Venoux M et al. (2008), Gene organization, evolution and expression of ...

XB-ART-38352

BMC Genomics 2008 Sep 09;9:406. doi: 10.1186/1471-2164-9-406.

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Gene organization, evolution and expression of the microtubule-associated protein ASAP (MAP9).

Venoux M , Delmouly K , Milhavet O , Vidal-Eychenié S , Giorgi D , Rouquier S .

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BACKGROUND: ASAP is a newly characterized microtubule-associated protein (MAP) essential for proper cell-cycling. We have previously shown that expression deregulation of human ASAP results in profound defects in mitotic spindle formation and mitotic progression leading to aneuploidy, cytokinesis defects and/or cell death. In the present work we analyze the structure and evolution of the ASAP gene, as well as the domain composition of the encoded protein. Mouse and Xenopus cDNAs were cloned, the tissue expression characterized and the overexpression profile analyzed. RESULTS: Bona fide ASAP orthologs are found in vertebrates with more distantly related potential orthologs in invertebrates. This single-copy gene is conserved in mammals where it maps to syntenic chromosomal regions, but is also clearly identified in bird, fish and frog. The human gene is strongly expressed in brain and testis as a 2.6 Kb transcript encoding a approximately110 KDa protein. The protein contains MAP, MIT-like and THY domains in the C-terminal part indicative of microtubule interaction, while the N-terminal part is more divergent. ASAP is composed of approximately 42% alpha helical structures, and two main coiled-coil regions have been identified. Different sequence features may suggest a role in DNA damage response. As with human ASAP, the mouse and Xenopus proteins localize to the microtubule network in interphase and to the mitotic spindle during mitosis. Overexpression of the mouse protein induces mitotic defects similar to those observed in human. In situ hybridization in testis localized ASAP to the germ cells, whereas in culture neurons ASAP localized to the cell body and growing neurites. CONCLUSION: The conservation of ASAP indicated in our results reflects an essential function in vertebrates. We have cloned the ASAP orthologs in mouse and Xenopus, two valuable models to study the function of ASAP. Tissue expression of ASAP revealed a high expression in brain and testis, two tissues rich in microtubules. ASAP associates to the mitotic spindle and cytoplasmic microtubules, and represents a key factor of mitosis with possible involvement in other cell cycle processes. It may have a role in spermatogenesis and also represents a potential new target for antitumoral drugs. Possible involvement in neuron dynamics also highlights ASAP as a candidate target in neurodegenerative diseases.

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Species referenced: Xenopus
Genes referenced: actb actl6a gcg map1a map9 pafah1b2

