Sequences adjacent to the 5' splice site control U1A binding upstream of the IgM heavy chain secretory poly(A) site

We have recently shown that the stability of the alternatively expressed immunoglobulin M heavy chain secretory mRNA is developmentally regulated by U1A. U1A binds novel non-consensus sites upstream of the secretory poly(A) site and inhibits poly(A) tail addition in undifferentiated cells. U1A's dependence for binding and function upon a stem-loop structure has been extensively characterized for the consensus sites. We therefore probed the structure surrounding the novel U1A binding sites. We show that two of the three novel binding sites represent the major single-stranded regions upstream of the secretory poly(A) site, consistent with a major role at this site. The strength of binding and ability of U1A to inhibit poly(A) polymerase correlate with the accessibility of the novel sites. However, long range interactions are responsible for maintaining them in an open configuration. Mutation of an RNase V1-sensitive site 102 nucleotides upstream, directly adjacent to the competing 5' splice site, changes the structure of one the U1A binding sites and thus abolishes the binding of the second U1A molecule and the ability of U1A ability to inhibit poly(A) polymerase activity at this site. These sites bind U1A via its N-terminal domain but with a 10-fold lower affinity than U1 small nuclear RNA. This lower binding affinity is more conducive to U1A's regulation of poly(A) tail addition to heterologous mRNA.     

Mutations in poly(A) site downstream elements affect in vitro cleavage activity.

Previous studies have shown that a sequence element downstream of the poly(A) addition site is required for efficient cleavage in vivo. We tested a group of downstream element point mutations in an in vitro reaction using HeLa cell nuclear extract as a source of cleavage activity. In close agreement with earlier studies (M. A. McDevitt, R. P. Hart, W. W. Wong, and J. R. Nevins, EMBO J. 5:2907-2913, 1986), a downstream element from the adenovirus E2a gene directed a higher level of cleavage activity than one from the simian virus 40 early gene. Furthermore, a single-base change in the downstream element could result in a decrease in cleavage activity of about 50-fold. That these mutations have similar effects in vivo and in vitro indicates that the HeLa cell nuclear extract system contains all of the factors required to study the mechanism of sequence recognition.     

B-lineage regulated polyadenylation occurs on weak poly(A) sites regardless of sequence composition at the cleavage and downstream regions.

Early/memory and plasma B-cell lines and fibroblasts were analyzed for their ability to use a 5' proximal (variant) versus a 3' distal (constant) poly(A) site, in the absence of a competing splice, from a set of related constructs. The proximal:distal poly(A) site use (P:D ratio) of the resulting cytoplasmic poly(A)+ mRNA is a measure of poly(A) site strength. In this context the immunoglobulin gamma2b secretory-specific poly(A) site showed a P:D ratio of 1:1 in plasma cells, 0.43:1 in early/memory B-cells and an intermediate value in fibroblasts. Meanwhile, a construct with a proximal SV40 early-like poly(A) site produced mRNA with a P:D ratio of >>50:1 in all cell types. Alterations in the region downstream of the proximal poly(A) addition site and at the site itself resulted in changes in the P:D ratio. However, these poly(A) sites, all with a P:D ratio of < or = 5:1, were used most efficiently in plasma cells. Constructs totally devoid of immunoglobulin sequences, but containing heterologous poly(A) sites producing mRNA with P:D ratios of < or = 5:1, were also used more efficiently in plasma cells. We therefore conclude that weak poly(A) sites, regardless of sequence composition, are used more efficiently in plasma cells than in the other cell types.     

Regulation of nuclear poly(A) addition controls the expression of immunoglobulin M secretory mRNA.

B-cell differentiation is accompanied by a dramatic increase in cytoplasmic accumulation and stability of the IgM heavy chain (mu) secretory mRNA. Despite considerable effort, the mechanism is unknown. We have identified three short motifs upstream of the secretory poly(A) site, which, when mutated in the mu heavy chain gene, significantly increase the accumulation of the secretory form of poly(A)(+) mRNA relative to the membrane form and regulate the expression of the secretory poly(A) site in a developmental manner. We show that these motifs bind U1A and inhibit polyadenylation in vitro and in vivo. Overexpression of U1A in vivo results in the selective inhibition of the secretory form. Thus, this novel mechanism selectively controls post-cleavage expression of the mu secretory mRNA. We present evidence that this mechanism is used to regulate alternative expression of other genes.     

