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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 13, 2004 as doi:10.1096/fj.04-1763fje. |
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* Biochimie Cellulaire, CNRS UMR 7098, Université Paris-6, Paris, France;
CRBM, UPR 1086, Montpellier, France;
Neurobiologie des Signaux Intercellulaires, UMR 7101, Université Paris-6, Paris, France; and
Department of Neurobiology, The Weizmann Institute, Rehovot, Israel
1Correspondence: Biochimie Cellulaire-CNRS UMR 7098, Université Paris-6, 9 quai Saint-Bernard, Bâtiment C-Case 265, Paris 75252, Cedex 05, France. E-mail: jclarche{at}snv.jussieu.fr
SPECIFIC AIM
We aimed to explore the molecular mechanisms that control translocation of tau mRNA toward the axon, a process by which mRNA prelocalization helps the corresponding microtubule (MT)-associated protein (MAP) tau to be compartmentalized, thus contributing to asymmetrical morphology of neuronal cells, essential for their functions. As MT-dependent axonal targeting of tau mRNA was shown to depend upon the presence of a 91 nucleotide segment located in its 3’-untranslated region and referred as the axonal targeting element (ATE), we searched for ATE-interacting proteins to understand how tau mRNA can be selectively recognized and escorted during its transport. Our approach involved Northwestern blot experiments using radioactive ATE as a probe to detect ATE binding proteins (ATE-BP), biochemical purification and immunodetection with specific antibodies.
PRINCIPAL FINDINGS
1. Detection of ATE-BPs by Northwestern blot using radioactive ATE RNA as probe
It has been previously shown that a segment of 91 nucleotides present in the 3’-untranslated region of tau mRNA is responsible for its axonal targeting. This ATE was then used as a probe in Northwestern blot experiments to detect interacting proteins (ATE-BPs) that could escort tau mRNA and mediate different aspects of its transport (recognition, axonal targeting, transport along MTs, translation regulation, and/or stability). Total proteins from embryonic to adult mouse brain were separated by 1-D PAGE, transferred onto nitrocellulose and overlaid with [32P]-labeled ATE probe. The level of ATE-BPs detected under these conditions was found to be maximal before birth and to decrease thereafter. For practical reasons, further experiments were carried out with 15-day-old mouse brains. At this stage, if ATE-BPs are at 50% of their maximal level, they are qualitatively identical to those found before birth. Quantitatively, by overlaying increasing amounts of labeled ATE onto a constant amount of brain proteins, the apparent Kd for ATE binding was estimated at 
5 nM. In addition, radioactive ATE probe was efficiently displaced from ATE-BPs after addition of excess nonradioactive probe.
2. Attempt to purify ATE-BPs directly from neuronal microtubules and further solubilization procedures
Since it has been shown that tau mRNA is transported onto axonal MT, we first tried to recover ATE-BPs by directly purifying MTs from brain. After MT assembly and Taxol stabilization, MT-bound proteins were salt-washed. Proteins from the different purification fractions were separated by 1-D PAGE and submitted to Northwestern blot with the radioactive ATE probe. In total brain extracts, three major ATE-BPs were detected (p110, p95, and p80) along with a few other minor ones. Upon fractionation, most of these proteins remained associated with the insoluble crude pellet, an expected behavior for proteins belonging to RNA granules tightly bound to stable, nondepolymerized MTs. Only faint traces of ATE-BPs were detected in the crude soluble supernatant fraction. However, when this fraction was submitted to MT assembly, scarce ATE-BPs were efficiently recruited onto MTs. If these experiments claim for an increased solubilization of ATE-BPs, the results confirm that ATE-BPs interact with MTs.
To further characterize ATE-BPs present mostly in the insoluble fraction, the homogenization buffer was modified to enhance their solubility. When brains were homogenized into Bicine buffer at pH 9 in the presence of 0.5 M NaCl, 70–80% of ATE-BPs were rendered soluble, as judged by their presence in the soluble fraction (S2-BEM fraction) revealed by Northwestern blot after 2-D PAGE (Fig. 2
A). Superposition of Northwestern signals over a silver-stained duplicate 2-D gel showed that the p95 signal colocalized with a well defined spot, whereas the p110 and p80 signals did not match any detectable protein spot, indicative of their very low abundance.
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3. Biochemical purification of p110 and sequence identification
Partial purification of p110 was achieved through a five step procedure. Proteins from S2-BEM were precipitated, resuspended, and chromatographed on four successive columns (anion exchange, cation exchange, hydrophobic interactions, and reversed phase). At each step, proteins from each fraction were separated by SDS-PAGE and probed for their ability to bind the radioactive ATE probe by Northwestern assays. Starting from 130 mouse brains, p110 was estimated to have been enriched 5,000 fold, with an overall yield of 2.5%. Figure 1
A shows Northwestern detection of p110 in fractions eluted from the last column. Proteins from fraction 14 were analyzed by 2-D PAGE and silver-stained (Fig. 1B
) or probed by Northwestern blot (Fig. 1C
). Substantial heterogeneity displayed by p110 in the pI dimension of the 2-D gel suggests that it likely undergoes complex posttranscriptional/posttranslational modifications.
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p110 (3 µg) was then digested with endolysine-C. Proteolytic peptides were separated by HPLC chromatography and four were microsequenced. They displayed a complete identity to previously cloned RNA binding protein interleukin enhancer binding factor 3 (Ilf3). This 911 amino-acid long protein comprises two double-stranded RNA binding motifs (dsRBM) located at positions 417–478 and 540–601. Since the ATE sequence was predicted to adopt a stem-loop structure, a large U-rich loop held at the tip of a long double-stranded RNA stem, these dsRBM therefore represent potent interacting sites for the ATE probe. The identity of p110 as Ilf3 was also confirmed by Western blot experiments using various antibodies (Fig. 2B-D
).
