Vertebrate Poly Research Articles

Vertebrate GLD2 poly(A) polymerases in the germline and the brain

Cytoplasmic polyadenylation is important in the control of mRNA stability and translation, and for early animal development and synaptic plasticity. Here, we focus on vertebrate poly(A) polymerases that are members of the recently described GLD2 family. We identify and characterize two closely related GLD2 proteins in Xenopus oocytes, and show that they possess PAP activity in vivo and in vitro and that they bind known polyadenylation factors and mRNAs known to receive poly(A) during development. We propose that at least two distinct polyadenylation complexes exist in Xenopus oocytes, one of which contains GLD2; the other, maskin and Pumilio. GLD2 protein interacts with the polyadenylation factor, CPEB, in a conserved manner. mRNAs that encode GLD2 in mammals are expressed in many tissues. In the brain, mouse, and human GLD2 mRNAs are abundant in anatomical regions necessary for long-term cognitive and emotional learning. In the hippocampus, mouse GLD2 mRNA colocalizes with CPEB1 and Pumilio1 mRNAs, both of which are likely involved in synaptic plasticity. We suggest that mammalian GLD2 poly(A) polymerases are important in synaptic translation, and in polyadenylation throughout the soma.

Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition

At least half of all human pre-mRNAs are subject to alternative 3' processing that may modulate both the coding capacity of the message and the array of post-transcriptional regulatory elements embedded within the 3' UTR. Vertebrate poly(A) site selection appears to rely primarily on the binding of CPSF to an A(A/U)UAAA hexamer upstream of the cleavage site and CstF to a downstream GU-rich element. At least one-quarter of all human poly(A) sites, however, lack the A(A/U)UAAA motif. We report that sequence-specific RNA binding of the human 3' processing factor CFI(m) can function as a primary determinant of poly(A) site recognition in the absence of the A(A/U)UAAA motif. CFI(m) is sufficient to direct sequence-specific, A(A/U)UAAA-independent poly(A) addition in vitro through the recruitment of the CPSF subunit hFip1 and poly(A) polymerase to the RNA substrate. ChIP analysis indicates that CFI(m) is recruited to the transcription unit, along with CPSF and CstF, during the initial stages of transcription, supporting a direct role for CFI(m) in poly(A) site recognition. The recognition of three distinct sequence elements by CFI(m), CPSF, and CstF suggests that vertebrate poly(A) site definition is mechanistically more similar to that of yeast and plants than anticipated.

The carboxyl terminus of vertebrate poly(A) polymerase interacts with U2AF 65 to couple 3′-end processing and splicing

Although it has been established that the processing factors involved in pre-mRNA splicing and 3'-end formation can influence each other positively, the molecular basis of this coupling interaction was not known. Stimulation of pre-mRNA splicing by an adjacent cis-linked cleavage and polyadenylation site in HeLa cell nuclear extract is shown to occur at an early step in splicing, the binding of U2AF 65 to the pyrimidine tract of the intron 3' splice site. The carboxyl terminus of poly(A) polymerase (PAP) previously has been implicated indirectly in the coupling process. We demonstrate that a fusion protein containing the 20 carboxy-terminal amino acids of PAP, when tethered downstream of an intron, increases splicing efficiency and, like the entire 3'-end formation machinery, stimulates U2AF 65 binding to the intron. The carboxy-terminal domain of PAP makes a direct and specific interaction with residues 17-47 of U2AF 65, implicating this interaction in the coupling of splicing and 3'-end formation.

Deregulation of poly(A) polymerase interferes with cell growth.

Vertebrate poly(A) polymerase (PAP) contains a catalytic domain and a C-terminal Ser-Thr-rich regulatory region. Consensus and nonconsensus cyclin-dependent kinase (cdk) sites are conserved in the Ser-Thr-rich region in vertebrate PAPs. PAP is phosphorylated by cdc2-cyclin B on these sites in vitro and in vivo and is inactivated by hyperphosphorylation in M-phase cells, when cdc2-cyclin B is active. In the experiments described here, we undertook a genetic approach in chicken DT40 cells to study the function of PAP phosphorylation. We found that PAP is highly conserved in chicken and is essential in DT40 cells. While cells could tolerate reduced levels of PAP, even modest overexpression of either wild-type PAP or a mutant PAP with two consensus cdk sites mutated (cdk- PAP) was highly deleterious and at a minimum resulted in reduced growth rates. Importantly, cells that expressed cdk- PAP had a significantly lower growth rate than did cells that expressed similar levels of wild-type PAP, which was reflected in increased accumulation of cells in the G0-G1 phase of the cell cycle. We propose that the lower growth rate is due to the failure of hyperphosphorylation and thus M-phase inactivation of cdk- PAP.

Involvement of the carboxyl terminus of vertebrate poly(A) polymerase in U1A autoregulation and in the coupling of splicing and polyadenylation.

