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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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Localization of hMex-3B in Dcp-containing bodies. (A) MCF7 cells expressing hMex-3B or hMex-3B/KH and GFP-hDcp1a proteins were stained with anti-hM3Bα antibody and revealed with ALEXA555-conjugated secondary antibody (left). Central panel shows GFP signal. Right panel shows overlay of the two signals. (B) MCF7 cells expressing hMex-3B protein treated with or without 5 μg/ml of cycloheximide were stained with anti-myc antibody (left) and anti-hDcp1a antibody (middle) and revealed with ALEXA488 and ALEXA555-conjugated secondary antibodies. Right panels show overlay of the two signals. Scale bar: 20 μm. (C) Sections of mouse duodenum (magnification: ×630 or ×1260) were stained with anti-hMex-3Bβ and revealed with an ALEXA555-conjugated secondary antibody. Tissues were mounted and observed by a confocal microscope, as described in the materials and methods section.
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Figure 5: Localization of hMex-3B in Dcp-containing bodies. (A) MCF7 cells expressing hMex-3B or hMex-3B/KH and GFP-hDcp1a proteins were stained with anti-hM3Bα antibody and revealed with ALEXA555-conjugated secondary antibody (left). Central panel shows GFP signal. Right panel shows overlay of the two signals. (B) MCF7 cells expressing hMex-3B protein treated with or without 5 μg/ml of cycloheximide were stained with anti-myc antibody (left) and anti-hDcp1a antibody (middle) and revealed with ALEXA488 and ALEXA555-conjugated secondary antibodies. Right panels show overlay of the two signals. Scale bar: 20 μm. (C) Sections of mouse duodenum (magnification: ×630 or ×1260) were stained with anti-hMex-3Bβ and revealed with an ALEXA555-conjugated secondary antibody. Tissues were mounted and observed by a confocal microscope, as described in the materials and methods section.

Mentions: The human mRNA decapping enzyme, hDcp1a, is concentrated in P bodies (28). MCF7 cells were co-transfected with vectors expressing hMex-3B and a GFP-hDcp1a fusion protein (Figure 5A). Indirect immunofluorescence and confocal analyses revealed that hMex-3B colocalized with hDcp1 in cytoplasmic foci, although we repeatedly observed that some of the hMex-3B-containing granules were devoid of hDcp1. hMex-3A, but not hMex-3C, was also found in Dcp-containing granules (data not shown). As suggested by the results displayed in Figure 5A, the hMex-3B KH mutant was no longer localized in P bodies and this was not due to the disruption of the P bodies’ integrity, since the punctuate distribution of Dcp1a was not affected by the expression of the hMex-3B KH mutant (Figure 5A).Figure 5.


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)

Localization of hMex-3B in Dcp-containing bodies. (A) MCF7 cells expressing hMex-3B or hMex-3B/KH and GFP-hDcp1a proteins were stained with anti-hM3Bα antibody and revealed with ALEXA555-conjugated secondary antibody (left). Central panel shows GFP signal. Right panel shows overlay of the two signals. (B) MCF7 cells expressing hMex-3B protein treated with or without 5 μg/ml of cycloheximide were stained with anti-myc antibody (left) and anti-hDcp1a antibody (middle) and revealed with ALEXA488 and ALEXA555-conjugated secondary antibodies. Right panels show overlay of the two signals. Scale bar: 20 μm. (C) Sections of mouse duodenum (magnification: ×630 or ×1260) were stained with anti-hMex-3Bβ and revealed with an ALEXA555-conjugated secondary antibody. Tissues were mounted and observed by a confocal microscope, as described in the materials and methods section.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 5: Localization of hMex-3B in Dcp-containing bodies. (A) MCF7 cells expressing hMex-3B or hMex-3B/KH and GFP-hDcp1a proteins were stained with anti-hM3Bα antibody and revealed with ALEXA555-conjugated secondary antibody (left). Central panel shows GFP signal. Right panel shows overlay of the two signals. (B) MCF7 cells expressing hMex-3B protein treated with or without 5 μg/ml of cycloheximide were stained with anti-myc antibody (left) and anti-hDcp1a antibody (middle) and revealed with ALEXA488 and ALEXA555-conjugated secondary antibodies. Right panels show overlay of the two signals. Scale bar: 20 μm. (C) Sections of mouse duodenum (magnification: ×630 or ×1260) were stained with anti-hMex-3Bβ and revealed with an ALEXA555-conjugated secondary antibody. Tissues were mounted and observed by a confocal microscope, as described in the materials and methods section.
Mentions: The human mRNA decapping enzyme, hDcp1a, is concentrated in P bodies (28). MCF7 cells were co-transfected with vectors expressing hMex-3B and a GFP-hDcp1a fusion protein (Figure 5A). Indirect immunofluorescence and confocal analyses revealed that hMex-3B colocalized with hDcp1 in cytoplasmic foci, although we repeatedly observed that some of the hMex-3B-containing granules were devoid of hDcp1. hMex-3A, but not hMex-3C, was also found in Dcp-containing granules (data not shown). As suggested by the results displayed in Figure 5A, the hMex-3B KH mutant was no longer localized in P bodies and this was not due to the disruption of the P bodies’ integrity, since the punctuate distribution of Dcp1a was not affected by the expression of the hMex-3B KH mutant (Figure 5A).Figure 5.

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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