4. Use of specific antibodies to further characterize p95 and p110
Rabbit polyclonal antibodies Ab78 and Ab83 were raised against either a mixture of two synthetic amino-terminal and central peptides (Ab78, aa 60–70 and 522–536) or a carboxyl-terminal peptide (Ab83, aa 898-911) of mIlf3. As control, we also used an anti-Ilf3 antibody from Braun’s laboratory raised against a large fragment of mouse Ilf3 (aa 468–796) comprising the region of the two dsRBM and the downstream region. S2-BEM proteins were separated by 2-D PAGE and submitted to Northwestern blot analysis with the radioactive ATE probe (Fig. 2A
) or to Western immunoblot using either Braun’s anti-Ilf3 antibody (Fig. 2B
), Ab78 (Fig. 2C
), or Ab83 (Fig. 2D
). Among p110, p95, and p80 detected by Northwestern blot analysis (Fig. 2A
), only p110 and p95 were probed with Braun’s Ab and Ab78 (Fig. 2B, C
). Only p110 was recognized by Ab83 (Fig. 2D
). These results confirm that p110 is Ilf3 and indicate that Ab83 is quite specific to Ilf3.
Experiments performed with Braun’s Ab and Ab78 (Fig. 2B, C
) showed that p95 was co-detected with p110-Ilf3. These results indicate that Ilf3 and p95 share common amino-terminal and central epitopes. The use of Ab83 (Fig. 2D
) showed that p95 differs from Ilf3 in its carboxyl-terminal domain. Altogether, these results are in good agreement with works reporting that Ilf3 primary gene transcripts are alternatively spliced into at least two variants: Ilf3/NF110/NFAR2 and NF90/NFAR1. Two protein variants share the first 701 aa residues but differ in their C-terminal sequences (aa 702-716 for NF90 and aa 702-911 for Ilf3). These data strongly suggest that p95 is NF90. p95 was further shown to react with the commercial monoclonal anti-NF90 antibody. In addition, the p95 protein species immunodepleted from a neuronal soluble fraction by Ab78 was shown to bind the ATE RNA probe by Northwestern blot analysis.
5. Immunolocalization of p110-Ilf3 in neurons
Specific anti-Ilf3 Ab83 antibody was used to immunolocalize Ilf3 in cultured mouse cortical neurons. Cells were processed for indirect double immunofluorescence with either Ab83 and GT335, a monoclonal anti-polyglutamylated tubulin antibody, or Ab83 and monoclonal antibody tau-1. After a 3 day culture, neuronal cell bodies and the proximal part of axons were intensively stained with Ab83; nuclei were barely decorated. Continuing parts of the axonal processes stained by tau-1 and dendritic branches decorated by GT335 were not stained at all by Ab83. Localization of Ilf3 is thus in good agreement with the reported localization of tau mRNA in neuronal cell bodies and axon hillock. When Ab78 (which recognizes both Ilf3 and NF90) was used, neuronal nuclei were stained almost exclusively.
On the other hand, Ilf3 and NF90 were clearly detected by Western and Northwestern blot analysis in both nuclear and cytoplasmic fractions purified from brain. Given the obvious presence of Ilf3 and NF90 within both nuclear and cytoplasmic compartments, we determined from immunolocalization experiments the existence of an alternative epitope availability between the two cellular compartments: the Ab83 epitope of Ilf3 is masked in the nucleus and the Ab78 epitope of Ilf3 and NF90 is masked in the cytoplasm. This modulated availability probably reflects selective interactions of these protein domains with associated RNA or nuclear/cytoplasmic proteins.
CONCLUSIONS AND SIGNIFICANCE
Neurons are highly polarized cells that exhibit sophisticated architectures comprising one long single axon and several dendritic branches. Essential functions of neurons rely on this particular polarity. In turn, formation and maintenance of axonal and dendritic processes depend on the assembly of differentiated microtubular networks that support neurite outgrowth and orientated vesicle trafficking, feeding cell processes, and synaptic transmission. One marked difference between axonal and dendritic MTs is their content in MAPs, which are, in the majority, distributed asymmetrically (MAP2 in dendrites and tau in axons). Besides signal-directed trafficking of proteins, mRNAs are also actively transported in cells, routing them to specific destinations and ensuring synthesis of specific proteins in particular cell areas. The aim of this study was to analyze molecular mechanisms underlying tau mRNA targeting through the identification of proteins that specifically bind the ATE sequence and could thus regulate and escort tau mRNA during its transport.
Figure 3
displays the expected roles of Ilf3/NF90 in tau mRNA transport. These proteins may first recognize tau mRNA within the nucleus through binding to the ATE RNA sequence via their dsRBM. They could then assist nuclear export of tau mRNA and, with the help of additional recruited proteins, escort tau mRNA toward axon. The different functions allotted to these escort proteins should comprise loading of RNA onto MT en route for axons and its motor-driven translocation (likely the kinesin-related KIF3A anterograde motor), regulation of mRNA stability (with the RNA-stabilizing HuD protein) and its translational capacity. Control of the different steps and interactions with RNA and escort proteins could be regulated by posttranslational modifications of Ilf3/NF90. These points are under investigation.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1763fje;
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