Messenger RNA is produced by RNA polymerase II (pol II) transcription, followed by processing of the primary transcript. Transcription, splicing and cleavage-polyadenylation can occur independently in vitro, but we demonstrate here that these processes are intimately linked in vivo. We show that the carboxy-terminal domain (CTD) of the pol II large subunit is required for efficient RNA processing. Splicing, processing of the 3' end and termination of transcription downstream of the poly(A) site, are all inhibited by truncation of the CTD. We found that the cleavage-polyadenylation factors CPSF and CstF specifically bound to CTD affinity columns and copurified with pol II in a high-molecular-mass complex. Our demonstration of an association between the CTD and 3'-processing factors, considered together with reports of a similar interaction with splicing factors, suggests that an mRNA 'factory' exists which carries out coupled transcription, splicing and cleavage-polyadenylation of mRNA precursors.

GLD-2 is a cytoplasmic poly(A) polymerase present in the Caenorhabditis elegans germ line and embryo. It is a divergent member of the DNA polymerase ␤ nucleotidyl transferase superfamily, which includes CCA-adding enzymes, DNA polymerases and eukaryotic nuclear poly(A) polymerases. The polyadenylation activity of GLD-2 is stimulated by physical interaction with an RNA binding protein, GLD-3. To test whether GLD-3 might stimulate GLD-2 by recruiting it to RNA, we tethered C. elegans GLD-2 to mRNAs in Xenopus oocytes by using MS2 coat protein. Tethered GLD-2 adds poly(A) and stimulates translation of the mRNA, demonstrating that recruitment is sufficient to stimulate polyadenylation activity. We use the same tethered assay to identify human and mouse poly(A) polymerases related to GLD-2. This may provide entrees to previously uncharacterized modes of polyadenylation in mammalian cells.

Intensity modulated photocurrent (IMPS) and photovoltage (IMVS) spectroscopies were used to study the mechanism of photoprocesses in P3HT:PCBM bulk heterojunction organic solar cells at various light intensities. The use of the frequency domain techniques allowed us to separate the bulk and interfacial processes and gain a valuable insight into the mechanism of losses in these devices. The results provide direct evidence that interfacial nongeminate recombination is one of the dominant loss and aging mechanisms in bulk heterojunction organic solar cells. The trapping of photoexcited holes in the P3HT phase was found to contribute to the increased recombination rate. The results suggest that promising ways of improving the efficiency of bulk heterojunction solar cells may be reducing the charge trapping both at and near the P3HT:PCBM interface, as well as improving the efficiency of charge extraction at contacts.

During oocyte maturation and early development, mRNAs receive poly(A) in the cytoplasm at distinct times relative to one another and to the cell cycle. These cytoplasmic polyadenylation reactions do not occur during oogenesis, but begin during oocyte maturation and continue throughout early development. In this report, we focus on the link between cytoplasmic polyadenylation and control of the cell cycle during meiotic maturation. Activation of maturation promoting factor, a complex of CDK1 and cyclin, is required for maturation and dependent on c-mos protein kinase. We demonstrate here that two classes of polyadenylation exist during oocyte maturation, defined by their dependence of c-mos and CDK1 protein kinases. Polyadenylation of the first class of mRNAs (class I) is independent of c-mos and CDK1 kinase activities, whereas polyadenylation of the second class (class II) requires both of these activities. Class I polyadenylation, through its effects on c-mos mRNA, is required for class II polyadenylation. cis-acting elements responsible for this distinction reside in the 3'-untranslated region, upstream of the polyadenylation signal AAUAAA. Cytoplasmic polyadenylation elements (CPEs) are sufficient to specify class I polyadenylation, and subtle changes in the CPE can substantially, though not entirely, shift an RNA from class I to class II. Activation of class I polyadenylation events is independent of hyperphosphorylation of CPE-binding protein or poly(A) polymerase, and requires cellular protein synthesis. The two classes of polyadenylation and of mRNA define a dependent pathway, in which polyadenylation of certain mRNAs requires the prior polyadenylation of another. We propose that this provides one method of regulating the temporal order of polyadenylation events, and links polyadenylation to the control of the meiotic cell cycle.

Cytoplasmic polyadenylation of specific mRNAs commonly is correlated with their translational activation during development. Here, we focus on links between cytoplasmic polyadenylation, translational activation and the control of meiotic maturation in Xenopus oocytes. We manipulate endogenous c-mos mRNA, which encodes a protein kinase that regulates meiotic maturation. We determined that translational activation of endogenous c-mos mRNA requires a long poly(A) tail per se, rather than the process of polyadenylation. For this, we injected 'prosthetic' poly(A)_synthetic poly(A) tails designed to attach by base pairing to endogenous c-mos mRNA that has had its own polyadenylation signals removed. This prosthetic poly(A) tail activates c-mos translation and restores meiotic maturation in response to progesterone. Thus the role of polyadenylation in activating c-mos mRNA differs from its role in activating certain other mRNAs, for which the act of polyadenylation is required. In the absence of progesterone, prosthetic poly(A) does not stimulate c-mos expression, implying that progesterone acts at additional steps to elevate c-Mos protein. By using a general inhibitor of polyadenylation together with prosthetic poly(A), we demonstrate that these additional steps include polyadenylation of at least one other mRNA, in addition to that of c-mos mRNA. These other mRNAs, encoding regulators of meiotic maturation, act upstream of c-Mos in the meiotic maturation pathway.