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Identification and characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNA-binding proteins differentially localized to processing bodies.

Buchet-Poyau K, Courchet J, Le Hir H, Séraphin B, Scoazec JY, Duret L, Domon-Dell C, Freund JN, Billaud M - Nucleic Acids Res. (2007)

Bottom Line: The hMex-3 are phosphoproteins that bind RNA through their KH domains and shuttle between the nucleus and the cytoplasm via the CRM1-dependent export pathway.Our analysis further revealed that hMex-3A and hMex-3B, but not hMex-3C, colocalize with both the hDcp1a decapping factor and Argonaute (Ago) proteins in processing bodies (P bodies), recently characterized as centers of mRNA turnover.Taken together, these findings indicate that hMex-3 proteins constitute a novel family of evolutionarily conserved RNA-binding proteins, differentially recruited to P bodies and potentially involved in post-transcriptional regulatory mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Université de Lyon, Lyon, F-69003, France.

ABSTRACT
In Caenorhabditis elegans, the Mex-3 protein is a translational regulator that specifies the posterior blastomere identity in the early embryo and contributes to the maintenance of the germline totipotency. We have now identified a family of four homologous human Mex-3 genes, called hMex-3A to -3D that encode proteins containing two heterogeneous nuclear ribonucleoprotein K homology (KH) domains and one carboxy-terminal RING finger module. The hMex-3 are phosphoproteins that bind RNA through their KH domains and shuttle between the nucleus and the cytoplasm via the CRM1-dependent export pathway. Our analysis further revealed that hMex-3A and hMex-3B, but not hMex-3C, colocalize with both the hDcp1a decapping factor and Argonaute (Ago) proteins in processing bodies (P bodies), recently characterized as centers of mRNA turnover. Taken together, these findings indicate that hMex-3 proteins constitute a novel family of evolutionarily conserved RNA-binding proteins, differentially recruited to P bodies and potentially involved in post-transcriptional regulatory mechanisms.

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Subcellular localization of hMex-3 proteins. (A) MCF7 cells expressing hMex-3A, -3B and -3C were stained with anti-myc antibody (top) or with specific anti-hM3Aβ or anti-hM3Bβ antibodies (bottom) and revealed by FITC-conjugated secondary antibodies. (B) MCF7 cells expressing hMex-3A, -3B, -3C proteins or hMex-3C mutated within the nuclear export signal (NES) sequence were treated with or without 20 ng/ml of Leptomycin B (LMB) and were stained as above. pEGFP-Rev-NES construct was used as a positive control. Scale bar: 20 μm.
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Figure 4: Subcellular localization of hMex-3 proteins. (A) MCF7 cells expressing hMex-3A, -3B and -3C were stained with anti-myc antibody (top) or with specific anti-hM3Aβ or anti-hM3Bβ antibodies (bottom) and revealed by FITC-conjugated secondary antibodies. (B) MCF7 cells expressing hMex-3A, -3B, -3C proteins or hMex-3C mutated within the nuclear export signal (NES) sequence were treated with or without 20 ng/ml of Leptomycin B (LMB) and were stained as above. pEGFP-Rev-NES construct was used as a positive control. Scale bar: 20 μm.

Mentions: To examine the subcellular distribution of hMex-3 proteins, we transiently expressed hMex-3A, -3B and -3C into human breast cancer MCF7 cells and carried out indirect immunofluorescence analysis with the anti-myc tag antibody. Confocal microscopy analysis revealed that hMex-3 proteins are located in the cytoplasm and excluded from the nucleus. However, we noticed a clear, specific labeling pattern, with hMex-3A and -3B showing a punctuated staining whereas hMex-3C was distributed more homogenously throughout the cytoplasm (Figure 4A). Strikingly, a mutation in the KH domain eliminated the spotted localization of hMex-3B, while a mutation in the RING finger domain did not modify the topology of the staining (Figure 4A). Using our polyclonal antibodies directed against hMex-3A and -3B we observed an expression pattern similar to the one observed with the anti-myc antibody (Figure 4A), thus further confirming the specificity of these anti-hMex-3 sera. However, we were unable to detect the endogenous hMex-3 proteins, probably due to their weak expression in MCF7 cells (see Figure 2A).Figure 4.


