Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules

Central nervous system myelination requires the synthesis of large amounts of myelin basic protein (MBP) at the axon–glia contact site. MBP messenger RNA (mRNA) is transported in RNA granules to oligodendroglial processes in a translationally silenced state. This process is regulated by the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binding to the cis-acting A2 response element (A2RE). Release of this repression of MBP mRNA translation is thus essential for myelination. Mice deficient in the Src family tyrosine kinase Fyn are hypomyelinated and contain reduced levels of MBP. Here, we identify hnRNP A2 as a target of activated Fyn in oligodendrocytes. We show that active Fyn phosphorylates hnRNP A2 and stimulates translation of an MBP A2RE–containing reporter construct. Neuronal adhesion molecule L1 binding to oligodendrocytes results in Fyn activation, which leads to an increase in hnRNP A2 phosphorylation. These results suggest that Fyn kinase activation results in the localized translation of MBP mRNA at sites of axon–glia contact and myelin deposition

Fyn kinase targets in oligodendroglial physiology and myelination

In the central nervous system (CNS), oligodendrocytes form the multilamellar and compacted myelin sheath by spirally wrapping around defined axons with their specialised plasma membrane. Myelin is crucial for the rapid saltatory conduction of nerve impulses and for the preservation of axonal integrity. The absence of the major myelin component Myelin Basic Protein (MBP) results in an almost complete failure to form compact myelin in the CNS. The mRNA of MBP is sorted to cytoplasmic RNA granules and transported to the distal processes of oligodendrocytes in a translationally silent state. A main mediator of MBP mRNA localisation is the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 which binds to the cis-acting A2 response element (A2RE) in the 3’UTR of MBP mRNA. A signalling cascade had been identified that triggers local translation of MBP at the axon-glial contact site, involving the neuronal cell adhesion molecule (CAM) L1, the oligodendroglial plasma membrane-tethered Fyn kinase and Fyn-dependent phosphorylation of hnRNP A2. This model was confirmed here, showing that L1 stimulates Fyn-dependent phosphorylation of hnRNP A2 and a remodelling of A2-dependent RNA granule structures. Furthermore, the RNA helicase DDX5 was confirmed here acting together with hnRNP A2 in cytoplasmic RNA granules and is possibly involved in MBP mRNA granule dynamics.rnLack of non-receptor tyrosine kinase Fyn activity leads to reduced levels of MBP and hypomyelination in the forebrain. The multiadaptor protein p130Cas and the RNA-binding protein hnRNP F were verified here as additional targets of Fyn in oligodendrocytes. The findings point at roles of p130Cas in the regulation of Fyn-dependent process outgrowth and signalling cascades ensuring cell survival. HnRNP F was identified here as a novel constituent of oligodendroglial cytoplasmic RNA granules containing hnRNP A2 and MBP mRNA. Moreover, it was found that hnRNP F plays a role in the post-transcriptional regulation of MBP mRNA and that defined levels of hnRNP F are required to facilitate efficient synthesis of MBP. HnRNP F appears to be directly phosphorylated by Fyn kinase what presumably contributes to the initiation of translation of MBP mRNA at the plasma membrane.rnFyn kinase signalling thus affects many aspects of oligodendroglial physiology contributing to myelination. Post-transcriptional control of the synthesis of the essential myelin protein MBP by Fyn targets is particularly important. Deregulation of these Fyn-dependent pathways could thus negatively influence disorders involving the white matter of the nervous system.rnrnIm zentralen Nervensystem (ZNS) bilden Oligodendrozyten die mehrschichtige und kompaktierte Myelinscheide, indem sie bestimmte Axone mit spezialisierten Abschnitten ihrer Zytoplasmamembran spiralförmig umwickeln. Das Myelin ist für die schnelle, saltatorische Leitung von