Phosphorylation of CPEB by Eg2 mediates the recruitment of CPSF into an active cytoplasmic polyadenylation complex.
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The release of Xenopus oocytes from prophase I arrest is largely driven by the cytoplasmic polyadenylation-induced translation of dormant maternal mRNAs. Two cis elements, the CPE and the hexanucleotide AAUAAA, and their respective binding factors, CPEB and a cytoplasmic form of CPSF, control polyadenylation. The most proximal stimulus for polyadenylation is Eg2-catalyzed phosphorylation of CPEB serine 174. Here, we show that this phosphorylation event stimulates an interaction between CPEB and CPSF. This interaction is direct, does not require RNA tethering, and occurs through the 160 kDa subunit of CPSF. Eg2-stimulated and CPE-dependent polyadenylation is reconstituted in vitro using purified components. These results demonstrate that the molecular function of Eg2-phosphorylated CPEB is to recruit CPSF into an active cytoplasmic polyadenylation complex. (+info)
Poly(A) polymerase and the regulation of cytoplasmic polyadenylation.
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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. (+info)
Posttranscriptional regulation of cyclin A1 and cyclin A2 during mouse oocyte meiotic maturation and preimplantation development.
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A shift from a meiotic cell cycle to a mitotic cell cycle occurs following fertilization. The molecular basis for this transition, however, is poorly understood. Although cyclin A1 is proposed to regulate M phase in the meiotic cell cycle, and cyclin A2 is proposed to regulate S and M phases in the mitotic cell cycle, little is known about changes in the expression levels of cyclin A1 and A2 during meiotic and mitotic cell cycles in mammalian oocytes. We report that the mRNA levels of both cyclins A1 and A2 decrease during oocyte maturation. The amount of cyclin A1 mRNA then increases between the one-cell and blastocyst stages, whereas that of cyclin A2 remains relatively constant. The amount of cyclin A1 protein declines during maturation and is not readily detected from the two-cell to the blastocyst stage. In contrast, cyclin A2 is not readily detected in the oocyte and metaphase II-arrested egg but is detected following fertilization and throughout the subsequent stages of preimplantation development. The appearance of cyclin A2 protein following fertilization positively correlates with an increase in the size of the mRNA. This increase, as well as the increase in the amount of cyclin A2 protein, is prevented by 3'-deoxyadenosine (3'-dA), an inhibitor of polyadenylation. Consistent with a role for cyclin A2 in regulating the G1/S transition, 3'-dA also inhibits DNA replication in treated one-cell embryos. These results suggest that regulation of expression of cyclins A1 and A2 is under posttranscriptional regulation and that the observed changes in their expression may be involved in the transformation of a meiotic cell cycle to a mitotic cell cycle following fertilization. (+info)
Expression of a green fluorescent protein variant in mouse oocytes by injection of RNA with an added long poly(A) tail.
(4/746)
In oocytes, cytoplasmic 3' polyadenylation regulates translational activation of dormant mRNA during meiotic maturation. Thus exogenous proteins are hardly expressed after injection of conventional RNA. To circumvent this, we synthesized a long polyadenylated (approximately 250 A) tail to encode RNA with an enhanced yellow fluorescent protein targeted to mitochondria (EYFP-mito), and injected it into mouse oocytes at the germinal vesicle (GV) stage. From this transcript, EYFP-mito was clearly expressed in approximately 80% of oocytes, while scarce expression from a transcript with only 30 A was observed. In strongly expressing oocytes, fluorescence was detected within 1-3 h after RNA injection, increased linearly up to 12 h, and reached a maximum at 12-15 h. The distribution of EYFP-mito matched the staining of mitochondria in these oocytes. About 80% of these oocytes underwent GV breakdown and 60% matured in vitro, comparable to non-expressing or non-RNA-injected oocytes. Some of the oocytes which strongly expressed EYFP-mito remained at the GV stage. Thus, the expression was not always accompanied by meiotic maturation, nor did it suppress the maturation process. Mature oocytes expressing EYFP-mito possessed normal fertilizability associated with intracellular Ca(2+) oscillations, and developed into 2-cell embryos. Thus, polyadenylated RNA is a useful tool applicable to the expression of EYFP-fused functional proteins or of indicator protein probes for studies of mammalian fertilization. (+info)
Rna15 interaction with the A-rich yeast polyadenylation signal is an essential step in mRNA 3'-end formation.
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In Saccharomyces cerevisiae, four factors [cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP)] are required for maturation of the 3' end of the mRNA. CF I and CF II are required for cleavage; a complex of PAP and PF I, which includes CF II subunits, participates in polyadenylation, along with CF I. These factors are directed to the appropriate site on the mRNA by two sequences: one A-rich and one UA-rich. CF I contains five proteins, two of which, Rna15 and Hrp1, interact with the mRNA through RNA recognition motif-type RNA binding motifs. Previous work demonstrated that the UV cross-linking of purified Hrp1 to RNA required the UA-rich element, but the contact point of Rna15 was not known. We show here that Rna15 does not recognize a particular sequence in the absence of other proteins. However, in complex with Hrp1 and Rna14, Rna15 specifically interacts with the A-rich element. The Pcf11 and Clp1 subunits of CF I are not needed to position Rna15 at this site. This interaction is essential to the function of CF I. A mutant Rna15 with decreased affinity for RNA is defective for in vitro RNA processing and lethal in vivo, while an RNA with a mutation in the A-rich element is not processed in vitro and can no longer be UV cross-linked to the Rna15 subunit assembled into CF I. Thus, the recognition of the A-rich element depends on the tethering of Rna15 through an Rna14 bridge to Hrp1 bound to the UA-rich motif. These results illustrate that the yeast 3' end is defined and processed by a mechanism surprisingly different from that used by the mammalian system. (+info)
Mpe1, a zinc knuckle protein, is an essential component of yeast cleavage and polyadenylation factor required for the cleavage and polyadenylation of mRNA.