Non-snRNP U1A levels decrease during mammalian B-cell differentiation and release the IgM secretory poly(A) site from repression

A regulated shift from the production of membrane to secretory forms of Immunoglobulin M (IgM) mRNA occurs during B cell differentiation due to the activation of an upstream secretory poly(A) site. U1A plays a key role in inhibiting the expression of the secretory poly(A) site by inhibiting both cleavage at the poly(A) site and subsequent poly(A) tail addition. However, how the inhibitory effect of U1A is alleviated in differentiated cells, which express the secretory poly(A) site, is not known. Using B cell lines representing different stages of B cell differentiation, we show that the amount of U1A available to inhibit the secretory poly(A) site is reduced in differentiated cells. Undifferentiated B cells have more total U1A than differentiated cells and a greater proportion of this is not associated with the U1snRNP. We show that this is available to inhibit poly(A) addition at the secretory poly(A) site using cold competitor RNA oligos to de-repress poly(A) addition in nuclear extracts from the respective cell lines. In addition, endogenous non-snRNP associated U1A-immunopurified from the different cell lines-inhibits poly(A) polymerase activity proportional to U1A recovered, suggesting that available U1A level alone is responsible for changes in its inhibitory effect at the secretory IgM poly (A) site.     

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.     

A single polypyrimidine tract binding protein (PTB) binding site mediates splicing inhibition at mouse IgM exons M1 and M2

Splicing of mouse immunoglobulin (IgM) exons M1 and M2 is directed by two juxtaposed regulatory elements, an enhancer and an inhibitor, located within the M2 exon. A primary function of the enhancer is to counteract the inhibitor, allowing splicing to occur. Here we show that the inhibitor contains two binding sites for polypyrimidine tract binding protein (PTB). Mutational analysis indicates that only one of these sites is necessary and sufficient to direct splicing inhibition both in vitro and in vivo. We demonstrate that the difference in activity of the two sites is explained by proximity to the intron. We further show that the presence of the enhancer results in the disruption of the PTB-inhibitor interaction, enabling splicing to occur. In the absence of the enhancer, splicing can be artificially activated by immuno-inhibition of PTB. Collectively, our results indicate that a single PTB binding site can function as an inhibitor that regulates alternative splicing both in vitro and in vivo.     

Proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer exist in multiple oligomeric forms.

We describe the purification to near homogeneity of proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer. Proteins binding to this site produce four protein-DNA complexes which are distinguished by their mobility in gel retardation assays and their elution properties in an anion exchange column. DNA affinity-purified preparations of three chromatographically separated pools, containing different subsets of the four complexes, each contained three polypeptides of 42.5, 44, and 45 kilodaltons (kDa). UV crosslinking of protein to enhancer DNA demonstrated that site C2-binding activities in the three different pools bound DNA through proteins of similar sizes (about 45 kDa), even though the protein-DNA complexes formed by these binding activities were quite distinct. Gel exclusion chromatography and equilibrium binding analyses indicated that the distinct protein-DNA complexes were due to different oligomeric forms of the individual subunits and that a larger multimeric form bound with high affinity to the heavy-chain enhancer site C2, while a smaller species had a much lower affinity for heavy-chain enhancer sequences. Purified protein has been used to map high-affinity binding sites for site C2-binding proteins within an immunoglobulin heavy-chain promoter and at site KE3 in the kappa light-chain enhancer.     

An RNA polymerase pause site is associated with the immunoglobulin mus poly(A) site

Immunoglobulin mu alternative RNA processing is regulated during B-cell maturation and requires balanced efficiencies of the competing splice (mum) and cleavage-polyadenylation (mus) reactions. When we deleted sequences 50 to 200 nucleotides beyond the mus poly(A) site, the mus/mum mRNA ratio decreased three- to eightfold in B, plasma, and nonlymphoid cells. The activity could not be localized to a smaller fragment but did function in heterologous contexts. Our data suggest that this region contains an RNA polymerase II pause site that enhances the use of the mus poly(A) site. First, known pause sites replaced the activity of the deleted fragment. Second, the mu fragment, when placed between tandem poly(A) sites, enhanced the use of the upstream poly(A) site. Finally, nuclear run-ons detected an increase in RNA polymerase loading just downstream from the mus poly(A) site, even when the poly(A) site was inactivated. When this mu fragment and another pause site were inserted 1 kb downstream from the mus poly(A) site, they no longer affected the mRNA expression ratio, suggesting that pause sites affect poly(A) site use over a limited distance. Fragments from the immunoglobulin A gene were also found to have RNA polymerase pause site activity.     

Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site

Flavopiridol (L86-8275) ((-)-cis-5, 7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)-piperidinyl] -4H-benzopyran-4-one), a potential antitumor drug, currently in phase II trials, has been shown to be an inhibitor of muscle glycogen phosphorylase (GP) and to cause glycogen accumulation in A549 non-small cell lung carcinoma cells (Kaiser, A., Nishi, K., Gorin, F.A., Walsh, D.A., Bradbury, E. M., and Schnier, J. B., unpublished data). Kinetic experiments reported here show that flavopiridol inhibits GPb with an IC(50) = 15.5 microm. The inhibition is synergistic with glucose resulting in a reduction of IC(50) for flavopiridol to 2.3 microm and mimics the inhibition of caffeine. In order to elucidate the structural basis of inhibition, we determined the structures of GPb complexed with flavopiridol, GPb complexed with caffeine, and GPa complexed with both glucose and flavopiridol at 1.76-, 2.30-, and 2.23-A resolution, and refined to crystallographic R values of 0.216 (R(free) = 0.247), 0.189 (R(free) = 0.219), and 0.195 (R(free) = 0.252), respectively. The structures provide a rational for flavopiridol potency and synergism with glucose inhibitory action. Flavopiridol binds at the allosteric inhibitor site, situated at the entrance to the catalytic site, the site where caffeine binds. Flavopiridol intercalates between the two aromatic rings of Phe(285) and Tyr(613). Both flavopiridol and glucose promote the less active T-state through localization of the closed position of the 280s loop which blocks access to the catalytic site, thereby explaining their synergistic inhibition. The mode of interactions of flavopiridol with GP is different from that of des-chloro-flavopiridol with CDK2, illustrating how different functional parts of the inhibitor can be used to provide specific and potent binding to two different enzymes.     

Paip1 interacts with poly(A) binding protein through two independent binding motifs

The 3' poly(A) tail of eukaryotic mRNAs plays an important role in the regulation of translation. The poly(A) binding protein (PABP) interacts with eukaryotic initiation factor 4G (eIF4G), a component of the eIF4F complex, which binds to the 5' cap structure. The PABP-eIF4G interaction brings about the circularization of the mRNA by joining its 5' and 3' termini, thereby stimulating mRNA translation. The activity of PABP is regulated by two interacting proteins, Paip1 and Paip2. To study the mechanism of the Paip1-PABP interaction, far-Western, glutathione S-transferase pull-down, and surface plasmon resonance experiments were performed. Paip1 contains two binding sites for PABP, PAM1 and PAM2 (for PABP-interacting motifs 1 and 2). PAM2 consists of a 15-amino-acid stretch residing in the N terminus, and PAM1 encompasses a larger C-terminal acidic-amino-acid-rich region. PABP also contains two Paip1 binding sites, one located in RNA recognition motifs 1 and 2 and the other located in the C-terminal domain. Paip1 binds to PABP with a 1:1 stoichiometry and an apparent K(d) of 1.9 nM.     

Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein

Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.     

Identification of a stem-loop structure important for polyadenylation at the murine IgM secretory poly(A) site.

We have previously shown that a distal GU-rich downstream element of the mouse IgM secretory poly(A) site is important for polyadenylation in vivo and for polyadenylation specific complex formation in vitro. This element can be predicted to form a stem-loop structure with two asymmetric internal loops. As stem-loop structures commonly define protein RNA binding sites, we have probed the biological activity of the secondary structure of this element. We show that mutations affecting the stem of the structure abolish the biological activity of this element in vivo and in vitro at the level of cleavage and polyadenylation specificity factor/cleavage stimulation factor complex formation and that both internal loops contribute to the enhancing effect of the sequence in vivo. Lead (II) cleavage patterns and RNase H probing of the sequence element in vitro are consistent with the predicted secondary structure. Furthermore, mobility on native PAGE suggests a bent structure. We propose that the secondary structure of this downstream element optimizes its interaction with components of the polyadenylation complex.