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	Figure 1. Comparison of the ASAP gene structure in human and mouse. The coding regions are represented by horizontal solid lines and the exons by closed boxes. The ATG start codons and the TGA stop codons are indicated, as well as the chromosome locations (cytogenetic bands and nucleotide locations in Mb). The transcribed mRNAs are represented below each gene.
	Figure 2. Phylogenetic analysis and pairwise comparison of ASAP from different species. (A) Phylogenetic tree of the ASAP protein from fish, frog, mouse, dog and human. The predicted protein sequences were compared using the ClustalW software to generate a rooted phylogenetic tree using the bootstrap method. Branch length and bootstrap numbers are indicated. (B) Pairwise comparison (%) of ASAP proteins between the species depicted in A. The values above the diagonal show protein sequence identities, those under the diagonal show protein sequence similarities. These values were estimated using the Gap program of the GCG package. (C) Clustal alignment of the phylogenetic analysis performed in (A) plus a partial chicken sequence, visualized using Jalview and Clustal X colour codes [63]. On top of the aligment, colour bars indicate conserved domains; blue, MIT-like; green, MAP; red, NLS. Phosphoserines 132 (SQ motif) and 625 are squared. Canonical (+) and (-) residues of the MIT domain are squared.
	Figure 3. Dot plot of the percent identity plot (PIP) analysis of human and mouse ASAP gene regions. The human ASAP sequence (hASAP) is represented on the x-axis, and the mouse sequence (mASAP) on the y-axis. The ATG start codon and the 5' upstream region are indicated, showing that the two genes are conserved for ~1 kb upstream the coding region. Introns differ between the two species by homology losses due to large insertion/deletion of repeats as examplified between exons 3 and 4, and exons 5 and 6.
	Figure 4. Domains and motifs of the ASAP protein. (A) Top: General structure of ASAP. The residues are numbered 1 (N-terminal end) to 647 (C-terminal end). The MAP domain is indicated as well as the coiled-coil regions (springs). The blue rectangle indicates the region that is the most conserved during evolution in the N-terminal part (Fig. 2), orange boxes indicate moderatly conserved regions and uncoloured portions represent variable regions. Bottom: Detail of the MAP domain. This domain contains a self similarity (SS) region as MAP1A, an MIT-like and an THY domain. The nuclear localization signals (NLS) are boxed in red. MAP, microtubule-associated protein; MIT, microtubule interacting and trafficking domain; THY, thymosin beta actin-binding motif. (B) Comparison of ASAP with MAP1A. Blast comparison of ASAP with protein databases showing a similarity region with human MAP1A containing a high fraction of Glu and Lys (43, 21.8% and 52, 26.4% respectively in ASAP). (C) Protein self matrix of human ASAP. The comparison was performed using the Strider software (window 23 amino-acids, stringency 5 amino-acids). One region with self-similarity (SS-ASAP, amino-acids 420–610) corresponding to the region homologous to MAP1A, could be identified.
	Figure 5. ASAP possesses an MIT-like domain. Multiple sequence alignment of selected MIT domain containing proteins [34] with ASAP. The consensus is indicated below the alignment: h, p, + and - indicate hydrophobic, polar, positively and negatively charged residues. Hydrophobic residues are highlighted in blue, polar residues in orange, positive residues in red and negative residues in purple. The secondary structure prediction at the bottom shows that this ASAP region is almost exclusively composed of helix (H). Conserved positions of (+) and (-) amino acids are also indicated in Fig. 2C.
	Figure 6. Overexpression of Cter and Nter ASAP deletion mutants. Forty-eight hours after transfection with 1 μg of EYFP-ASAP-Cter or EYFP-ASAP-Nter, U2-OS cells were analyzed by immunofluorescence (A) and western-blot (B). (A) Cells were fixed with PFA and stained with an anti-α-tubulin (red) and Hoechst dye 33258 (DNA). EYFP signals are in green. (Bar, 10 μm). (B) Immunoblot of EYFP, EYFP-ASAP-FL (full-length), EYFP-ASAP-Cter or EYFP-ASAP-Nter U2-OS transfected cells using an anti-GFP antibody (top); NT: non-transfected cells. The same blot was probed with an anti-α-tubulin to demonstrate equal loading (bottom).
	Figure 7. Characterization of the mouse ASAP. (A) Characterization of anti-mouse ASAP antibodies by immunoblotting. Non-transfected (NT) or transfected U2-OS (with a human ASAP siRNA) or NIH3T3 (with EYFP or EYFP-mASAP) cells were blotted first with an anti-GFP (left), then with the anti-mASAP (right). The specificity of the affinity-purified anti-mASAP antibody was assessed by using cell extracts from NIH3T3 and U2-OS (right), and compared to the profile observed 1) after ASAP-depletion by siRNA in the U2-OS cells (right) and 2) with the anti-GFP antibody against EYFP-mASAP transfected NIH3T3 cells (left). * : non specific bands observed with the α-GFP and that still show up in the α-mASAP blot since the same membrane was used. The same blot was finally probed with an anti-α-tubulin to demonstrate equal loading (bottom). The sizes of molecular weight marker are shown on the left. (B) Subcellular localization of endogenous mASAP during the cell cycle. Exponentially growing NIH3T3 cells were fixed with PFA and processed for immunofluorescence with the affinity-purified antibody against mASAP (green), and an antibody against α-tubulin (red). DNA was stained with Hoechst dye 33258 (blue). (C) Overexpression of mASAP leads to MTs bundles and monopolar spindle formation in NIH3T3 cells. Forty-eight hours after transfection with 2 μg of EYFP-mASAP, cells were fixed with PFA and stained with an anti-α-tubulin (1) or an anti-GT335 (2) (red) and Hoechst 33258 (DNA). Insets show a higher magnification of GT-335 foci. EYFP signals are in green. (Bars, 10 μm).
	Figure 8. Expression of ASAP in human and murine tissues. (A) Northern-blot analysis of ASAP transcript in adult human tissues was carried out with pre-made Northern blots purchased from Clontech. The blots were hybridized with a 32P-labelled full-length ASAP cDNA probe. The sizes of molecular weight marker are shown on the left. (B) RT-PCR analysis of ASAP in murine tissues. Total RNA was prepared from various mouse tissues. A 279-bp ASAP RT-PCR product is visualized. Expression of GAPD was determined as a control of RNA integrity. (C) Western-blot analysis of mASAP protein was carried out using the mASAP antibody on testis, brain, spleen and colon mouse tissue extracts and compared to the profile observed on NIH3T3 and U2-OS (positive controls) or HT29 (negative control derived from colon) cell extracts. α-tubulin levels served as loading control.
	Figure 9. ASAP expression in testis. Immunohistochemical staining of testis cross-sections of adult mouse using ASAP-antibody (A, B) or preimmune serum (C). Nuclei are stained with Hoechst 33258. Scale bars: 100 μM (A) and 50 μM (B, C).
	Figure 10. Expression of ASAP in differenciated neural stem cells. (A-D) After 9 days of differentiation neural stem cells differentiate into neurons and astrocytes. (A) Nuclei stained with DAPI (blue), (B) astrocytes revealed by GFAP immunostaining (green), (C) neuronal cells identified by detection of β-III tubulin (red), (D) merged visualization of A-C. (E-H) ASAP is expressed in neurons but not in astrocytes after 9 days of differentiation of neural stem cells. (E) Nuclei stained with DAPI, (F) detection of ASAP, (G) β-III tubulin positive neuronal cells, (H) merged visualization of E-G. Scale Bar: (A-D), 100 μm; (E-H), 50 μm.
	Figure 11. Characterization of Xenopus ASAP. Asynchronous XL2 cells were transfected with the EYFP-X-ASAP cDNA and analyzed (A) by immunoblot using an anti-GFP antibody or (B) by immunofluorescence. Cells were fixed in PAF/MTSB and co-stained with the anti α-tubulin (red) and Hoechst 33258 (blue).

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