Localization and association to cytoskeleton of COLL1alpha mRNA in Paracentrotus lividus egg requires cis- and trans-acting factors

COLL1alpha mRNA is asymmetrically distributed in the Paracentrotus lividus egg. Here we examine the involvement of the cytoskeleton in the localization process of collagen mRNA. The use of drugs such as colchicine and cytochalasin B reveals a perturbation of localization collagen mRNA. Moreover, the presence of specific cis-and trans-acting factors involved in cytoskeleton binding and the localization process was investigated. By Northwestern experiment we found that the 3'UTR of COLL1alpha mRNA is also able to bind two proteins of 54 and 40 kDa in a cellular fraction containing the cytoskeleton. Finally, we found that the protein of 54 kDa is LP54, a protein that binds the 3'UTRs of P. lividus maternal bep messengers and is necessary for their localization.     

RNA-protein interactions within the 3 ' untranslated region of vimentin mRNA.

Several functions have been attributed to protein binding within the 3'untranslated region (3'UTR) of mRNA, including mRNA localization, stability, and translational repression. Vimentin is an intermediate filament protein whose 3'untranslated sequence is highly conserved between species. In order to identify sequences that might play a role in vimentin mRNA function, we synthesized32P-labeled RNA from different regions of vimentin's 3'UTR and assayed for protein binding with HeLa extracts using band shift assays. Sequences required for binding are contained within a region 61-114 nucleotides downstream of the stop codon, a region which is highly conserved from Xenopus to man. As judged by competition assays, binding is specific. Solution probing studies of 32P-labeled RNA with various nucleases and lead support a complex stem and loop structure for this region. Finally, UV cross-linking of the RNA-protein complex identifies an RNA binding protein of 46 kDa. Fractionation of a HeLa extract on a sizing column suggests that in addition to the 46 kDa protein, larger complexes containing additional protein(s) can be identified. Vimentin mRNA has been shown to be localized to the perinuclear region of the cytoplasm, possibly at sites of intermediate filament assembly. To date, all sequences required for localization of various mRNAs have been confined to the 3'UTR. Therefore, we hypothesize that this region and associated protein(s) might be important for vimentin mRNA function such as in localization.     

The AU-rich 3' untranslated region of human MDR1 mRNA is an inefficient mRNA destabilizer.

The human multidrug resistance gene MDR1 encodes a membrane-bound protein, referred to as P-glycoprotein, that acts as a pump to extrude toxins from cells. The 3' untranslated region (3'UTR) of the human MDR1 mRNA is very AU-rich (70%) and contains AU-rich sequences similar to those shown to confer rapid decay on c-myc, c-fos, and lymphokine mRNAs. We tested the ability of the MDR1 3'UTR to act as an mRNA destabilizing element in the human hepatoma cell line HepG2. The MDR1 mRNA has an intermediate half-life of 8 h in HepG2 cells compared to a half-life of 30 min for c-myc mRNA. The MDR1 mRNA half-life was prolonged to >20 h upon treatment with the protein synthesis inhibitor cycloheximide. We constructed expression vectors containing the human beta-globin coding region with the 3'UTR from either MDR1 or c-myc. The c-myc 3'UTR increased the decay of the chimeric mRNA, but the MDR1 3'UTR had no effect. We tested the ability of MDR1 3'UTR sequences to compete for interaction with AU-binding proteins in cell extracts; MDR1 RNA probes had a fivefold lower affinity for AU-binding proteins that interact with the c-myc AU-rich 3'UTR. Overall, our data suggest that the MDR1 3'UTR does not behave as an active destabilizing element in HepG2 cells.     

Nuclear import of metallothionein requires its mRNA to be associated with the perinuclear cytoskeleton

The influence of mRNA localization on metallothionein-1 protein distribution was studied by immunocytochemistry. We used Chinese hamster ovary cells that had been transfected with either a native metallothionein-1 gene construct or metallothionein-1 5'-untranslated region and coding sequences linked to the 3'-untranslated region from glutathione peroxidase. The change in the 3'-untranslated region caused the delocalization of the mRNA with a loss of the perinuclear localization and association with the cytoskeleton. Clones were selected which expressed similar levels of metallothionein-1 protein, as assessed by radioimmunoassay. The results showed that loss of metallothionein-1 mRNA localization was associated with a loss of metallothionein-1 protein localization, most notably with a lack of metallothionein-1 protein in the nucleus of synchronized cells which were beginning to synthesize DNA. This indicates that the association of metallothionein-1 mRNA with the cytoskeleton around the nucleus is essential for efficient shuttling of the protein into the nucleus during the G(1) to S phase transition. This is the first demonstration of a physiological role for perinuclear mRNA localization and we propose that such localization may be important for a wide range of nuclear proteins, including those that shuttle between nucleus and cytoplasm in a cell cycle dependent manner.     

Okadaic acid-mediated induction of the c-fos gene in estrogen receptor-negative human breast carcinoma cells utilized, in part, posttranscriptional mechanisms involving adenosine-uridine-rich elements.

