Modulation of microfilament protein composition by transfected cytoskeletal actin genes.

HuT-14T is a highly tumorigenic fibroblast cell line which exhibits a reduced steady-state level of beta-actin due to coding mutations in one of two beta-actin alleles. The normal rate of total actin synthesis could be restored in some clones of cells following transfection of the functional beta-actin gene but not following transfection of the functional gamma-actin gene. In gamma-actin gene-transfected substrains that have increased rates of gamma-actin synthesis, beta-actin synthesis is further reduced in a manner consistent with an autoregulatory mechanism, resulting in abnormal ratios of actin isoforms. Thus, both beta- and gamma-actin proteins can apparently regulate the synthesis of their coexpressed isoforms. In addition, decreased synthesis of normal beta-actin seems to correlate with a concomitant down-regulation of tropomyosin isoforms.     

Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin cDNA.

We report the complete nucleotide sequence of a human beta actin cDNA. Both the 5' and 3' untranslated regions of the sequence are similar (greater than 80%) to the analogous regions of the rat beta-actin gene reported by Nudel et al (1983). When a segment of the 3' untranslated region is used as a radiolabelled probe, strong hybridization to chick beta actin mRNA is seen. This conservation of sequences suggests that strong selective pressures operate on non-translated segments of beta actin mRNA.     

Human cytoplasmic actin proteins are encoded by a multigene family

We characterized nine human actin genes that we isolated (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981) from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and alpha-, beta-, and gamma-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria we show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken beta-actin cDNA. We conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.     

Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.     

Identification of sequences within the murine granulocyte-macrophage colony-stimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells

Phorbol esters (TPA) and concanavalin A (ConA) are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF) production in murine thymoma EL-4 cells by mRNA stabilization. The role of the 3'-untranslated region (3'-UTR) in GM-CSF mRNA stabilization induced by TPA and ConA in EL-4 cells was examined by transfection studies using chloramphenicol acetyltransferase (CAT) constructions. The GM-CSF 3'-UTR contains a 63-nucleotide region at its 3' end with repeating ATTTA motifs which is responsible for mRNA degradation in a variety of cell types (Shaw, G., and Kamen, R. (1986) Cell 46, 659-666). We produced constructs containing most of the GM-CSF 3'-UTR (303 nucleotides, pRSV-CATgm) or the 3'-terminal AT-rich region (116 nucleotides, pRSV-CATau) and measured CAT enzyme activity and CAT mRNA after transient transfection into EL-4 and NIH 3T3 cells. Low levels of CAT activity were seen in both cells with either plasmid compared with levels of CAT activity obtained with pRSV-CAT. TPA treatment caused an approximately 10-fold increase in CAT activity and mRNA in EL-4 cells transfected with pRSV-CATgm. No increases were seen in EL-4 cells transfected with pRSV-CATau or pRSV-CAT. No response to TPA was detected in transfected NIH 3T3 cells, indicating that the response to TPA is relatively cell-specific. There was no increase in CAT activity after ConA treatment in EL-4 or NIH 3T3 cells transfected with any of the constructs suggesting that the GM-CSF 3'-UTR lacks elements that can respond alone to ConA. Nuclear run-on and actinomycin D chase experiments in EL-4 cells showed that TPA induces CAT activity via mRNA stabilization. By linker-substitution mutagenesis we show that TPA inducibility depends on a 60-nucleotide region of the 3'-UTR whose 5' end is located 160 nucleotides upstream of the 5' end of the AU-rich region.     

Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype

We have characterized the structure and function of RNA sequences that direct beta-cytoplasmic actin mRNA to the cell periphery were mapped to two segments of 3'-untranslated region by expression of LacZ/beta-actin chimeric mRNAs in chicken embryo fibroblasts (CEFs). A 54-nt segment, the "RNA zipcode," and a homologous but less active 43-nt segment each localized beta-galactosidase activity to the leading lamellae. This zipcode contains the full activity, and mutations or deletions within it reduce, but do not eliminate, its activity, indicating that several motifs contribute to the activity. Two of these motifs, when multimerized, can regenerate almost full activity. These sequences are highly conserved in evolution, since the human beta-actin zipcode, positioned identically in the 3'UTR localizes equally well in chicken cells. Complementary phosphorothioate oligonucleotides against the zipcode delocalized endogenous beta-actin mRNA, whereas those complementary to the region just outside the zipcode, or sense oligonucleotides, did not. Actin mRNA or protein levels were unaffected by the antisense treatments, but a dramatic change in lamellipodia structure, and actin stress fiber organization was observed using the same antizipcode oligonucleotides which delocalized the mRNA. Hence, discrete 3'UTR sequences direct beta-actin isoform synthesis to the leading lamellae and affect cell morphology, presumably through the actin cytoskeleton.     

Construction of a plasmid containing the complete coding region of human elongation factor 2.

A plasmid pUChEF-2 containing the coding sequence as well as the complete 3'-untranslated region (3'UTR) of human EF-2 mRNA was constructed. The plasmid construct was assembled from a cDNA insert of pHGR81 (Rapp et al., (1988) Biol. Chem. Hoppe-Seyler 369, 247-250) comprising the C-terminal portion of the coding region and the 3'UTR, as well as a polymer chain reaction PCR fragment (Rapp et al., (1989) Biol. Chem. Hoppe-Seyler 370, 1071-1075) covering the missing part of the coding region from the amino-terminus.     

