scholarly journals Two ZBP1 KH domains facilitate β-actin mRNA localization, granule formation, and cytoskeletal attachment

2002 ◽  
Vol 160 (1) ◽  
pp. 77-87 ◽  
Author(s):  
Kim L. Farina ◽  
Stefan Hüttelmaier ◽  
Kiran Musunuru ◽  
Robert Darnell ◽  
Robert H. Singer
Keyword(s):  
Rna Binding ◽  
Leading Edge ◽  
Mrna Localization ◽  
Binding Studies ◽  
Granule Formation ◽  
Rna Recognition Motifs ◽  
Hnrnp K ◽  
Actin Mrna ◽  
Recognition Motifs ◽  
Localization Element

Chicken embryo fibroblasts (CEFs) localize β-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of β-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of β-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized β-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both β-actin mRNA localization and cell motility.

Cytoplasmic trafficking of IGF-II mRNA-binding protein by conserved KH domains

2002 ◽  
Vol 115 (10) ◽  
pp. 2087-2097 ◽  
Author(s):  
Finn C. Nielsen ◽  
Jacob Nielsen ◽  
Mette A. Kristensen ◽  
Grete Koch ◽  
Jan Christiansen
Keyword(s):  
Rna Binding ◽  
Leading Edge ◽  
Cortical Region ◽  
Structural Determinants ◽  
Granule Formation ◽  
Rna Recognition Motifs ◽  
Motile Cells ◽  
Recognition Motifs ◽  
Rnp Granules ◽  
Mrna Binding

The IGF-II mRNA-binding proteins (IMPs), which are composed of two RNA recognition motifs, (RRM) and four hnRNP K homology (KH) domains, have been implicated in subcytoplasmic localization of mRNAs during embryogenesis. The IMP family originated via two gene duplications before the divergence of vertebrates, and IMP homologues consisting of only the four KH motifs have been identified in Drosophila and Caenorhabditis elegans. Here we characterise the trafficking of GFP-IMP1 fusion proteins and determine the structural determinants for proper cytoplasmic localization. GFP-IMP1 is present in large 200-700 nm RNP granules, which are distributed along microtubules. In motile cells, GFP-IMP1 is transported towards the leading edge into the cortical region of the lamellipodia where it is connected to microfilaments. Granules travel in an ATP-dependent fashion at an average speed of 0.12 μm/s (range 0.04-0.22 μm/s), and cells switch from a delocalized to a localized pattern within 15-20 minutes. Both granule formation and localization are unaffected by removal of the two RRMs, whereas deletion of the KH domains, which mediate RNA-binding, impairs these functions. We conclude that IMP1 localization is associated with motility and that the major functions of IMP1 are carried out by the phylogenetically conserved KH domains.

scholarly journals Nuclear transit of human zipcode-binding protein IMP1

2003 ◽  
Vol 376 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Jacob NIELSEN ◽  
Sidsel K. ADOLPH ◽  
Ewa RAJPERT-De MEYTS ◽  
Jens LYKKE-ANDERSEN ◽  
Grete KOCH ◽  
...  
Keyword(s):  
Translational Control ◽  
Nuclear Export ◽  
Rna Binding ◽  
Binding Protein ◽  
Leptomycin B ◽  
Granule Formation ◽  
Rna Recognition Motifs ◽  
3T3 Fibroblasts ◽  
Recognition Motifs ◽  
Homology Domains

The human IMPs (insulin-like growth factor II mRNA-binding proteins) belong to a vertebrate zipcode-binding protein family consisting of two RNA recognition motifs and four K homology domains and have been implicated in cytoplasmic mRNA localization, turnover and translational control. In the present study, we show that IMP1 is capable of translocating into nuclei of NIH 3T3 fibroblasts and its immunoreactivity is present in the nuclei of human spermatogenic cells. IMP1 does not contain a simple import signal, but nuclear entry was facilitated by disruption of RNA binding and cytoplasmic granule formation. IMP1 contains two NESs (nuclear export signals) within the RNA-binding K homology domains 2 and 4. The former is a leucine-rich leptomycin B-sensitive NES, whereas the latter is a leptomycin B-insensitive NES. Taken together, these results indicate that IMP1 may attach to its target mRNAs in the nucleus and thereby define the cytoplasmic fate of the transcripts.

