Conservation defines functional motifs in the squint/nodal-related 1 RNA dorsal localization element

ABSTRACT Immediate-early protein IE1 is a principal regulator of viral transcription and a contributor to origin-specific DNA replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Since these viral functions involve interaction of dimeric IE1 with palindromic homologous region (hr) enhancer-origin elements of the AcMNPV genome within the nucleus, it is presumed that proper nuclear transport of IE1 is essential for productive infection. To investigate the mechanisms of IE1 nuclear import, we analyzed the effect of site-directed mutations on IE1 subcellular distribution. As demonstrated by fluorescence microscopy and biochemical fractionation of plasmid-transfected cells, wild-type IE1 localized predominantly to the nucleus. Substitution or deletion of amino acid residues within a positively charged domain (residues 534 to 538) adjacent to IE1's oligomerization motif impaired nuclear import and caused loss of transactivation. Moreover, upon coexpression, these import-defective mutations prevented nuclear entry of wild-type IE1. In contrast, double-mutated IE1 defective for both nuclear import and dimerization failed to block nuclear entry or transactivation by wild-type IE1. Thus, import-defective IE1 dominantly interfered with wild-type IE1 by direct interaction and cytosolic trapping. Collectively, our data indicate that the small basic domain encompassing residues R537 and R538 constitutes a novel nuclear localization element that functions only upon IE1 dimerization. These findings support a model wherein IE1 oligomerizes within the cytosol as a prerequisite for nuclear entry and subsequent high-affinity interaction with the symmetrical binding sites comprising AcMNPV hr enhancer-origin elements.

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The Targeting of Xcat2 mRNA to the Germinal Granules Depends on a cis-Acting Germinal Granule Localization Element within the 3′UTR

Developmental Biology ◽

10.1006/dbio.1999.9554 ◽

2000 ◽

Vol 217 (2) ◽

pp. 221-229 ◽

Cited By ~ 52

Author(s):

Malgorzata Kloc ◽

Szczepan Bilinski ◽

Agnes Pui-Yee Chan ◽

Laurence D Etkin

Keyword(s):

Cis Acting ◽

Germinal Granules ◽

Localization Element

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A putative stem-loop structure in Drosophila crumbs is required for mRNA localisation in epithelia and germline cells

Journal of Cell Science ◽

10.1242/jcs.236497 ◽

2020 ◽

pp. jcs.236497

Author(s):

Srija Bhagavatula ◽

Elisabeth Knust

Keyword(s):

Epithelial Cells ◽

Transmembrane Protein ◽

Loop Structure ◽

Stem Loop ◽

Stem Loop Structure ◽

Evolutionarily Conserved ◽

Mrna Enrichment ◽

Egg Chambers ◽

Protein Localisation ◽

Localization Element

Crumbs (Crb) is an evolutionarily conserved transmembrane protein localised in the apical membrane of epithelial cells. Loss or mis-localisation of Crb is often associated with disruption of apico-basal cell polarity. crb mRNA is also apically enriched in epithelial cells, and, as shown here, accumulates in the oocyte of developing egg chambers. We narrowed down the Localization Element (LE) of crb mRNA to 47 nucleotides forming a putative stem-loop structure, suggesting to be recognised by Egalitarian (Egl). Mutations in conserved nucleotides abrogate apical transport. crb mRNA enrichment in the oocyte is affected in egl mutant egg chambers. A CRISPR based genomic deletion of the crb locus that includes the LE disrupts asymmetric crb mRNA localisation in epithelia and prevents its accumulation in the oocyte during early stages of oogenesis, but does not affect Crb protein localisation in embryonic and follicular epithelia. However, flies lacking the LE show ectopic Crb protein expression in the nurse cells. These data suggest an additional role of the Drosophila 3’-UTR in regulating translation in a tissue specific manner.

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

The Journal of Cell Biology ◽

10.1083/jcb.200206003 ◽

2002 ◽

Vol 160 (1) ◽

pp. 77-87 ◽

Cited By ~ 144

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.

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Isolation and identification of a protein binding to the localization element of Metallothionein-1 mRNA

Biochemical Society Transactions ◽

10.1042/bst0320705 ◽

2004 ◽

Vol 32 (5) ◽

pp. 705-706 ◽

Cited By ~ 7

Author(s):

I. Mickleburgh ◽

B. Burtle ◽

D. Nury ◽

H. Chabanon ◽

Z. Chrzanowska-Lightowlers ◽

...

Keyword(s):

Mammalian Cells ◽

Chinese Hamster ◽

Elongation Factor ◽

Ovary Cell ◽

Isolation And Identification ◽

Flight Mass Spectrometry ◽

Cell Extracts ◽

Perinuclear Cytoplasm ◽

Paramagnetic Beads ◽

Localization Element

mRNA localization provides a mechanism for localized protein synthesis. mRNAs encoding certain proteins, including c-MYC, c-FOS, MT-1 (Metallothionein-1) and vimentin, are localized around the nuclei of mammalian cells and are associated with the cytoskeleton. Targeting of these mRNAs to the perinuclear cytoplasm is mediated by elements within their 3′-UTRs (3′-untranslated regions), but many of the trans-acting proteins remain unidentified. UV cross-linking assays using radiolabelled transcripts indicated that a protein of approx. 50 kDa (from the Chinese-hamster ovary cell extracts) bound to the MT-1 3′-UTR sequence. Competition experiments using unlabelled mutant 3′-UTR RNAs revealed that the binding of this protein is specific to localization-positive mutants. Isolation of a 50 kDa protein was achieved by an RNA affinity-based method in which biotinylated MT-1 3′-UTR RNA was anchored to paramagnetic beads. Bound proteins were eluted and analysed by SDS/PAGE. The 50 kDa protein was extracted from the gel, subjected to trypsin digestion and identified by matrix-assisted laser-desorption/ionization–time-of-flight mass spectrometry as eukaryote elongation factor 1α.

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An RNA-binding tropomyosin recruits kinesin-1 dynamically to oskar mRNPs

10.1101/067876 ◽

2016 ◽

Cited By ~ 1

Author(s):

Imre Gáspár ◽

Vasily Sysoev ◽

Artem Komissarov ◽

Anne Ephrussi

Keyword(s):

Nuclear Export ◽

Rna Binding ◽

Rna Localization ◽

Posterior Pole ◽

Transport Processes ◽

Nurse Cells ◽

Motor Activation ◽

Cargo Transport ◽

Dependent Transport ◽

Localization Element

AbstractLocalization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport-competent mRNPs. We show that the posterior-targeting kinesin-1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein-dependent transport from the nurse cells into the oocyte. We demonstrate that kinesin-1 recruitment requires the DmTropomyosin1-I/C isoform, an atypical RNA-binding tropomyosin that binds directly to dimerizing oskar 3’UTRs. Finally, we show that a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin-1 and that the motor is activated during mid-oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo-dependent motor activation constitutes an optmized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo-transport processes and between the cargo associated dynein and kinesin-1.

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