Interactions required for inhibition of poly(A) polymerase (PAP) by the U1 snRNP-specific U1A protein, a reaction whose function is to autoregulate U1A protein production, are examined. PAP inhibition requires a substrate RNA to which at least two molecules of U1A protein can bind tightly, but we demonstrate that the secondary structure of the RNA is not highly constrained. A mutational analysis reveals that the carboxy-terminal 20 amino acids of PAP are essential for its inhibition by the U1A-RNA complex. Remarkably, transfer of these amino acids to yeast PAP, which is otherwise not affected by U1A protein, is sufficient to confer U1A-mediated inhibition onto the yeast enzyme. A glutathione S-transferase fusion protein containing only these 20 PAP residues can interact in vitro with an RNA-U1A protein complex containing two U1A molecules, but not with one containing a single U1A protein, explaining the requirement for two U1A-binding sites on the autoregulatory RNA element. A mutational analysis of the U1A protein demonstrates that amino acids 103-119 are required for PAP inhibition. A monomeric synthetic peptide consisting of the conserved U1A amino acids from this region has no detectable effect on PAP activity. However, the same U1A peptide, when conjugated to BSA, inhibits vertebrate PAP. In addition to this activity, the U1A peptide-BSA conjugate specifically uncouples splicing and 3'-end formation in vitro without affecting uncoupled splicing or 3'-end cleavage efficiencies. This suggests that the carboxy-terminal region of PAP with which it interacts is involved not only in U1A autoregulation but also in the coupling of splicing and 3'-end formation.

3'-end-forming signals of yeast mRNA.

It was previously shown that three distinct but interdependent elements are required for 3' end formation of mRNA in the yeast Saccharomyces cerevisiae: (i) the efficiency element TATATA and related sequences, which function by enhancing the efficiency of positioning elements; (ii) positioning elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation. In this study, we have shown that several A-rich sequences, including the vertebrate poly(A) signal AATAAA, are also positioning elements. Saturated mutagenesis revealed that optimum sequences of the positioning element were AATAAA and AAAAAA and that this element can tolerate various extents of replacements. However, the GATAAA sequence was completely ineffective. The major cleavage sites determined in vitro corresponded to the major poly(A) sites observed in vivo. Our findings support the assumption that some components of the basic polyadenylation machinery could have been conserved among yeasts, plants, and mammals, although 3' end formation in yeasts is clearly distinct from that of higher eukaryotes.

Characterization of an Arabidopsis thaliana cDNA homologue to animal poly(ADP-ribose) polymerase

A full-length Arabidopsis thaliana cDNA ( app) encoding a protein with high similarity (about 60%) to the catalytic domain of vertebrate poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) has been cloned. The N-terminal extension of the Arabidopsis protein shows similarities with domains of different nuclear and DNA binding proteins in agreement with nuclear localization and putative function of a plant PARP. APP is encoded by a single gene mapped at the top of chromosome 4 of the Arabidopsis genome and mRNA is abundant in cell suspension culture compared to its accumulation in whole plant.

A dissection of the cauliflower mosaic virus polyadenylation signal.

Mutagenesis analysis of the polyadenylation [poly(A)] signal from the cauliflower mosaic virus (CaMV), a plant pararetrovirus, revealed striking differences to known vertebrate poly(A) signals. Our results show that (1) the AATAAA sequence is necessary for efficient cleavage at the poly(A) site, although the requirement for an authentic AATAAA might be less stringent in plant than in vertebrate cells; (2) surprisingly and in contrast to the majority of vertebrate poly(A) signals, the sequences downstream of the CaMV poly(A) site do not influence processing efficiency drastically although they affect the precision of cleavage; and (3) deletion of sequences upstream of the CaMV AATAAA sequence decreased processing at the CaMV site dramatically, suggesting the presence of one or several positively acting upstream elements. An oligonucleotide consisting of CaMV upstream sequences could induce the recognition of a normally silent exogenous poly(A) signal when inserted upstream of its AATAAA motif.

Are U4 small nuclear ribonucleoproteins involved in polyadenylation?

The mechanism whereby eukaryotic pre-messenger RNAs are polyadenylated is unknown. Most models for polyadenylation invoke cleavage of precursor transcripts at the site of poly(A) addition followed by polymerization of A residues by poly(A) polymerase. Analysis of the sequences surrounding poly(A) addition sites has identified the consensus recognition sequence element AAUAAA as necessary but not sufficient for polyadenylation. A second recognition sequence element CACUG , was observed by Benoit et al. to be adjacent to the site of poly(A) addition in several sequenced RNAs. Here, we analyse 61 vertebrate poly(A) addition sequences, define a more extensive recognition sequence for polyadenylation than previously recognized and suggest how the site of poly(A) addition may be chosen. Furthermore, we find that the defined recognition sequence has elements which are complementary to regions within the small nuclear RNA U4, suggesting that U4 small nuclear ribonucleoproteins (snRNPs) may mediate polyadenylation in a fashion similar to the role of U1 snRNPs in splicing. The model invokes hybridization of U4 RNA to AAUAAA recognition elements as related to primary site selection, and hybridization to CAYUG recognition elements as related to cleavage site selection.