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The PXDLS linear motif regulates circadian rhythmicity through protein-protein interactions.

Shalev M, Aviram R, Adamovich Y, Kraut-Cohen J, Shamia T, Ben-Dor S, Golik M, Asher G - Nucleic Acids Res. (2014)

Bottom Line: Our bioinformatics analysis of short linear motifs, implicated in protein interactions, reveals an enrichment of the Pro-X-Asp-Leu-Ser (PXDLS) motif within circadian transcripts.Remarkably, the motif is evolutionary conserved in the core clock protein REV-ERBα, and additional proteins implicated in the clock's function (NRIP1, CBP).Furthermore, we demonstrate that the PXDLS motifs of NRIP1 and CBP are involved in circadian rhythmicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

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The PXDLS motif disrupts circadian rhythmicity in single cells. (A) NIH3T3 cells stably expressing Venus-REV-ERBα reporter were transfected with Cherry expression vector either with the PXDLS*3 or the mutant PXDLS*3 transgene. Cells were synchronized with a short Dex treatment and single cells either expressing the Cherry fluorescent protein (i.e. PXDLS*3 or mutant PXDLS*3) or non-expressing cells (PXDLS*3 neighboring cells) were monitored and analyzed for their circadian oscillations using time-lapse fluorescence microscopy for three consecutive days at 1 h resolution. Each plot represents fluorescence intensity profile for an individual cell (for additional plots see also Supplementary Figure S3). (B) An average plot of single cell profiles expressing the PXDLS*3 (red), the mutant PXDLS*3 transgene (green) or non-expressing cells (PXDLS*3 neighboring cells) (blue) (n = 25 for each condition). Arbitrary Units (AU).
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Figure 3: The PXDLS motif disrupts circadian rhythmicity in single cells. (A) NIH3T3 cells stably expressing Venus-REV-ERBα reporter were transfected with Cherry expression vector either with the PXDLS*3 or the mutant PXDLS*3 transgene. Cells were synchronized with a short Dex treatment and single cells either expressing the Cherry fluorescent protein (i.e. PXDLS*3 or mutant PXDLS*3) or non-expressing cells (PXDLS*3 neighboring cells) were monitored and analyzed for their circadian oscillations using time-lapse fluorescence microscopy for three consecutive days at 1 h resolution. Each plot represents fluorescence intensity profile for an individual cell (for additional plots see also Supplementary Figure S3). (B) An average plot of single cell profiles expressing the PXDLS*3 (red), the mutant PXDLS*3 transgene (green) or non-expressing cells (PXDLS*3 neighboring cells) (blue) (n = 25 for each condition). Arbitrary Units (AU).

Mentions: Hitherto, the involvement of the PXDLS motif was assessed in a cell population, causing shallow circadian oscillations. Low-amplitude oscillations in a cell population might be due to inefficient synchronization of circadian clocks within individual cells that leads to rapid dampening, or the result of an inherent cell-autonomous defect in the clock's function. To discriminate between the two scenarios, we performed fluorescence recording of NIH3T3-Rev-VNP cells, stably expressing the circadian Venus-Rev reporter (26). Cells were transfected with Cherry expression vector either with the PXDLS*3 or the mutant PXDLS*3 transgene as a control. Single cells expressing the Cherry fluorescent protein or neighboring cells that do not express the Cherry fluorescent protein were monitored and analyzed for their circadian oscillations (Figure 3, Supplementary Figure S3, and Supplementary Movie S1). In contrast to the mutant PXDLS*3 and the PXDLS*3 non-expressing cells (neighboring cells), overexpression of the PXDLS*3 transgene disrupted the circadian rhythmicity in single cells (Figure 3, Supplementary Figure S3, and Supplementary Movie S1).


The PXDLS linear motif regulates circadian rhythmicity through protein-protein interactions.

Shalev M, Aviram R, Adamovich Y, Kraut-Cohen J, Shamia T, Ben-Dor S, Golik M, Asher G - Nucleic Acids Res. (2014)

The PXDLS motif disrupts circadian rhythmicity in single cells. (A) NIH3T3 cells stably expressing Venus-REV-ERBα reporter were transfected with Cherry expression vector either with the PXDLS*3 or the mutant PXDLS*3 transgene. Cells were synchronized with a short Dex treatment and single cells either expressing the Cherry fluorescent protein (i.e. PXDLS*3 or mutant PXDLS*3) or non-expressing cells (PXDLS*3 neighboring cells) were monitored and analyzed for their circadian oscillations using time-lapse fluorescence microscopy for three consecutive days at 1 h resolution. Each plot represents fluorescence intensity profile for an individual cell (for additional plots see also Supplementary Figure S3). (B) An average plot of single cell profiles expressing the PXDLS*3 (red), the mutant PXDLS*3 transgene (green) or non-expressing cells (PXDLS*3 neighboring cells) (blue) (n = 25 for each condition). Arbitrary Units (AU).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 3: The PXDLS motif disrupts circadian rhythmicity in single cells. (A) NIH3T3 cells stably expressing Venus-REV-ERBα reporter were transfected with Cherry expression vector either with the PXDLS*3 or the mutant PXDLS*3 transgene. Cells were synchronized with a short Dex treatment and single cells either expressing the Cherry fluorescent protein (i.e. PXDLS*3 or mutant PXDLS*3) or non-expressing cells (PXDLS*3 neighboring cells) were monitored and analyzed for their circadian oscillations using time-lapse fluorescence microscopy for three consecutive days at 1 h resolution. Each plot represents fluorescence intensity profile for an individual cell (for additional plots see also Supplementary Figure S3). (B) An average plot of single cell profiles expressing the PXDLS*3 (red), the mutant PXDLS*3 transgene (green) or non-expressing cells (PXDLS*3 neighboring cells) (blue) (n = 25 for each condition). Arbitrary Units (AU).
Mentions: Hitherto, the involvement of the PXDLS motif was assessed in a cell population, causing shallow circadian oscillations. Low-amplitude oscillations in a cell population might be due to inefficient synchronization of circadian clocks within individual cells that leads to rapid dampening, or the result of an inherent cell-autonomous defect in the clock's function. To discriminate between the two scenarios, we performed fluorescence recording of NIH3T3-Rev-VNP cells, stably expressing the circadian Venus-Rev reporter (26). Cells were transfected with Cherry expression vector either with the PXDLS*3 or the mutant PXDLS*3 transgene as a control. Single cells expressing the Cherry fluorescent protein or neighboring cells that do not express the Cherry fluorescent protein were monitored and analyzed for their circadian oscillations (Figure 3, Supplementary Figure S3, and Supplementary Movie S1). In contrast to the mutant PXDLS*3 and the PXDLS*3 non-expressing cells (neighboring cells), overexpression of the PXDLS*3 transgene disrupted the circadian rhythmicity in single cells (Figure 3, Supplementary Figure S3, and Supplementary Movie S1).

Bottom Line: Our bioinformatics analysis of short linear motifs, implicated in protein interactions, reveals an enrichment of the Pro-X-Asp-Leu-Ser (PXDLS) motif within circadian transcripts.Remarkably, the motif is evolutionary conserved in the core clock protein REV-ERBα, and additional proteins implicated in the clock's function (NRIP1, CBP).Furthermore, we demonstrate that the PXDLS motifs of NRIP1 and CBP are involved in circadian rhythmicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

Show MeSH
Related in: MedlinePlus