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.     

Plasma cell-regulated polyadenylation at the Ig gamma 2b secretion-specific poly(A) site.

We found that the sequences downstream of the Ig gamma 2b secretory-specific (sec) poly(A) site play an important role in the preferential production of sec Ig mRNA during plasma B cell development. The Ig gamma 2b mRNA production in a deletion mutant (delta-Kpn) lacking the Ig sec poly(A) site and downstream consensus element (dsc) has been previously shown to default to the use of the downstream membrane-specific (mb) poly(A) site. In this study restoration of the Ig sec poly(A) site and dsc to the delta-Kpn gene causes a significant increase in the use of the sec poly(A) site vs mb poly(A) site in stable transfectants of plasma but not memory B cell tumors, indicating plasma cell-specific recognition of the Ig sec dsc. Restoration of the poly(A) cleavage site alone to delta-Kpn did not restore regulation. Substitution of an SV40 downstream poly(A) element for the Ig dsc in the delta-Kpn gene also does not restore regulation. The data further indicate that although the Ig dsc is clearly very important in the plasma cell-regulated expression, the difference in the processing ratios of the restored vs the intact Ig gamma 2b gene in plasma cells suggests that there are other yet to be defined sequences that may also play a role in the intact gene. Insertion of a 130-nucleotide segment of the gene containing the Ig sec poly(A) site and dsc into a heterologous, guanosyl phosphotransferase gene resulted in plasma cell-regulated polyadenylation of the sec poly(A) site. Neither the mb nor the SV40 early poly(A) sites and their respective dscs, in similar gpt chimeras, were regulated. Therefore the region downstream of the Ig sec poly(A) site plays an essential role in regulating polyadenylation at the sec poly(A) site in plasma cells but not memory cells. A model involving a plasma cell-specific recognition factor for the Ig sec dsc is presented.     

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.     

Regulation of stage-specific gene expression during early mouse development: effect of cytochalasin B and aphidicolin on stage-specific protein synthesis in mouse eggs

Mechanisms regulating stage-specific translation in mouse embryos were studied by inhibitor experiments. When fertilized eggs were continuously treated with cytochalasin B, cleavage was prevented, whereas karyokinesis proceeded, resulting in protein synthesis patterns changing stage-specifically as in control embryos through preimplantation development. When fertilized eggs were continuously exposed to aphidicolin, cleavage and DNA synthesis were inhibited, thus keeping their protein synthesis at the level of fertilized eggs with few new polypeptides appearing after one day. The next day these eggs stopped translation almost completely. Stage-specific translation therefore might be controlled by nuclear replications rather than by cytoplasmic clock.     

A novel poly(A)-binding protein acts as a specificity factor in the second phase of messenger RNA polyadenylation

Polyadenylation of mRNA precursors by poly(A) polymerase depends on a specificity factor, CPF, recognizing the polyadenylation signal AAUAAA. This paper describes an apparently novel poly(A)-binding protein that acts as a second specificity factor, mediating the recognition of the growing poly(A) tail. A transition from a slow initiation phase of polyadenylation to rapid elongation occurs when the growing tail is long enough to serve as a binding site for the poly(A)-binding protein. Elongation of an RNA carrying a tail of 10 or more adenylate residues can occur independently of CPF. A sharp decrease in the poly(A) chain growth rate after the addition of approximately 200 adenylate residues invites speculations about a role of the poly(A)-binding protein in poly(A) tail length control.     

Xenopus embryonic poly(A) binding protein 2 (ePABP2) defines a new family of cytoplasmic poly(A) binding proteins expressed during the early stages of vertebrate development

We describe a new RNA binding protein from Xenopus we have named ePABP2 (embryonic poly(A) binding protein type II). Based on amino acid similarity, ePABP2 is closely related to the ubiquitously expressed nuclear PABP2 protein that directs the elongation of mRNA poly(A) tails during pre-mRNA processing. However, in contrast to known PABP2 proteins, Xenopus ePABP2 is a cytoplasmic protein that is predominantly expressed during the early stages of Xenopus development and in adult ovarian tissue. Biochemical experiments indicate ePABP2 binds poly(A) with specificity and that this binding requires the RRM domain. Mouse and human ePABP2 proteins were also identified and mouse ePABP2 expression is also confined to the earliest stages of mouse development and adult ovarian tissue. We propose that Xenopus ePABP2 is the founding member of a new class of poly(A) binding proteins expressed in vertebrate embryos. Possible roles for this protein in regulating mRNA function in early vertebrate development are discussed.     

Cell- and stage-specific high-level expression of TBP-related factor 2 (TRF2) during mouse spermatogenesis

Mice lacking the TBP-related factor 2 (TRF2) gene, which is highly expressed in the testis, have a severe defect in spermiogenesis. Here we show that the expression of TRF2 is both cell type- and stage-specific. TRF2 expression was first detected in the late pachytene spermatocytes at stage VIII and increased throughout the subsequent stages. After meiotic divisions, the TRF2 expression declined continuously in round spermatids during progression from stage I to stage V. This observation is consistent with an essential regulatory role of TRF2 in male germ cell differentiation during spermatogenesis.     

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.     

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.     

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.