Nervenimpulsen und den Erhalt der axonalen Integrität von entscheidender Bedeutung. Das Fehlen des Myelin Basischen Proteins (MBP), eines der Hauptbestandteile des Myelins, führt zu beinahe vollständigem Verlust des kompakten Myelins im ZNS. Die mRNA von MBP wird in zytoplasmatische RNA Granulen sortiert und in diesen, in translationell inaktivem Zustand, bis zu den äußeren Fortsätzen der Oligodendrozyten transportiert. Eine der für diese Lokalisation der mRNA von MBP wichtigsten Komponenten ist der trans-agierende Faktor heterogenes nukleäres Ribonukleoprotein (hnRNP) A2, der an die cis-agierende A2 Erkennungssequenz in der 3’UTR der MBP mRNA bindet. Es wurde eine Signalkaskade entdeckt, die das neuronale Zelladhäsionsmolekül L1, die oligodendrogliale, an die Plasmamembran gebundene Fyn Kinase und die von Fyn abhängige Phosphorylierung von hnRNP A2 einschließt und die lokale Translation von MBP an der Axon-Glia Kontaktstelle initiiert. Dieses Modell wurde hier bestätigt. Es wurde gezeigt, dass L1 die von Fyn abhängige Phosphorylierung von hnRNP A2 und strukturelle Veränderungen in von A2 abhängigen RNA Granulen stimuliert. Des weiteren wurde der Verdacht bestätigt, dass die RNA Helikase DDX5 mit hnRNP A2 in zytoplasmatischen RNA Granulen interagiert und möglicherweise eine Rolle in MBP mRNA Granulen spielt.rnVerlust der nicht-Rezeptor Tyrosin Kinase Fyn führt zu reduzierten Mengen an MBP und zu Hypomyelinisierung im Vorderhirn. Das Multiadapterprotein p130Cas und das RNA-bindende Protein hnRNP F wurden in dieser Arbeit als weitere Substrate von Fyn in Oligodendrozyten verifiziert. Die Befunde deuten auf Funktionen von p130Cas in der Regulation des von Fyn abhängigen Fortsatzwachstums und in Signalkaskaden, die das Zellüberleben steuern, hin. HnRNP F wurde hier als neuer Bestandteil oligodendroglialer, zytoplasmatischer RNA Granulen, die hnRNP A2 und MBP mRNA enthalten, identifiziert. Darüber hinaus wurde herausgefunden, dass hnRNP F eine Rolle bei der posttranskriptionellen Regulation von MBP mRNA spielt und dass definierte Mengen an hnRNP F notwendig sind, um eine effiziente Synthese von MBP zu ermöglichen. HnRNP F scheint direkt von Fyn phosphoryliert zu werden, was wahrscheinlich zur Initiation der Translation von MBP mRNA an der Zytoplasmamembran beiträgt.rnDie Fyn Kinase beeinflusst somit viele Aspekte der Physiologie von Oligodendrozyten und trägt damit zur Myelinisierung bei. Die posttranskriptionelle Kontrolle der Synthese des essenziellen Myelinproteins MBP durch Fyn-Substrate ist hier besonders zu erwähnen. Eine Deregulation dieser Fyn-abhängigen Signalwege könnte somit negative Auswirkungen auf krankhafte Veränderungen der weißen Substanz des Nervensystems haben.r

Heterogeneous nuclear ribonucleoprotein (hnRNP) F is a novel component of oligodendroglial RNA transport granules contributing to regulation of myelin basic protein (MBP) synthesis

Myelin basic protein (MBP) is a major component of central nervous system (CNS) myelin. The absence of MBP results in the loss of almost all compact myelin in the CNS. MBP mRNA is sorted into RNA granules that are transported to the periphery of oligodendrocytes in a translationally inactive state. A central mediator of this transport process is the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 that binds to the cis-acting A2-response element in the 3'UTR of MBP mRNA. Recently, we found that activation of the Src family nonreceptor tyrosine kinase Fyn in oligodendrocytes leads to phosphorylation of hnRNP A2 and to increased translation of MBP mRNA. Here, we identify the RNA-binding protein hnRNP F as a novel component of MBP mRNA transport granules. It is associated with hnRNP A2 and MBP mRNA in cytoplasmic granular structures and is involved in post-transcriptional regulation of MBP expression. Fyn kinase activity results in phosphorylation of hnRNP F in the cytoplasm and its release from MBP mRNA and RNA granules. Our results define hnRNP F as a regulatory element of MBP expression in oligodendrocytes and imply an important function of hnRNP F in the control of myelin synthesis

