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A Loss of Function Allele for Murine Staufen1 Leads to Impairment of Dendritic Staufen1-RNP Delivery and Dendritic Spine Morphogenesis

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A Loss of Function Allele for Murine Staufen1 Leads to Impairment of Dendritic Staufen1-RNP Delivery and Dendritic Spine Morphogenesis A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis John P. Vessey*†‡, Paolo Macchi*†‡§, Joel M. Stein‡¶, Martin Mikl†, Kelvin N. Hawkerʈ**, Petra Vogelsang*††, Krzysztof Wieczorek†, Georgia Vendra†, Julia Riefler†, Fabian Tu¨ bing*†‡‡, Samuel A. J. Aparicioʈ§§, Ted Abel¶, and Michael A. Kiebler*†¶¶ *The-Max Planck Institute for Developmental Biology, Spemannstrasse 35, D-72076, Tu¨bingen, Germany; †Center for Brain Research, Medical University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria; ¶Department of Biology, University of Pennsylvania, Philadelphia, PA 19104; and ʈCambridge Institute for Medical Research, Wellcome Trust/MRC Building, Department of Oncology, Hills Road, Cambridge, CB2 2XY, United Kingdom Edited by Ruth Lehmann, New York University Medical Center, New York, NY, and approved September 5, 2008 (received for review May 12, 2008) The dsRNA-binding protein Staufen was the first RNA-binding Results and Discussion protein proven to play a role in RNA localization in Drosophila.A Targeted Disruption of the Stau1 Gene Leads to a Mutant Stau1 mammalian homolog, Staufen1 (Stau1), has been implicated in Protein Deficient in RNA-Binding. To generate a loss of function dendritic RNA localization in neurons, translational control, and allele in Stau1, we inserted an IRES␤geo cassette into exon 5 mRNA decay. However, the precise mechanisms by which it fulfills (10). This exon encodes for most of the dsRNA-binding domain these specific roles are only partially understood. To determine its 3 (dsRBD3), which is crucial for RNA-binding (11). Translation physiological functions, the murine Stau1 gene was disrupted by Ј tm1Apa of any truncated 5 transcript would result in dsRBD1 and homologous recombination. Homozygous stau1 mutant dsRBD2 and lack the critical functions encoded by dsRBD3. The mice express a truncated Stau1 protein lacking the functional targeting vector introduces a translational stop upstream of the RNA-binding domain 3. The level of the truncated protein is IRES sequence and a cassette with a polyA into the BglII site of significantly reduced. Cultured hippocampal neurons derived from exon 5 (Fig. 1A). This allele was designated stau1tm1Apa (12). stau1tm1Apa homozygous mice display deficits in dendritic delivery Targeting was carried out in 129SvEv (129S7) ES cells. This was of Stau1-EYFP and ␤-actin mRNA-containing ribonucleoprotein verified via Southern blotting using a 5Ј external probe (Fig. 1B particles (RNPs). Furthermore, these neurons have a significantly Ј reduced dendritic tree and develop fewer synapses. Homozygous Bottom) and by 3 PCR (Fig. 1B Top) using primers stauR5 and stau1tm1Apa mutant mice are viable and show no obvious deficits neo236, yielding a 2.9kb PCR product. Two correctly targeted in development, fertility, health, overall brain morphology, and a clones were selected that transmitted through the germline. The variety of behavioral assays, e.g., hippocampus-dependent learn- F1 founders were backcrossed once to parental 129SvEv mice tm1Apa/tm1Apa ing. However, we did detect deficits in locomotor activity. Our data before intercrossing. The resulting stau1 mice were suggest that Stau1 is crucial for synapse development in vitro but viable, reproduced normally, and showed no obvious anatomical not critical for normal behavioral function. abnormalities [supporting information (SI) Fig. S1]. Mice were genotyped by multiplex PCR as shown in Fig. 1C. tm1Apa dendrite ͉ RNA transport ͉ ribonucleoprotein particles ͉ To determine the effect of the stau1 allele on expression double-stranded RNA-binding protein of Stau1 protein, forebrain extracts were prepared from both WT and homozygous stau1tm1Apa mice and subjected to Western blotting. Probing with an immunopurified polyclonal anti-Stau1 rotein synthesis-dependent synaptic plasticity is considered a Ϸ Pfundamental mechanism facilitating learning and memory in antibody (13) revealed a truncated Stau1 protein of 50 kDa the central nervous system. Local translation of mRNA stored in that is substantially reduced in expression levels (Fig. 1D). No dendrites has emerged as an important mechanism contributing protein of predicted full-length was observed. In contrast, Stau2 to this synapse-specific event (1–2). Although a number of components have been recently implicated in dendritic mRNA Author contributions: J.P.V., P.M., J.M.S., M.M., K.N.H., K.W., G.V., S.A.J.A., T.A., and M.A.K. localization, the underlying molecular mechanisms are only designed research; J.P.V., P.M., J.M.S., M.M., K.N.H., P.V., K.W., J.R., and F.T. performed partially understood. A key protein involved in this process is research; J.P.V., P.M., J.M.S., M.M., K.N.H., P.V., K.W., G.V., J.R., F.T., S.A.A., T.A., and M.A.K. Staufen, a dsRNA-binding protein originally identified in Dro- analyzed data; and J.P.V. and M.A.K. wrote the paper. sophila (3). Drosophila embryos