scholarly journals Sla1, a Schizosaccharomyces pombe Homolog of the Human La Protein, Induces Ectopic Meiosis when Its C Terminus Is Truncated

2003 ◽  
Vol 2 (6) ◽  
pp. 1274-1287 ◽  
Author(s):  
Kaori Tanabe ◽  
Noriko Ito ◽  
Tomomi Wakuri ◽  
Fumiyo Ozoe ◽  
Makoto Umeda ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Small Region ◽  
Full Length ◽  
Rna Recognition Motifs ◽  
La Protein ◽  
C Terminus ◽  
Localization Signal ◽  
Recognition Motifs

ABSTRACT Sla1 is a Schizosaccharomyces pombe homolog of the human La protein. La proteins are known to be RNA-binding proteins that bear conserved RNA recognition motifs (La and RRMs), but their biological functions still have not been fully resolved. In this study, we show that the S. pombe La homolog (Sla1) is involved in regulating sexual development. Sla1 truncated in the C terminus (Sla1ΔC) induced ectopic sporulation in the ras1Δ strain and several other sporulation-deficient mutants. The C terminus contains a nuclear localization signal. While full-length Sla1 localizes in the nucleus, Sla1ΔC is found throughout the cell, suggesting the cytoplasmic localization of Sla1ΔC is involved in its sporulation-inducing activity. Further deletion analysis of Sla1 indicated that a small region (35 amino acids) that includes a portion of RRM2 is sufficient to induce sporulation. The La motif (RRM1) is not involved in this activity. Strikingly, Sla1ΔC induced haploid meiosis in a heterothallic strain, similar to the pat1-114 or mei2-SATA mutation. Sla1ΔC induced sporulation in a mei3 disruptant but not in a mei2 disruptant, indicating that Sla1ΔC requires Mei2 to induce haploid meiosis. Deletion of the chromosomal sla1 gene lowered the temperature sensitivity of the pat1-114 mutant. Two-hybrid analysis indicated that Pat1 interacts with Sla1ΔC but not full-length Sla1. Thus, Sla1ΔC may block Pat1 activity. This block would remove the inhibition on Mei2, which would then drive the cell into haploid meiosis. Finally, Sla1 was degraded prior to the start of meiosis when we monitored Sla1 in cells in which meiosis was synchronously induced. The ability of truncated Sla1 to induce ectopic meiosis represents a very novel function that has hitherto not been suspected for the La family of proteins.

scholarly journals Specificity of RNA Binding by CPEB: Requirement for RNA Recognition Motifs and a Novel Zinc Finger

1998 ◽  
Vol 18 (2) ◽  
pp. 685-693 ◽  
Author(s):  
Laura E. Hake ◽  
Raul Mendez ◽  
Joel D. Richter
Keyword(s):  
Zinc Finger ◽  
Rna Binding ◽  
Rna Recognition ◽  
Cytoplasmic Polyadenylation ◽  
Functional Motif ◽  
Rna Recognition Motifs ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Maternal Mrnas ◽  
Recognition Motifs

