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. 2007 Jan 8;27(6):2324–2342. doi: 10.1128/MCB.02300-06

FIG. 10.

FIG. 10.

The carboxy terminus of Caprin-1 selectively binds mRNAs associated with cellular proliferation through a mechanism dependent on the RGG motifs. (A) mRNA for c-Myc and cyclin D2 coprecipitate with endogenous Caprin-1 and G3BP-1. 293T cells were lysed in polysome lysis buffer. The supernatants of these lysates were precleared as described using beads coated with normal rabbit serum or mouse IgG1 for the anti-Caprin-1 and anti-G3BP-1 precipitations, respectively. Precleared polysome lysates were then incubated with protein A-Sepharose beads, which had been conjugated with anti-Caprin-1 rabbit serum, control rabbit serum, or protein G-Sepharose beads conjugated with the anti-G3BP-1 mouse MAb or an isotype-matched control mouse IgG1 MAb. RNA was extracted from the proteins bound to the beads after IP, as described above. RT-PCR was performed as described to detect the presence of c-Myc, cyclin D2, and GAPDH transcripts. (B) A carboxy-terminal fragment of Caprin-1 (381 to 709) that fails to bind G3BP-1 selectively binds mRNA for c-Myc and cyclin D2. As before, 293T cells were transfected with the indicated Flag-tagged fragments comprising the carboxy termini of Caprin-1 (381 to 709) or G3BP-1 (142 to 466) or the amino-terminal HR1 region of Caprin-1, Caprin-1 (47 to 380). The fragments were immunoprecipitated with anti-Flag as described above, and associated mRNA were assayed as before. Analysis of IP by SDS-PAGE and immunoblotting with antibody to Caprin-1 and G3BP-1 confirmed that fragments did not interact with endogenous protein (data not shown). (C) The RGG motifs in Caprin-1 are essential for selective binding of c-Myc and cyclin D2 mRNA. As before, 293T cells were transfected with a Flag-tagged carboxy-terminal fragment of Caprin-1 (381 to 709), a fragment in which each of the RGG motif had been mutated to AGG (AGGX3), or a Caprin-1 fragment that was truncated at residue 606 so it lacked the RGG motifs entirely, Caprin-1 (381to 605). The fragments were immunoprecipitated with anti-Flag and associated mRNAs were assayed as before.