Oligodendroglial p130Cas is a target of Fyn kinase involved in process formation, cell migration and survival

Oligodendrocytes are the myelinating glial cells of the central nervous system. In the course of brain development, oligodendrocyte precursor cells migrate, scan the environment and differentiate into mature oligodendrocytes with multiple cellular processes which recognize and ensheath neuronal axons. During differentiation, oligodendrocytes undergo dramatic morphological changes requiring cytoskeletal rearrangements which need to be tightly regulated. The non-receptor tyrosine kinase Fyn plays a central role in oligodendrocyte differentiation and myelination. In order to improve our understanding of the role of oligodendroglial Fyn kinase, we have identified Fyn targets in these cells. Purification and mass-spectrometric analysis of tyrosine-phosphorylated proteins in response to overexpressed active Fyn in the oligodendrocyte precursor cell line Oli-neu, yielded the adaptor molecule p130Cas. We analyzed the function of this Fyn target in oligodendroglial cells and observed that reduction of p130Cas levels by siRNA affects process outgrowth, the thickness of cellular processes and migration behavior of Oli-neu cells. Furthermore, long term p130Cas reduction results in decreased cell numbers as a result of increased apoptosis in cultured primary oligodendrocytes. Our data contribute to understanding the molecular events taking place during oligodendrocyte migration and morphological differentiation and have implications for myelin formation

(A) Oli- cells and primary oligodendrocytes were transfected with full-length hnRNP A2 and immunostained for hnRNP A2 or MBP

Images were acquired by confocal microscopy, and either a single slice (Oli-) or the complete stack (primary oligodendrocytes) is depicted. HnRNP A2–containing granules are present in the processes of Oli- cells as well as primary oligodendrocytes. Insets show an enlarged view of the boxed sections. Bars, 10 μm. (B) A granule-free supernatant was analyzed by Western blotting for hnRNP E1 and A2 proteins after RNase A treatment or L1-Fc binding. Western blot bands were analyzed densitometrically from 8 and 15 experiments for RNase treatment and L1-Fc binding, respectively. The control values (RNase A and C-Fc) were set to 1 and the mean relative increase of hnRNP E1 and A2 in the granule-free fraction was plotted in response to RNase A treatment or L1-Fc binding. Error bars indicate SEM; significance was tested with tests: *, P ≤ 0.05; **, P ≤ 0.01. = 8 (RNase A) and = 15 (L1-Fc). (C) The model illustrates the proposed events: During initial axon–glial contacts, neuronal L1 binds glial F3 (1), leading to an activation of Fyn (2), which phosphorylates hnRNP A2 (3). This leads to a release of hnRNP A2 and E1 from the granule and liberation of MBP mRNA (4) at the axon–glial contact site, allowing localized synthesis of the MBP protein (5) required for generation of the myelin sheath. The dotted lines illustrate potential alternative activation pathways of Fyn kinase mediated by L1 binding.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) Oli- cells were transfected with control EGFP or a constitutively active Fyn (Fyn) construct