ABSTRACT CPEB is an RNA binding protein that interacts with the maturation-type cytoplasmic polyadenylation element (CPE) (consensus UUUUUAU) to promote polyadenylation and translational activation of maternal mRNAs in Xenopus laevis. CPEB, which is conserved from mammals to invertebrates, is composed of three regions: an amino-terminal portion with no obvious functional motif, two RNA recognition motifs (RRMs), and a cysteine-histidine region that is reminiscent of a zinc finger. In this study, we investigated the physical properties of CPEB required for RNA binding. CPEB can interact with RNA as a monomer, and phosphorylation, which modifies the protein during oocyte maturation, has little effect on RNA binding. Deletion mutations of CPEB have been overexpressed inEscherichia coli and used in a series of RNA gel shift experiments. Although a full-length and a truncated CPEB that lacks 139 amino-terminal amino acids bind CPE-containing RNA avidly, proteins that have had either RRM deleted bind RNA much less efficiently. CPEB that has had the cysteine-histidine region deleted has no detectable capacity to bind RNA. Single alanine substitutions of specific cysteine or histidine residues within this region also abolish RNA binding, pointing to the importance of this highly conserved domain of the protein. Chelation of metal ions by 1,10-phenanthroline inhibits the ability of CPEB to bind RNA; however, RNA binding is restored if the reaction is supplemented with zinc. CPEB also binds other metals such as cobalt and cadmium, but these destroy RNA binding. These data indicate that the RRMs and a zinc finger region of CPEB are essential for RNA binding.

Structural properties and RNA-binding activities of two RNA recognition motifs of a mouse neural RNA-binding protein, mouse-Musashi-1

Gene ◽  
1997 ◽  
Vol 186 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Yasuyuki Kurihara ◽  
Takashi Nagata ◽  
Takao Imai ◽  
Ado Hiwatashi ◽  
Masataka Horiuchi ◽  
...  
Keyword(s):  
Structural Properties ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Recognition Motifs

Domain necessary for Drosophila ELAV nuclear localization: function requires nuclear ELAV

1999 ◽  
Vol 112 (24) ◽  
pp. 4501-4512 ◽  
Author(s):  
Y.M. Yannoni ◽  
K. White
Keyword(s):  
Nuclear Localization ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Localization Sequence ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Mutant Proteins ◽  
Localization Sequence ◽  
Recognition Motifs

The neuron specific Drosophila ELAV protein belongs to the ELAV family of RNA binding proteins which are characterized by three highly conserved RNA recognition motifs, an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. Despite their highly conserved RNA recognition motifs the ELAV family members are a group of proteins with diverse posttranscriptional functions including splicing regulation, mRNA stability and translatability and have a variety of subcellular localizations. The role of the ELAV hinge in localization and function was examined using transgenes encoding ELAV hinge deletions, in vivo. Subcellular localization of the hinge mutant proteins revealed that residues between amino acids 333–374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human ELAV family member, HuR. This defined sequence, however, was insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays revealed that mutant proteins that fail to localize to the nucleus are unable to provide ELAV vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag.

scholarly journals Visualization of mRNA translation in living cells

2006 ◽  
Vol 175 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Alexis J. Rodriguez ◽  
Shailesh M. Shenoy ◽  
Robert H. Singer ◽  
John Condeelis
Keyword(s):  
Mrna Translation ◽  
Leading Edge ◽  
Intercellular Junctions ◽  
Mrna Localization ◽  
Living Cells ◽  
C2c12 Cells ◽  
Cell Periphery ◽  
Actin Mrna ◽  
Cell Asymmetry ◽  
Cellular Compartments

The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of β-actin mRNA. Constructs coding for β-actin, containing tetracysteine motifs, were transfected into C2C12 cells, and sites of nascent polypeptide chains were detected using the biarsenial dyes FlAsH and ReAsH, a technique we call translation site imaging. These sites colocalized with β-actin mRNA at the leading edge of motile myoblasts, confirming that they were translating. β-Actin mRNA lacking the sequence (zipcode) that localizes the mRNA to the cell periphery, eliminated the translation there. A pulse-chase experiment on living cells showed that the recently synthesized protein correlated spatially with the sites of its translation. Additionally, localization of β-actin mRNA and translation activity was enhanced at cell contacts and facilitated the formation of intercellular junctions.

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