Chromosoma

, Volume 119, Issue 2, pp 205–215 | Cite as

Coilin phosphorylation mediates interaction with SMN and SmB′

  • Cory G. Toyota
  • Misty D. Davis
  • Angela M. Cosman
  • Michael D. Hebert
Research Article

Abstract

Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB′ binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB′ than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB.

Supplementary material

412_2009_249_Fig7_ESM.gif (21 kb)
Supplemental Fig. 1

SmB′ and SMN interaction with full length GST–coilin and GST–coilin DDE. GST-proteins were incubated with decreasing amounts of SmB′ (top panel) or SMN (lower panel). Reactions were subject to SDS-PAGE and western transfer, followed by detection of the various proteins with appropriate antibodies. There is no significant difference between SmB′ and SMN binding with full length coilin or DDE phosphomimic. GST-protein levels are shown to demonstrate that similar amounts were used throughout the experiment. Input lanes represent 5% of the maximum amount of total protein used in the CoIP experiments (lanes 2 and 6) (GIF 20 kb)

412_2009_249_Fig7_ESM.tif (1.4 mb)
High Resolution Image. (TIFF 1399 kb)
412_2009_249_Fig8_ESM.gif (5 kb)
Supplemental Fig. 2

The GFP-coilin Δ3nt construct is resistant to siRNA treatment. Constructs were designed, including silent mutations comprising either a single or three nucleotide change, to be resistant to the siRNA mediated depletion of endogenous coilin. Endogenous coilin was knocked down by transfecting HeLa cells with duplex coilin (+) and control siRNA (-). After incubating 48 hours, cells were transfected with equal amounts of GFP-coilin, GFP-coilin Δ1nt, or GFP-coilin Δ3nt DNA. At 72 hours post-siRNA treatment, cells were harvested and subjected to western analysis. Proteins were detected with anti-GFP antibodies. Unprotected GFP-coilin was clearly depleted (lane 1) compared to the control (lane 2). The single nucleotide modification conferred some protection (lanes 3 and 4), but replacement of three nucleotides was required for complete resistance (lanes 4 and 5) (GIF 4 kb)

412_2009_249_Fig8_ESM.tif (426 kb)
High Resolution Image. (TIFF 426 kb)

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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Cory G. Toyota
    • 1
  • Misty D. Davis
    • 1
  • Angela M. Cosman
    • 1
  • Michael D. Hebert
    • 1
  1. 1.Department of BiochemistryUniversity of Mississippi Medical CenterJacksonUSA

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