antibody incubations were performed for 1 h at room temperature+ Rabbit polyclonal anti-U1A antibodies (Lutz & Alwine, 1994) were diluted 1:750 in PBS containing 5% nonfat dry milk; rabbit polyclonal anti-PSF antibodies were diluted 1:1,000+ Bound antibodies were detected by chemiluminescence (ECL system, Amersham) using a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG, Cappel) diluted 1:8,000 in PBS+…”

Adenosine Augments IL-10 Production by Macrophages through an A2B Receptor-Mediated Posttranscriptional Mechanism

“…Following the binding reaction, samples were irradiated with UV light (2500 J) (CL-1000, UV Crosslinker; UVP) on ice for 15 min (32). Samples were briefly boiled in SDS sample buffer and RNA-protein complexes resolved on 8 -16% Trisglycine gels (Invitrogen Life Technologies).…”

Interaction between the U1 snRNP-A protein and the 160-kD subunit of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro.

“…) Valsamakis et al 1991, 1992~ Gilmartin et al 1992~ Schek et al 1992. Previously, we have described the USE motifs found in SV40 late mRNA Alwine 1989~ Schek et al 1992} and have shown that the UIA protein utilizes these motifs to interact with the RNA (Lutz and Alwine 1994). This interaction significantly affects the efficiency of utilization of the SV40 late polyadenylation signal in vitro and has led us to suggest that the interaction of the UIA protein with the USE motifs, close to the AAUAAA, could increase the possibility of interactions between the U1 snRNP and the cleavage and polyadenylation complex associated with the AAUAAA.…”