Signal transduction via modulation of phosphorylation after selective inhibition of protein phosphatase (PP) 1 and/or PP2A appears to play a role in okadaic acid (OA)-mediated effects. Treatment of several estrogen receptor-negative human breast carcinoma (HBC) cells with 100 nM OA resulted in induction of c-fos, c-myc, and cyclin-dependent kinase inhibitor p21WAF1/CIP1 genes. Transfections of various luciferase reporter constructs in HBC cells revealed involvement of activator protein-1-dependent as well as -independent pathways in induction of the c-fos gene by OA. MDA-MB-468 HBC cells were stably transfected with plasmids expressing luciferase, chimeric luciferase- c-fos 3' untranslated region (3'UTR), or chimeric luciferase-p21WAF1/CIP 3'UTR mRNAs. Expression of chimeric luciferase-c-fos and luciferase-p21WAF1/CIP1 mRNAs was elevated by OA in several independent sublines. Actinomycin D chase experiments revealed an enhanced rate of decay of luciferase-c-fos mRNA, whereas treatment with OA caused approximately 3.5-fold enhanced stability of the chimeric luciferase-c-fos mRNA only. By transfecting different plasmids containing deletions of c-fos 3'UTR, OA-responsive sequences were mapped to an 86-nucleotide, AU-rich region. UV cross-linking experiments using HBC cell cytosolic proteins showed multiple complexes with the AU-rich region subfragments of c-fos, as well as c-myc and p21WAF1/CIP1 mRNAs. OA enhanced binding of a novel Mr approximately 75,000 protein present in the cytosolic extracts of HBC cells to the AU-rich RNA probes of all of the above three genes. Taken together, OA regulation of HBC cell gene expression involves the activator protein-1 pathway, as well as enhanced binding of a novel Mr approximately 75,000 protein to an AU-rich region of the 3'UTRs of the target genes.     

Annexin A2 recognises a specific region in the 3'-UTR of its cognate messenger RNA

Annexin A2 is a multifunctional Ca(2+)- and lipid-binding protein. We previously showed that a distinct pool of cellular Annexin A2 associates with mRNP complexes or polysomes associated with the cytoskeleton. Here we report in vitro and in vivo experiments showing that Annexin A2 present in this subset of mRNP complexes interacts with its cognate mRNA and c-myc mRNA, but not with beta(2)-microglobulin mRNA translated on membrane-bound polysomes. The protein recognises sequence elements within the untranslated regions, but not within the coding region, of its cognate mRNA. Alignment of the Annexin A2-binding 3'-untranslated regions of annexin A2 mRNA from several species reveals a five nucleotide consensus sequence 5'-AA(C/G)(A/U)G. The Annexin A2-interacting region of the 3'-untranslated region can be mapped to a sequence of about 100 nucleotides containing two repeats of the consensus sequence. The binding elements appear to involve both single and double stranded regions, indicating that a specific higher order mRNA structure is required for binding to Annexin A2. We suggest that this type of interaction is representative for a group of mRNAs translated on cytoskeleton-bound polysomes.     

The insulin-like growth factor mRNA binding-protein IMP-1 and the Ras-regulatory protein G3BP associate with tau mRNA and HuD protein in differentiated P19 neuronal cells

Tau mRNA is axonally localized mRNA that is found in developing neurons and targeted by an axonal localization signal (ALS) that is located in the 3'UTR of the message. The tau mRNA is trafficked in an RNA-protein complex (RNP) from the neuronal cell body to the distal parts of the axon, reaching as far as the growth cone. This movement is microtubule-dependent and is observed as granules that contain tau mRNA and additional proteins. A major protein contained in the granule is HuD, an Elav protein family member, which has an identified mRNA binding site on the tau 3'UTR and stabilizes the tau message and several axonally targeted mRNAs. Using GST-HuD fusion protein as bait, we have identified four proteins contained within the tau RNP, in differentiated P19 neuronal cells. In this work, we studied two of the identified proteins, i.e. IGF-II mRNA binding protein 1 (IMP-1), the orthologue of chick beta-actin binding protein-ZBP1, and RAS-GAP SH3 domain binding protein (G3BP). We show that IMP-1 associates with HuD and G3BP-1 proteins in an RNA-dependent manner and binds directly to tau mRNA. We also show an RNA-dependent association between G3BP-1 and HuD proteins. These associations are investigated in relation to the neuronal differentiation of P19 cells.     

Detection and characterization of a 3' untranslated region ribonucleoprotein complex associated with human alpha-globin mRNA stability.

