Platelets are specialized hemostatic cells that circulate in the blood as anucleate cytoplasts. We report that platelets unexpectedly possess a functional spliceosome, a complex that processes pre-mRNAs in the nuclei of other cell types. Spliceosome components are present in the cytoplasm of human megakaryocytes and in proplatelets that extend from megakaryocytes. Primary human platelets also contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. In response to integrin engagement and surface receptor activation, platelets precisely excise introns from interleukin-1beta pre-mRNA, yielding a mature message that is translated into protein. Signal-dependent splicing is a novel function of platelets that demonstrates remarkable specialization in the regulatory repertoire of this anucleate cell. While this mechanism may be unique to platelets, it also suggests previously unrecognized diversity regarding the functional roles of the spliceosome in eukaryotic cells.

Diacylglycerol kinases (DGKs) terminate signalling from diacylglycerol by converting it to phosphatidic acid. Diacylglycerol regulates cell growth and differentiation, and its transient accumulation in the nucleus may be particularly important in this regulation. Here we show that a fraction of DGK-zeta is found in the nucleus, where it regulates the amount of nuclear diacylglycerol. Reducing nuclear diacylglycerol levels by conditional expression of DGK-zeta attenuates cell growth. The nuclear-localization signal of DGK-zeta is located in a region that is homologous to the phosphorylation-site domain of the MARCKS protein. This is, to our knowledge, the first evidence that this domain, which is a major target for protein kinase C, can localize a protein to the nucleus. Two isoforms of protein kinase C, but not others, regulate the localization of DGK-zeta. Our results define a cycle in which diacylglycerol activates protein kinase C, which then regulates the metabolism of diacylglycerol by alternating the intracellular location of DGK-zeta. This may be a general mechanism to control mitogenic signals that depend on nuclear diacylglycerol.

A procedure is described that directs the self-induced deletion of DNA sequences as they pass through the male germ line of mice. The testes-specific promoter from the angiotensin-converting enzyme gene was used to drive expression of the Cre-recombinase gene. Cre was linked to the selectable marker Neo r , and the two genes flanked with loxP elements. This cassette was targeted to the Hoxa3 gene in mouse ES cells that were in turn used to generate chimeric mice. In these chimeras, somatic cells derived from the ES cells retained the cassette, but self-excision occurred in all ES-cell-derived sperm.

To further explore the role of diacylglycerol metabolism in endothelial responses, we used a degenerate reverse transcription-polymerase chain reaction method to identify diacylglycerol kinase isozymes expressed by human endothelial cells. We report the isolation of a 3.5-kilobase cDNA encoding a novel diacylglycerol kinase (hDGK) with a predicted molecular mass of 103.9 kDa. Human DGK contains two zinc fingers, an ATP binding site, and four ankyrin repeats near the carboxyl terminus. A unique feature, as compared with other diacylglycerol kinases, is the presence of a sequence homologous to the MARCKS phosphorylation site domain. From Northern blot analysis of multiple tissues, we observed that hDGK mRNA is expressed at highest levels in brain. COS-7 cells transfected with the hDGK cDNA express 117-kDa and 114-kDa proteins that react specifically with an antibody to a peptide derived from a unique sequence in hDGK. The transfected cells also express increased diacylglycerol kinase activity, which is not altered in the presence of R59949, an inhibitor of human platelet DGK activity. The hDGK displays stereoselectivity for 1,2-diacylglycerol species in comparison to 1,3-diacylglycerol, but does not exhibit any specificity for molecular species of long chain diacylglycerols.