Considerable evidence indicates that mRNA associates with structural filaments in the cell (cytoskeleton). This relationship would be an important mechanism to effect mRNA sorting since specific mRNAs could be sequestered at sites within the cell. In addition, it can provide a mechanism for spatial regulation of mRNA expression. However, the precise structural interactions between mRNA and the cytoskeleton have yet to be defined. An objective of this work was to visualize 'individual' poly(A) mRNA molecules in situ by electron microscopy to identify their relationship to individual filaments. Poly(A) RNA and filaments were identified simultaneously using antibodies to detect hybridized probe and filaments or actin-binding proteins. In human fibroblasts, most of the poly(A) mRNA (72%) was localized within 5 nm of orthogonal networks of F-actin filaments. Poly(A) mRNA also colocalized with vimentin filaments (29%) and microtubules (<10%). The sites of mRNA localization were predominantly at filament intersections. The majority of poly(A) mRNA and polysomes colocalized with the actin crosslinking proteins, filamin, and α-actinin, and the elongation factor, EF-1α (actin-binding protein; ABP-50). Evidence that intersections contained single mRNA molecules was provided by using a labeled oligo dT probe to prime the synthesis of cDNA in situ using reverse transcriptase. Both the poly(A) and cis sequences of the same mRNA molecule could then be visualized independently. We propose that the cytoskeletal intersection is a mRNA receptor and serves as a 'microdomain' where mRNA is attached and functionally expressed.
Background
Cultured spinal motor neurons are a valuable tool to study basic mechanisms of development, axon growth and pathfinding, and, importantly, to analyze the pathomechanisms underlying motor neuron diseases. However, the application of this cell culture model is limited by the lack of efficient gene transfer techniques which are available for other neurons. To address this problem, we have established magnetofection as a novel method for the simple and efficient transfection of mouse embryonic motor neurons. This technique allows for the study of the effects of gene expression and silencing on the development and survival of motor neurons.
Results
We found that magnetofection, a novel transfection technology based on the delivery of DNA-coated magnetic nanobeads, can be used to transfect primary motor neurons. Therefore, in order to use this method as a new tool for studying the localization and transport of axonal proteins, we optimized conditions and determined parameters for efficient transfection rates of >45% while minimizing toxic effects on survival and morphology. To demonstrate the potential of this method, we have used transfection with plasmids encoding fluorescent fusion-proteins to show for the first time that the spinal muscular atrophy-disease protein Smn is actively transported along axons of live primary motor neurons, supporting an axon-specific role for Smn that is different from its canonical function in mRNA splicing. We were also able to show the suitability of magnetofection for gene knockdown with shRNA-based constructs by significantly reducing Smn levels in both cell bodies and axons, opening new opportunities for the study of the function of axonal proteins in motor neurons.
Conclusions
In this study we have established an optimized magnetofection protocol as a novel transfection method for primary motor neurons that is simple, efficient and non-toxic. We anticipate that this novel approach will have a broad applicability in the study of motor neuron development, axonal trafficking, and molecular mechanisms of motor neuron diseases.
The localization of β-actin mRNA to the leading lamellae of chicken fibroblasts and neurite growth cones of developing neurons requires a 54-nt localization signal (the zipcode) within the 3′ untranslated region. In this study we have identified and isolated five proteins binding to the zipcode. One of these we previously identified as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate premRNA splicing. However, ZBP2 has a 47-amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of β-actin mRNA in both fibroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of β-actin mRNA.