Identification and characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNA-binding proteins differentially localized to processing bodies.

Buchet-Poyau K, Courchet J, Le Hir H, Séraphin B, Scoazec JY, Duret L, Domon-Dell C, Freund JN, Billaud M - Nucleic Acids Res. (2007)

Subcellular localization of hMex-3 proteins. (A) MCF7 cells expressing hMex-3A, -3B and -3C were stained with anti-myc antibody (top) or with specific anti-hM3Aβ or anti-hM3Bβ antibodies (bottom) and revealed by FITC-conjugated secondary antibodies. (B) MCF7 cells expressing hMex-3A, -3B, -3C proteins or hMex-3C mutated within the nuclear export signal (NES) sequence were treated with or without 20 ng/ml of Leptomycin B (LMB) and were stained as above. pEGFP-Rev-NES construct was used as a positive control. Scale bar: 20 μm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851655&req=5

Figure 4: Subcellular localization of hMex-3 proteins. (A) MCF7 cells expressing hMex-3A, -3B and -3C were stained with anti-myc antibody (top) or with specific anti-hM3Aβ or anti-hM3Bβ antibodies (bottom) and revealed by FITC-conjugated secondary antibodies. (B) MCF7 cells expressing hMex-3A, -3B, -3C proteins or hMex-3C mutated within the nuclear export signal (NES) sequence were treated with or without 20 ng/ml of Leptomycin B (LMB) and were stained as above. pEGFP-Rev-NES construct was used as a positive control. Scale bar: 20 μm.
Mentions: To examine the subcellular distribution of hMex-3 proteins, we transiently expressed hMex-3A, -3B and -3C into human breast cancer MCF7 cells and carried out indirect immunofluorescence analysis with the anti-myc tag antibody. Confocal microscopy analysis revealed that hMex-3 proteins are located in the cytoplasm and excluded from the nucleus. However, we noticed a clear, specific labeling pattern, with hMex-3A and -3B showing a punctuated staining whereas hMex-3C was distributed more homogenously throughout the cytoplasm (Figure 4A). Strikingly, a mutation in the KH domain eliminated the spotted localization of hMex-3B, while a mutation in the RING finger domain did not modify the topology of the staining (Figure 4A). Using our polyclonal antibodies directed against hMex-3A and -3B we observed an expression pattern similar to the one observed with the anti-myc antibody (Figure 4A), thus further confirming the specificity of these anti-hMex-3 sera. However, we were unable to detect the endogenous hMex-3 proteins, probably due to their weak expression in MCF7 cells (see Figure 2A).Figure 4.

Bottom Line: The hMex-3 are phosphoproteins that bind RNA through their KH domains and shuttle between the nucleus and the cytoplasm via the CRM1-dependent export pathway.Our analysis further revealed that hMex-3A and hMex-3B, but not hMex-3C, colocalize with both the hDcp1a decapping factor and Argonaute (Ago) proteins in processing bodies (P bodies), recently characterized as centers of mRNA turnover.Taken together, these findings indicate that hMex-3 proteins constitute a novel family of evolutionarily conserved RNA-binding proteins, differentially recruited to P bodies and potentially involved in post-transcriptional regulatory mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Université de Lyon, Lyon, F-69003, France.

ABSTRACT
In Caenorhabditis elegans, the Mex-3 protein is a translational regulator that specifies the posterior blastomere identity in the early embryo and contributes to the maintenance of the germline totipotency. We have now identified a family of four homologous human Mex-3 genes, called hMex-3A to -3D that encode proteins containing two heterogeneous nuclear ribonucleoprotein K homology (KH) domains and one carboxy-terminal RING finger module. The hMex-3 are phosphoproteins that bind RNA through their KH domains and shuttle between the nucleus and the cytoplasm via the CRM1-dependent export pathway. Our analysis further revealed that hMex-3A and hMex-3B, but not hMex-3C, colocalize with both the hDcp1a decapping factor and Argonaute (Ago) proteins in processing bodies (P bodies), recently characterized as centers of mRNA turnover. Taken together, these findings indicate that hMex-3 proteins constitute a novel family of evolutionarily conserved RNA-binding proteins, differentially recruited to P bodies and potentially involved in post-transcriptional regulatory mechanisms.

Show MeSH