Cleavage and polyadenylation define the 3 ends of almost all eukaryotic mRNAs and are thought to occur during transcription. We describe a human in vitro system utilizing an immobilized template, in which transcripts in RNA polymerase II elongation complexes are efficiently cleaved and polyadenylated. Because the cleavage rate of free RNA is much slower, we conclude that cleavage is functionally coupled to transcription. Inhibition of positive transcription elongation factor b (P-TEFb) had only a modest negative effect on cleavage, as long as transcripts were long enough to contain the polyadenylation signal. In contrast, removal of the carboxyl-terminal domain of the large subunit of RNA polymerase II had a dramatic negative effect on cleavage. Unexpectedly, the 5 portion of transcript after cleavage remained associated with the template in a functional, polyadenylation-competent complex. Efficient cleavage required 5 capping by the human capping enzyme, but the reduction of cleavage seen of transcripts in COOH-terminal domain-less polymerase elongation complexes, was not because of lack of capping.Processing of eukaryotic mRNA starts during transcription and is influenced by the RNA polymerase II elongation complex (1, 2). Capping, polyadenylation, and splicing have been seen to occur on nascent transcripts in vitro, and a variety of in vivo and in vitro approaches have strongly implicated the carboxyl-terminal domain (CTD) 2 of the large subunit of RNA polymerase II in connecting transcription with these events (3-6). Whereas many of the factors required for mRNA processing have been identified and characterized, much less is known about how the processing and transcription machinery functionally influence each other.3Ј End formation is linked to transcription. Although RNA polymerase II is capable of transcribing hundreds of kilobase pairs in a completely processive manner, after transcribing a functional polyadenylation signal the polymerase usually terminates within 1 kb (3, 7) in a process that requires the CTD (8, 9). Factors required for polyadenylation have been found to associate with isolated transcription complexes (10) and the CTD has been implicated in bringing in the factors (11, 12). There is some controversy about when polyadenylation factors associate with the transcription complex. Polyadenylation factors have been found to associate with promoter binding factors (13), and yeast chromatin immunoprecipitation experiments in one study have localized the factors throughout the gene (11), but in another only at the 3Ј end of genes after the passage of a functional polyadenylation signal (14). Recent studies in yeast (15), Drosophila (16), and Xenopus (17) have implicated the CTD kinase positive transcription elongation factor b (P-TEFb) in promoting efficient polyadenylation. Drosophila histone mRNA 3Ј end formation, involving a completely different set of factors, was found not to be stimulated by having the RNA in an elongation complex (18). However, a strong transcriptional pause was found a...

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Abstract. Ponticulin is a 17-kD glycoprotein that represents a major high affinity link between the plasma membrane and the cortical actin network of Dictyostelium. To assess the role of ponticulin in pseudopod extension and retraction, the motile behavior of two independently generated mutants lacking ponticulin was analyzed using computer-assisted two-and threedimensional motion analysis systems. More than half of the lateral pseudopods formed off the substratum by ponticulin-minus cells slipped relative to the substratum during extension and retraction. In contrast, all pseudopods formed off the substratum by wild-type cells were positionally fixed in relation to the substratum. Ponticulin-minus cells also formed a greater proportion of both anterior and lateral pseudopods off the substratum and absorbed a greater proportion of lateral pseudopods into the uropod than wild-type cells. In a spatial gradient of cAMP, ponticulin-minus cells were less efficient in tracking the source of chemoattractant. Since ponticulin-minus cells extend and retract pseudopods with the same time course as wild-type cells, these behavioral defects in ponticulin-minus cells appear to be the consequence of pseudopod slippage. These resuits demonstrate that pseudopods formed off the substratum by wild-type cells are positionally fixed in relation to the substratum, that ponticulin is required for positional stabilization, and that the loss of ponticulin and the concomitant loss of positional stability of pseudopods correlate with a decrease in the efficiency of chemotaxis.

Previous studies have demonstrated that overexpression of the carboxyl‐terminal fragment, CaD39, of human fibroblast caldesmon in Chinese hamster ovary cells protected endogenous tropomyosin from turnover and stabilized actin microfilament bundles [Warren et al., 1994: J. Cell Biol. 125:359–368]. To assess the consequences of having CaD39‐stabilized microfilaments in living cell, we characterized the motile behaviors of stable CaD39‐expressing lines. We here found that CaD39‐expressing cells adhered faster to plastic, glass, fibronectin‐coated glass, and collagen‐coated glass than control cells. Moreover, the CaD39‐expressing cells also exhibited enhanced spreading immediately after attachment. Despite these differences, overexpression of CaD39 had little effect on the velocity of intracellular granule movement, or the velocity and persistence of cellular translocation. However, CaD39‐expressing cells were more elongate and encompassed less area than non‐expressing cells during migration in a wound‐healing assay. In interphase cells, the expressed CaD39 fragments were found associated with tropomyosin‐enriched microfilaments. Like endogenous caldesmon, the CaD39 fragment was also modified at mitosis. Although a significant portion of CaD39 underwent only partial modification, the majority of the CaD39 was released from the microfilaments during mitosis. This is consistent with the finding that the CaD39‐induced advantage for attachment and spreading was lost during mitosis. In CaD39‐expressing cells, an incomplete release of the CaD39 from microfilaments at mitosis was found which may be responsible for the increase in the frequency of multinuclear cells in CaD39‐expressing lines. © 1996 Wiley‐Liss, Inc.

These observations support a role for VPAC1 during embryonic and neonatal development of intestines and endocrine pancreas.