In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including β-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the β-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs.
by
Tsuyoshi Udagawa;
Natalie G. Farny;
Mira Jakovcevski;
Hanoch Kaphzan;
Juan Marcos Alarcon;
Shobha Anilkumar;
Maria Ivshina;
Jessica A. Hurt;
Kentaro Nagaoka;
Vijayalaxmi C. Nalavadi;
Lori J. Lorenz;
Gary Bassell;
Schahram Akbarian;
Sumantra Chattarji;
Eric Klann;
Joel D. Richter
Fragile X syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of FMR1, which encodes the translational repressor fragile X mental retardation protein (FMRP). FMRP and cytoplasmic polyadenylation element-binding protein (CPEB), an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1-/y; Cpeb1-/-double-knockout mice displayed amelioration of biochemical, morphological, electrophysiological and behavioral phenotypes associated with FXS. Acute depletion of CPEB1 in the hippocampus of adult Fmr1-/y mice rescued working memory deficits, demonstrating reversal of this FXS phenotype. Finally, we find that FMRP and CPEB1 balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS and that the maintenance of this homeostasis by FMRP and CPEB1 is necessary for normal neurologic function.
The intracellular distribution of HIV-1 RNA transcripts in infected cells was studied using in situ hybridization detected by electron microscopy and cellular fractionation. Although viral RNA and core protein could be detected throughout the cytoplasm and nucleus, viral RNA was found in significantly increased amounts in mitochondria relative to the cytoplasm and nucleus. In contrast, cellular poly(A) RNA or viral gag proteins were not increased in the mitochondria. A cell line containing an integrated latent genome that could be induced to express viral RNA after phorbol ester stimulation showed an increase in viral RNA accumulation in mitochondria parallel with the increase in HIV expression levels. Concomitant with HIV expression, there was a decrease in mitochondrial viability. Using immunofluorescent markers to detect probes to HIV RNA transcripts and antibodies to mitochondrial proteins simultaneously in single cells, there was an inverse relationship between the amount of viral RNA and mitochondrial integrity. High levels of viral RNA in mitochondria were found in acutely (but not chronically) infected cells. We propose that HIV RNA import into mitochondria can compromise mitochondrial function.
Fragile X syndrome is the most common inherited form of cognitive deficiency in humans and perhaps the best-understood single cause of autism. A trinucleotide repeat expansion, inactivating the X-linked FMR1 gene, leads to the absence of the fragile X mental retardation protein. FMRP is a selective RNA-binding protein that regulates the local translation of a subset of mRNAs at synapses in response to activation of Gp1 metabotropic glutamate receptors (mGluRs) and possibly other receptors. In the absence of FMRP, excess and dysregulated mRNA translation leads to altered synaptic function and loss of protein synthesis-dependent plasticity. Recent evidence indicates the role of FMRP in regulated mRNA transport in dendrites. New studies also suggest a possible local function of FMRP in axons that may be important for guidance, synaptic development, and formation of neural circuits. The understanding of FMRP function at synapses has led to rationale therapeutic approaches.
Neurotrophins play an essential role in the regulation of actin- dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of β-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that β-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of β- actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of β-actin mRNA. Localization of β-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of β-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of β- actin mRNA within growth cones.
The phosphoinositide 3-kinase (PI3K) complex plays important roles in virtually all cells of the body. The enzymatic activity of PI3K to phosphorylate phosphoinositides in the membrane is mediated by a group of catalytic and regulatory subunits. Among those, the class I catalytic subunits, p110a, p1103, p110y, and p1108, have recently drawn attention in the neuroscience field due to their specific dysregulation in diverse brain disorders. While in non-neuronal cells these catalytic subunits may have partially redundant functions, there is increasing evidence that in neurons their roles are more specialized, and confined to distinct receptor-dependent pathways.This review will summarize the emerging role of class I PI3K catalytic subunits in neurotransmitter-regulated neuronal signaling, and their dysfunction in a variety of neurological diseases, including fragile X syndrome, schizophrenia, and epilepsy. We will discuss recent literature describing the use of PI3K subunit-selective inhibitors to rescue brain disease-associated phenotypes in in vitro and animal models. These studies give rise to the exciting prospect that these drugs, originally designed for cancer treatment, may be repurposed as therapeutic drugs for brain disorders in the future.