The highly stable nature of globin mRNA is of central importance to erythroid cell differentiation. We have previously identified cytidine-rich (C-rich) segments in the human alpha-globin mRNA 3' untranslated region (alpha-3'UTR) which are critical in the maintenance of mRNA stability in transfected erythroid cells. In the present studies, we have detected trans-acting factors which interact with these cis elements to mediate this stabilizing function. A sequence-specific ribonucleoprotein (RNP) complex is assembled after incubation of the alpha-3'UTR with a variety of cytosolic extracts. This so-called alpha-complex is sequence specific and is not formed on the 3'UTR of either beta-globin or growth hormone mRNAs. Furthermore, base substitutions within the C-rich stretches which destabilize alpha-globin mRNA in vivo result in a parallel disruption of the alpha-complex in vitro. Competition studies with a series of homoribopolymers reveals a striking sensitivity of alpha-complex formation to poly(C), suggesting the presence of a poly(C)-binding activity within the alpha-complex. Three predominant proteins are isolated by alpha-3'UTR affinity chromatography. One of these binds directly to poly(C). This cytosolic poly(C)-binding protein is distinct from previously described nuclear poly(C)-binding heterogeneous nuclear RNPs and is necessary but not sufficient for alpha-complex formation. These data suggest that a messenger RNP complex formed by interaction of defined segments within the alpha-3'UTR with a limited number of cytosolic proteins, including a potentially novel poly(C)-binding protein, is of functional importance in establishing high-level stability of alpha-globin mRNA.     

Immediate Translation of Formin DIAPH1 mRNA after Its Exiting the Nucleus Is Required for Its Perinuclear Localization in Fibroblasts

DIAPH1 is a formin protein which promotes actin polymerization, stabilizes microtubules and consequently is involved in cytoskeleton dynamics, cell migration and differentiation. In contrast to the relatively well-understood signaling cascades that regulate DIAPH1 activity, its spatial regulation of biogenesis is not understood. A recent report showed that synthesis of DIAPH1 is confined in the perinuclear ER compartment through translation-dependent mRNA targeting. However, the underlying mechanism of DIAPH1 local synthesis is yet to be elucidated. Here, we provide evidence to demonstrate that the 5′-cap-mediated immediate translation of DIAPH1 mRNA upon exiting nucleus is required for localizing the mRNA in the perinuclear ER compartment. This is supported by data: 1) Delayed translation of DIAPH1 mRNA resulted in loss of perinuclear localization of the mRNA; 2) Once delocalized, DIAPH1 mRNA could not be retargeted to the perinuclear region; and 3) The translation of DIAPH1 mRNA is 5′-cap dependent. These results provide new insights into the novel mechanism of DIAPH1 local synthesis. In addition, these findings have led to the development of new approaches for manipulating DIAPH1 mRNA localization and local protein synthesis in cells for functional studies. Furthermore, a correlation of DIAPH1 mRNA and DIAPH1 protein localization has been demonstrated using a new method to quantify the intracellular distribution of protein.     

Specific interaction between human parechovirus nonstructural 2A protein and viral RNA

The functional properties of the nonstructural 2A protein are variable among different picornaviruses. The 2A protein of the human parechovirus 1 (HPEV1) has been shown to lack the proteolytic activity found in many other picornaviruses, but no particular function has been identified for HPEV1 2A. To obtain information about the role of HPEV1 2A in the viral life cycle, the protein was expressed in Escherichia coli. A polyclonal antibody was then raised against the protein and employed to investigate its subcellular localization in the infected cells by immunofluorescence microscopy. Typically, a diffuse cytoplasmic staining pattern, concentrated to the perinuclear area, was observed in the infected cells. However, at late stages of infection some infected cells also exhibited diffuse nuclear staining. Viral RNA, visualized by fluorescent in situ hybridization, partly colocalized with 2A in the perinuclear region. Three experimental approaches including Northwestern blot, UV cross-linking, and gel retardation demonstrated that 2A possesses RNA binding activity. Competition experiments with various single-stranded RNA molecules addressed the specificity of 2A binding. These studies revealed that the 2A protein bound RNA corresponding to the 3'-untranslated region (UTR) of the viral genome with highest affinity. At the N- and C-terminal ends of the protein, two regions, necessary for RNA binding, were identified by mutagenesis. In addition, we demonstrated that 2A has affinity to double-stranded RNA containing 3'UTR(+)-3'UTR(-). In conclusion, our experiments showed that HPEV1 2A binds to viral 3'UTR RNA, a feature that could be important for the function of the protein during HPEV1 replication.     

The subcellular distribution of early endosomes is affected by the annexin II2p11(2) complex

The tyrosine kinase substrate annexin II is a member of a multigene family of Ca2+ and lipid-binding proteins which have been implicated in a number of membrane-related events. We have analyzed the subcellular distribution of annexin II in relation to other cellular components in normal and specifically manipulated MDCK cells. In a polarized monolayer of MDCK cells annexin II and its cellular ligand p11 are restricted almost exclusively to the cortical regions of the cells which also contain peripheral early endosomes. Treatment of the polarized cells with low Ca2+ medium leads to a disintegration of the cortical cytoskeleton and a translocation of both, the annexin II2p11(2) complex and early endosomes, to the cytoplasm. A similar translocation which is however specific for the annexin II2p11(2) complex and early endosomes and does not affect other elements of the cell cortex is observed in cells expressing a trans-dominant annexin II- p11 mutant. This chimeric mutant protein causes the aggregation of endogenous annexin II and p11 and the simultaneous detachment of early endosomes from the cell periphery resulting in the binding of the early endosomes but no other components of the endocytotic or biosynthetic pathways to the annexin II/p11 aggregates. The specificity of this effect argues for the association of the annexin II2p11(2) complex with early endosomes and suggests that this association contributes to establish the peripheral localization of early endosomal structures.