The removal of intervening sequences from transcripts is catalyzed by the spliceosome, a multicomponent complex that assembles on the newly synthesized pre-mRNA. Pre-mRNA translation in the cytoplasm leads to the generation of aberrant proteins that are potentially harmful. Therefore, tight control to prevent undesired pre-mRNA export from the nucleus and its subsequent translation is an essential requirement for reliable gene expression. Here, we show that the natural product FR901464 (1) and its methylated derivative, spliceostatin A (2), inhibit in vitro splicing and promote pre-mRNA accumulation by binding to SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein in the spliceosome. Importantly, treatment of cells with these compounds resulted in leakage of pre-mRNA to the cytoplasm, where it was translated. Knockdown of SF3b by small interfering RNA induced phenotypes similar to those seen with spliceostatin A treatment. Thus, the inhibition of pre-mRNA splicing during early steps involving SF3b allows unspliced mRNA leakage and translation.

lation with anti-CD28 enhanced NFATc nuclear accumulation (Fig. 4B), in keeping with the finding that T H 2 cytokine induction in wildtype T H cells requires costimulation (Fig. 2C). In contrast, anti-CD3 treatment alone led to an increase in nuclear NFATc in Jnk1-/-T H cells and a decrease in cytoplasmic NFATc (Fig. 4, A and B), consistent with the high T H 2 cytokine production by CD3-activated Jnk1-/cells (Fig. 2C). The enhanced accumulation of nuclear NFATc in Jnk1-/-T H cells was observed in cells 8, 24, and 48 hours after stimulation, but was not observed in nonactivated cells (10). NFATc accumulation was specific because the amount of nuclear NFATp, a proposed negative regulator of T H 2 cytokine genes (21), was the same in wild-type and Jnk1-/cells (Fig. 4A). Enhanced nuclear accumulation of NFATc in Jnk1-/-T cells was not blocked by anti-IL-4 (Fig. 4A); hence, increased IL-4 production and NFATc nuclear localization is intrinsic to T cell receptor signaling and is not secondary to IL-4 production. Because NFATc can bind to the IL-4 promoter and is required for IL-4 production and T H 2 differentiation (20, 22), the greatly enhanced amount of nuclear NFATc could account for the increased IL-4 production in CD3activated Jnk1-deficient mice. The mechanism by which JNK1 negatively regulates NFATc nuclear accumulation remains to be resolved. The isoform NFAT4 is phosphorylated and negatively regulated by JNK, leading to nuclear exclusion (23). This regulation appears to be specific to the NFAT4 isoform; evidence for JNK regulation of NFATc was not reported (23). An indirect mechanism may therefore account for the altered regulation of NFATc in Jnk1-/-T H cells. NFATc and NFATp can bind to the IL-4 promoter NFAT sites (22). Both Jnk1 and NFATp knockout mice have enhanced T cell proliferation and T H 2 cytokine production (21, 24), precisely the opposite of the NFATc knockout. It is therefore possible that these two NFAT factors antagonize each other in the regulation of the IL-4 gene. The apparent similarity between NFATp-/and Jnk1-/phenotypes supports a functional linkage between JNK1 and NFAT. Our results further reveal a novel mechanism by which TCR signaling negatively regulates T H 2 cytokines through JNK1. Positive and negative regulation of JNK1 activity may affect the decision of T H cells to differentiate into T H 1 or T H 2 effectors, and therefore may affect the type of immune response that is initiated. The function of JNK1 demonstrated in this study is distinct from that of JNK2, which is required for IFN-␥ production in T H 1 cells (14). Moreover, the related p38 mitogen-activated protein kinase pathway is T H 1 specific and drives IFN-␥ transcription (25). Together, these pathways potentiate the T H 1 response and provide a potential target for pharmaceutical intervention.

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