Sequential expression of chicken actin genes during myogenesis

Embryonic muscle development permits the study of contractile protein gene regulation during cellular differentiation. To distinguish the appearance of particular actin mRNAs during chicken myogenesis, we have constructed DNA probes from the transcribed 3' noncoding region of the single-copy alpha-skeletal, alpha-cardiac, and beta-cytoplasmic actin genes. Hybridization experiments showed that at day 10 in ovo (stage 36), embryonic hindlimbs contain low levels of actin mRNA, predominantly consisting of the alpha-cardiac and beta-actin isotypes. However, by day 17 in ovo (stage 43), the amount of alpha-skeletal actin mRNA/microgram total RNA increased more than 30-fold and represented approximately 90% of the assayed actin mRNA. Concomitantly, alpha-cardiac and beta-actin mRNAs decreased by 30% and 70%, respectively, from the levels observed at day 10. In primary myoblast cultures, beta-actin mRNA increased sharply during the proliferative phase before fusion and steadily declined thereafter. alpha-Cardiac actin mRNA increased to levels 15-fold greater than alpha-skeletal actin mRNA in prefusion myoblasts (36 h), and remained at elevated levels. In contrast, the alpha-skeletal actin mRNA remained low until fusion had begun (48 h), increased 25-fold over the prefusion level by the completion of fusion, and then decreased at later times in culture. Thus, the sequential accumulation of sarcomeric alpha-actin mRNAs in culture mimics some of the events observed in embryonic limb development. However, maintenance of high levels of alpha-cardiac actin mRNA as well as the transient accumulation of appreciable alpha-skeletal actin mRNA suggests that myoblast cultures lack one or more essential components for phenotypic maturation.     

Trypanosoma cruzi infection affects actin mRNA regulation in heart muscle cells

We have previously described alterations in the cytoskeletal organization of heart muscle cells (HMC) infected with Trypanosoma cruzi in vitro. Our aim was to investigate whether these changes also affect the regulation of the actin mRNAs during HMC differentiation. Northern blot analysis revealed that alpha-cardiac actin mRNA levels increased during cell differentiation while beta-actin mRNA levels declined. Nonmuscle cells displayed beta-actin mRNA signal localized at the cell periphery, while alpha-cardiac actin mRNA had a perinuclear distribution in myocytes. Trypanosoma cruzi-infected cells showed 50% reduction in alpha-cardiac actin mRNA expression after 72 h of infection. In contrast, beta-actin mRNA levels increased approximately 79% after 48 h of infection. In addition, in situ beta-actin mRNA was delocalized from the periphery into the perinuclear region. These observations support the hypothesis that Trypanosoma cruzi affects actin mRNA regulation and localization through its effect on the cytoskeleton of heart muscle cells.     

Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis

MicroRNAs (miRNAs) are approximately 21-nt-long RNAs involved in regulating development, differentiation, and other processes in eukaryotes. In metazoa, nearly all miRNAs control gene expression by imperfectly base-pairing with the 3'-untranslated region (3'-UTR) of target mRNAs and repressing protein synthesis by an unknown mechanism. It is also unknown whether miRNA-mRNA duplexes containing mismatches and bulges provide specific features that are recognized by factors mediating the repression. miRNAs form part of ribonucleoprotein complexes, miRNPs, that contain Argonaute (Ago) and other proteins. Here we demonstrate that effects of miRNAs on translation can be mimicked in human HeLa cells by the miRNA-independent tethering of Ago proteins to the 3'-UTR of a reporter mRNA. Inhibition of protein synthesis occurred without a change in the reporter mRNA level and was dependent on the number, but not the position, of the hairpins tethering hAgo2 to the 3'-UTR. These findings indicate that a primary function of miRNAs is to guide their associated proteins to the mRNA.     

The hepatitis C virus RNA 3'-untranslated region strongly enhances translation directed by the internal ribosome entry site

The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5'- and 3'-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5'-UTR, and subsequent viral RNA replication requires sequences in the 3'-UTR and in the 5'-UTR. Addressing previous conflicting reports on a possible function of the 3'-UTR for RNA translation in this study, we found that reporter construct design is an important parameter in experiments testing 3'-UTR function. A translation enhancer function of the HCV 3'-UTR was detected only after transfection of monocistronic reporter RNAs or complete RNA genomes having a 3'-UTR with a precise 3' terminus. The 3'-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. The variable region, the poly(U . C) tract, and the most 3' terminal stem-loop 1 of the highly conserved 3' X region contribute significantly to translation enhancement, whereas stem-loops 2 and 3 of the 3' X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minus-strand synthesis in the HCV 3'-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3'-5' end interaction and a switch between translation and RNA replication.     

Separate cis-trans pathways post-transcriptionally regulate murine CD154 (CD40 ligand) expression: a novel function for CA repeats in the 3'-untranslated region

We report a role for CA repeats in the 3'-untranslated region (3'-UTR) in regulating CD154 expression. Human CD154 is encoded by an unstable mRNA; this instability is conferred in cis by a portion of its 3'-UTR that includes a polypyrimidine-rich region and CA dinucleotide repeat. We demonstrate similar instability activity with the murine CD154 3'-UTR. This instability element mapped solely to a conserved 100-base CU-rich region alone, which we call a CU-rich response element. Surprisingly, the CA dinucleotide-rich region also regulated reporter expression but at the level of translation. This activity was associated with poly(A) tail shortening and regulated by heterogeneous nuclear ribonucleoprotein L levels. We conclude that the CD154 3'-UTR contains dual cis-acting elements, one of which defines a novel function for exonic CA dinucleotide repeats. These findings suggest a mechanism for the association of 3'-UTR CA-rich response element polymorphisms with CD154 overexpression and the subsequent risk of autoimmune disease.