Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

Abstract

Cells normally restrict the nuclear export and expression of intron-containing mRNA. In many cell lines, this restriction can be overcome by inclusion of cis-acting elements, such as the Mason-Pfizer monkey virus constitutive transport element (CTE), in the RNA. In contrast, we observed that CTE-mediated expression from human immunodeficiency virus Gag-Pol reporters was very inefficient in 293 and 293T cells. However, addition of Sam68 led to a dramatic increase in the amount of Gag-Pol proteins produced in these cells. Enhancement of CTE function was not seen when a Sam68 point mutant (G178E) that is defective for RNA binding was used. Additionally, the effect of Sam68 was inhibited in a dose-dependent manner by coexpression of an activated form of the nuclear kinase Sik/BRK that hyperphosphorylated Sam68. RNA analysis showed that cytoplasmic Gag-Pol-CTE RNA levels were only slightly enhanced by the addition of Sam68, compared to a 60- to 70-fold increase in the levels of Gag-Pol protein expression. Thus, in this system, Sam68 functioned to enhance the cytoplasmic utilization of RNA containing the CTE. These results suggest that Sam68 may interact with specific RNAs in the nucleus to provide a “mark” that affects their cytoplasmic fate. They also provide further evidence of links between signal transduction and RNA utilization.

ACKNOWLEDGMENTS

The first two authors contributed equally to this work.

We thank Angela Tyner for the gift of plasmids expressing wild-type and mutant forms of Sik/BRK and Sally Parsons, Joan Steitz, Doug Black, Gary Brewer, and Bryan Cullen for other materials used in this study. Susan Prasad and Joy Morgenegg provided expert technical assistance.

This work was supported by National Institutes of Health grants AI34721 to M.-L.H. and AI47008 to D.R. J.C. is the recipient of NSRA fellowship AI10630. Salary support for M.-L.H. and D.R. was provided by the Charles H. Ross Jr. and Myles H. Thaler Endowments at the University of Virginia.