Dual nuclear roles of an essential scavenger decapping enzyme

The broad findings of the current work focus on mammalian DcpS and provide an analysis of its role in pre-mRNA splicing, transcription, and its requirement in mouse development. Mutagenesis and heterokaryon assays reveal an N-terminal nuclear localization sequence and a central nuclear export sequence that account for its nucleocytoplasmic dynamics. Cell-based reporter assays reveal two novel DcpS nuclear roles that explain its prevalent nuclear presence. A cell-based mRNA assay, which detects reporter splicing patterns, indicates DcpS knockdown leads to pre-mRNA processing deficiency in the first intron. DcpS or Cbp20 overexpression partially and completely corrects this defect, respectively. Catalytically inactive DcpS displaces the Cbp20-cap interaction by competition in a reconstituted system. These results suggest DcpS is a positive regulator of pre-mRNA splicing and is in line with the idea that DcpS knockdown predictably upregulates intracellular cap structure levels. Another cell-based translation assay, which detects reporter enzyme activity, shows DcpS chemical repression or knockdown stimulates cap-dependent translation and reduces 4EBP1 protein level. The unexpected opposite effect on translation suggests DcpS catalysis does not directly regulate protein synthesis as previously thought. DcpS chemical repression by a competitive inhibitor leads to 4EBP1 and 4EBP2 transcriptional reductions that correlate with their steady state mRNA levels. These results point toward an additional DcpS nuclear role in transcription of specific targets. On a final note, the failure to generate DcpS homozygous progeny as early as 6 days post coitum (dpc) indicates this gene is essential for mouse embryogenesis. DcpS heterozygous intercross yields approximately two heterozygotes for each wildtype during 9.5 dpc and postnatal period. DcpS heterozygotes are normal with respect to fertility. Western blots show DcpS is ubiquitously expressed in an adult tissue panel comprised of brain, liver, kidney, and heart. The present study identifies at least two nuclear roles of DcpS in pre-mRNA first intron splicing and in transcription, and its essential role in mouse development.Ph.D.Includes bibliographical referencesIncludes vitaby Vincent She