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Regulated specific proteolysis of the Cajal body marker protein coilin

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Abstract

Cajal bodies (CB) are subnuclear domains that contain various proteins with diverse functions including the CB marker protein coilin. In this study, we investigate the proteolytic activity of calpain on coilin. Here, we report a 28-kDa cleaved coilin fragment detected by two coilin antibodies that is cell cycle regulated, with levels that are consistently reduced during mitosis. We further show that an in vitro calpain assay with full-length or C-terminal coilin recombinant protein releases the same size cleaved fragment. Furthermore, addition of exogenous RNA to purified coilin induces proteolysis by calpain. We also report that the relative levels of this cleaved coilin fragment are susceptible to changes induced by various cell stressors, and that coilin localization is affected by inhibition or knockdown of calpain both under normal and stressed conditions. Collectively, our data suggest that coilin is subjected to regulated specific proteolysis by calpain, and this processing may play a role in the regulation of coilin activity and CB formation.

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Acknowledgments

This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM081448.

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  1. Department of Biochemistry, The University of Mississippi Medical Center, 2500 North State Street, Jackson, MS, 39216-4505, USA

    Venkatramreddy Velma, Hanna J. Broome & Michael D. Hebert

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  1. Venkatramreddy Velma
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  2. Hanna J. Broome
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  3. Michael D. Hebert
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Correspondence to Michael D. Hebert.

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Supplemental Figure 1

Coilin siRNA treatment reduces full-length and cleaved coilin fragment levels. (A) U2OS cells were transfected with control or coilin siRNA. 24hr post-transfection, the cells were harvested and the lysates subjected to SDS-PAGE, Western transfer, then probed with anti-coilin and anti-β-tubulin antibodies. (B) Quantification of knockdown from (A) and Figure 1B (48 and 72 h time points). The full-length coilin and cleaved coilin signals were divided by the tubulin signal. The values for coilin siRNA were then normalized to the results for the control siRNA. (JPEG 22 kb)

High-resolution image (TIFF 172 kb)

Supplemental Figure 2

The cleaved coilin fragment does not arise from the extreme N- or C-terminus of full-length coilin. HeLa cells were transfected with GFP-coilin or coilin-GFP. 24hr post-transfection, the cells were harvested and the lysates were incubated with calpain as described in “Materials and methods.” The reactions were subjected to SDS-PAGE, Western transfer, and then probed with anti-GFP and anti-coilin antibodies. The letter ‘a’ indicates full-length GFP-coilin or Coilin-GFP, ‘b’ indicates endogenous full-length coilin and ‘c’ indicates cleaved coilin fragment. (JPEG 12 kb)

High-resolution image (TIFF 130 kb)

Supplemental Figure 3

Protein bands detected by anti-coilin H300 antibody from Figure 6C were quantified by densitometric analysis. Cleaved coilin fragment to full-length coilin ratios were calculated for U2OS (A) and WI38 (B) cells. We have observed similar results in three experiments. (C) ALLN treatment does not influence SMN levels. HeLa cells were treated with DMSO or ALLN for 16 hr. The harvested cell lysates subjected to SDS-PAGE, Western transfer, then probed with anti-SMN and anti-β-tubulin antibodies. (JPEG 13 kb)

High-resolution image (TIFF 94 kb)

Supplemental Figure 4

Calpain4 KD decreases nucleolar accumulation of coilin: quantification of Figures 7 and 8. U2OS (A) and HeLa (B) cells were counted for each treatment and the percentages of cells with nucleolar coilin were presented; more than fifty cells were counted for each treatment. (JPEG 10 kb)

High-resolution image (TIFF 65 kb)

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Velma, V., Broome, H.J. & Hebert, M.D. Regulated specific proteolysis of the Cajal body marker protein coilin. Chromosoma 121, 629–642 (2012). https://doi.org/10.1007/s00412-012-0387-4

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