lacking Staufen exhibit anterior The authors declare no conflict of interest. head defects, deletions of abdominal segments, and a lack of This article is a PNAS Direct Submission. germ cell formation resulting from the mislocalization of oskar Freely available online through the PNAS open access option. and bicoid mRNA (4). Interestingly, a recent study implicated ‡J.P.V., P.M., and J.M.S. contributed equally to this work. Drosophila Staufen in long-term memory formation (5). §Present address: Centre for Integrative Biology, University of Trento, Via delle Regole 101, In mammals, the function of the two mammalian Staufen 38060 Mattarello, Italy. proteins, termed Stau1 and Stau2, is much less clear. Both **Present address: NCIP Ropewalk House, 113 The Ropewalk, Nottingham NG1–6HA, UK. Staufen proteins are components of RNPs that travel along ††Present address: Broegelmann Research Laboratory, The Gade Institute, University of microtubules from the soma to distant dendritic sites. Whereas Bergen, Haukelandsveien 28, N-5021 Bergen, Norway. Stau1 is thought to function in translational control and mRNA ‡‡Present Address: Roche Diagnostics, TE-FC2, Nonnenwald 2, D-82377 Penzberg, Germany. decay (6, 7), the brain-specific Stau2 has been implicated in §§Present address: BC Cancer Agency, 675 West 10th Avenue, Vancouver, BC, Canada mRNA transport (8) and dendritic spine morphogenesis (9). To V5Z 1L3. address the role of the mammalian Staufen1 protein in vivo,we ¶¶To whom correspondence should be addressed. E-mail: michael.kiebler@meduniwien. developed a loss of function allele in murine Stau1 using gene ac.at. targeting. This allowed us to determine whether the lack of Stau1 This article contains supporting information online at www.pnas.org/cgi/content/full/ affects neuronal granule assembly, synapse formation, or learn- 0804583105/DCSupplemental. ing and memory. © 2008 by The National Academy of Sciences of the USA 16374–16379 ͉ PNAS ͉ October 21, 2008 ͉ vol. 105 ͉ no. 42 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804583105 Downloaded by guest on September 24, 2021 A expression was not affected by the disruption of the Stau1 locus B (Fig. 1E). To further characterize the low level truncated protein, we isolated and sequenced mRNA from forebrains of stau1tm1Apa homozygous mice. Transcripts isolated contain an in-frame deletion of 207 nucleotides (68 residues) coding for the dsRBD3 (Fig. 1F) as the result of skipping exon 5. We refer to this protein as ⌬Stau1. Since the dsRBD3 of Stau1 is crucial for RNA binding in Drosophila and mammals (11, 14), we next assessed the RNA-binding capacity of the ⌬Stau1 protein by a Northwestern assay. Untagged mouse Stau1 and ⌬Stau1 were expressed in HeLa cells at approximately equal levels (Fig. 1G Top). After renaturation of the blotted proteins, the nitrocellu- lose membrane was exposed to a synthetic 32P-labeled poly(rI/ rC) probe. Overexpressed Stau1 bound significant amounts of RNA (Fig. 1G Lower). A second band of lower molecular weight, presumably an endogenous RNA-binding protein comigrating C D with overexpressed ⌬Stau1 (Fig. 1G, arrowhead), prevents a E conclusive statement regarding the RNA-binding capacity of the ⌬Stau1 protein. However, since this lower comigrating band is of the same relative intensity in all three lanes, we conclude that overexpressed ⌬Stau1 does not bind RNA to the same extent as overexpressed WTStau1. Loss of WTStau1 Leads to Impairments in Dendritic RNP Localization. F G Although brain extracts from stau1tm1Apa homozygous mice contained significantly reduced levels of Stau1 (Fig. 1D), we next asked whether any residual protein could be detected in hippocampal neurons. Both pyramidal neurons in the CA1 (Fig. 2B) as well as dissociated hippocampal neurons in culture (Fig. 2D) derived from stau1tm1Apa homozygous mice fail to express significant amounts of Stau1 protein in contrast to their WT counterparts (Fig. 2 A and C). Staining of cultured neurons revealed that Stau2 is expressed at equal levels by both WT and stau1tm1Apa homozygous mouse neurons in a parti- culate pattern in both the cell body and distal dendrites (Fig. S2A). Since Drosophila Staufen heterooligomerizes with itself Fig. 1. Truncated stau1 lacking dsRBD3 leads to reduced Stau1 protein levels and several copies of oskar mRNA to form functional RNA and an impairment of RNA-binding capacity. (A) Targeting strategy for dis- particles (11), we investigated whether ⌬Stau1-EYFP protein rupting the Stau1 gene in mouse. WT genomic locus (Top) shows the location shows any impairment in particle formation and dendritic of the dsRBD3, the sites for homologous recombination with the targeting ␤ localization upon expression in both WT (Fig. 2G)or vector (IRES geopA, Middle) and the final targeted, mutated Stau1 locus tm1Apa (Bottom). Arrowheads indicate the primers used for genotyping. 5Ј probe, stau1 homozygous neurons (Fig. 2H). When WTStau1- indicates the probe used for Southern blotting. (B)3Ј PCR (Top) and 5Ј EYFP was transfected into neurons derived from ⌬Stau1 mice Southern blot (Bottom) analysis of WT (ϩ/ϩ), heterozygous (ϩ/-), and mutant (Fig. 2F), 68% of all transfected neurons display puncta in (Ϫ/Ϫ) Stau1 mice.
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