Tyrosine-phosphorylated proteins were immunoprecipitated (P-Tyr IP) from cell lysates (lysate) and analyzed for hnRNP A2 and hnRNP E1/E2. HnRNP A2 immunoprecipitates from cells transfected with active Fyn, whereas hnRNP E1/E2 is absent. Mouse brain lysate and antibody beads alone served as blotting controls. Note that the hnRNP A2 antibody also recognizes the splice variants hnRNP B1 and B0a. HC and LC, heavy and light chain of mouse anti-phosphotyrosine antibody used in the immunopreciptiation. Black lines indicate that intervening lines have been spliced out. (B) Endogenous hnRNP A2 was immunoprecipitated from Oli- cells and subjected to an in vitro kinase assay using purified recombinant Fyn kinase. The top shows a phosphotyrosine blot and a band at 36 kD only in the presence of recombinant Fyn. This was identified as hnRNP A2 by reblotting with an hnRNP A2 antibody (bottom). Numbers to the right of the gel blots indicate molecular mass in kD. (C) HnRNP A2 coimmunoprecipitates with Fyn from Oli- cells transfected with wild-type Fyn, whereas a control protein (α-tubulin) does not.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) L1-Fc was bound to Oli- cells in the presence of control or F3 siRNA and detected with an anti–human Fc Cy2 antibody (left)

F3 knockdown was analyzed by Western blotting of the cells used for L1-Fc binding. Mouse brain lysate was used as control and GAPDH served as a loading control (right). Bars, 10 μm. (B) Differentiated Oli- cells were incubated with 75 nM of control Fc (human IgG, C-Fc) or L1-Fc and 75 nM L1-Fc in the presence of monoclonal F3 or O4 (control) antibodies. Binding was quantified by cell ELISA (see Materials and methods; = 6). (C) Oli- cells were treated with 0 or 25 nM L1-Fc after treatment with control or F3 siRNA. Binding was quantified by cell ELISA and the ratio of 25 nM Li-Fc–treated cells/0 nM L1-Fc–treated cells was plotted to express the relative amount of bound L1-Fc in control and F3 siRNA-treated cells. Note that because of a different experimental setup, the reduction of F3 protein levels was not as efficient as in the experiment shown in A ( = 9). Error bars indicate SEM. Significance was assessed by tests: *, P < 0.02; **, P < 0.01.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) Differentiated Oli- cells or primary oligodendrocytes were treated with the indicated concentrations of L1-Fc

Equal amounts of cell lysates were analyzed by Western blotting using the indicated antibodies. Numbers on top indicate L1-Fc concentration in nM. (B, left) Oli- cells were incubated with control Fc (C-Fc, human IgG) or L1-Fc in the presence of control or Fyn siRNA. Tyrosine-phosphorylated proteins were immunoprecipitated (P-Tyr IP) and analyzed by Western blotting for hnRNP A2 (P-Tyr A2). Levels of tyrosine-phosphorylated A2 are strongly increased in L1-Fc–treated cells compared with control Fc–treated cells, and this effect is reduced in cells treated with Fyn siRNA. Total lysates (before IP) were analyzed by Western blotting with hnRNP A2, Fyn, and GAPDH antibodies and demonstrated unchanged levels of total hnRNP A2 and a reduction of Fyn protein by Fyn siRNA. GAPDH served as a loading control. (B, right) The diagram represents the data from three such experiments. Protein bands of tyrosine-phosphorylated hnRNP A2 were densitometrically quantified and the values of control Fc–treated cells were set to 1. The relative increase of tyrosine-phosphorylated hnRNP A2 in L1-Fc–treated cells in the presence of control and Fyn siRNA was plotted. Error bars indicate SEM; = 3.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) An A2RE-containing region of the 3′ UTR of MBP14 mRNA was cloned downstream of the luciferase coding sequence (CDS) and used as a translational reporter in the DualGlo luciferase assay

(B) Oli- cells were nucleofected with an A2RE containing luciferase reporter, a luciferase control, and either wild-type Fyn (Fyn WT), constitutively active Fyn (Fyn), or EGFP. was normalized to luciferase activity for every measurement in all experiments ( = 3). (C) Total RNA was isolated from the cells used in the luciferase assay shown in B. qRT-PCR was performed to compare A2RE luciferase mRNA from Fyn (WT and Fyn)-transfected cells with EGFP-transfected cells ( was used for normalization). Error bars indicate SEM. Significance was assessed with tests: *, P < 0.05; **, P < 0.01; ns, not significant.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p