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In Saccharomyces cerevisiae, in vitro mRNA cleavage and polyadenylation require the poly(A) binding protein, Pab1p, and two multiprotein complexes: CFI (cleavage factor I) and CPF (cleavage and polyadenylation factor). We characterized a novel essential gene, MPE1 (YKL059c), which interacts genetically with the PCF11 gene encoding a subunit of CFI. Mpe1p is an evolutionarily conserved protein, a homolog of which is encoded by the human genome. The protein sequence contains a putative RNA-binding zinc knuckle motif. MPE1 is implicated in the choice of ACT1 mRNA polyadenylation site in vivo. Extracts from a conditional mutant, mpe1-1, or from a wild-type extract immunoneutralized for Mpe1p are defective in 3'-end processing. We used the tandem affinity purification (TAP) method on strains TAP-tagged for Mpe1p or Pfs2p to show that Mpe1p, like Pfs2p, is an integral subunit of CPF. Nevertheless a stable CPF, devoid of Mpe1p, was purified from the mpe1-1 mutant strain, showing that Mpe1p is not directly involved in the stability of this complex. Consistently, Mpe1p is also not necessary for the processive polyadenylation, nonspecific for the genuine pre-mRNA 3' end, displayed by the CPF alone. However, a reconstituted assay with purified CFI, CPF, and the recombinant Pab1p showed that Mpe1p is strictly required for the specific cleavage and polyadenylation of pre-mRNA. These results show that Mpe1p plays a crucial role in 3' end formation probably by promoting the specific link between the CFI/CPF complex and pre-mRNA. (+info)
The complete gene sequence of titin, expression of an unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-line to I-band linking system.
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Titin is a giant vertebrate striated muscle protein with critical importance for myofibril elasticity and structural integrity. We show here that the complete sequence of the human titin gene contains 363 exons, which together code for 38 138 residues (4200 kDa). In its central I-band region, 47 novel PEVK exons were found, which contribute to titin's extensible spring properties. Additionally, 3 unique I-band titin exons were identified (named novex-1 to -3). Novex-3 functions as an alternative titin C-terminus. The novex-3 titin isoform is approximately 700 kDa in size and spans from Z1-Z2 (titin's N-terminus) to novex-3 (C-terminal exon). Novex-3 titin specifically interacts with obscurin, a 721-kDa myofibrillar protein composed of 57 Ig/FN3 domains, followed by one IQ, SH3, DH, and a PH domain at its C-terminus. The obscurin domains Ig48/Ig49 bind to novex-3 titin and target to the Z-line region when expressed as a GFP fusion protein in live cardiac myocytes. Immunoelectron microscopy detected the C-terminal Ig48/Ig49 obscurin epitope near the Z-line edge. The distance from the Z-line varied with sarcomere length, suggesting that the novex-3 titin/obscurin complex forms an elastic Z-disc to I-band linking system. This system could link together calcium-dependent, SH3-, and GTPase-regulated signaling pathways in close proximity to the Z-disc, a structure increasingly implicated in the restructuring of sarcomeres during cardiomyopathies. (+info)
Agonist-mediated down-regulation of rat beta1-adrenergic receptor transcripts: role of potential post-transcriptional degradation factors.
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The human beta1-adrenergic receptor (AR) and hamster beta2-AR transcripts can be post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation via interaction of their 3' untranslated regions (UTR) with RNA binding proteins. Using RNase protection assays, we have determined that chronic isoproterenol exposure of rat C6 glioma cells results in the accelerated reduction of beta1-AR mRNAs. To determine the role of cellular environment on the agonist-independent and agonist-mediated degradation of beta1-AR mRNAs, we transfected rat beta1-AR expression recombinants into both hamster DDT1MF2 cells and rat L6 cells. The rat beta1-AR mRNAs in the two transfectant cell pools retain longer agonist-independent half-lives than in the C6 environment and undergo accelerated degradation upon chronic agonist exposure. Using UV-cross-linking/immunoblot and immunoprecipitation analyses, we have determined that the rat beta1-AR 3' UTR recognizes a predominant M(r) 39,000 component, identified as the mammalian elav-like protein HuR, and several other minor components, including the heteronuclear protein hnRNP A1. HuR levels are more highly expressed in C6 cells than in DDT1MF2 and L6 cells and are induced after chronic isoproterenol treatment. Furthermore, C6 transfectants containing an HuR expression recombinant exhibit reduced beta1-AR mRNA half-lives that were statistically comparable with half-lives identified in isoproterenol-treated C6 cells. These results imply that HuR plays a potential role in the agonist-independent and agonist-mediated down-regulation of beta1-AR mRNAs. (+info)