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We have identified an amino acid sequence in the Drosophila Transformer (Tra) protein that is capable of directing a heterologous protein to nuclear speckles, regions of the nucleus previously shown to contain high concentrations of spliceosomal small nuclear RNAs and splicing factors. This sequence contains a nucleoplasmin-like bipartite nuclear localization signal (NLS) and a repeating arginine/serine (RS) dipeptide sequence adjacent to a short stretch of basic amino acids. Sequence comparisons from a number of other splicing factors that colocalize to nuclear speckles reveal the presence of one or more copies of this motif. We propose a two-step subnuclear localization mechanism for splicing factors. The first step is transport across the nuclear envelope via the nucleoplasmin-like NLS, while the second step is association with components in the speckled domain via the RS dipeptide sequence.Eukaryotic pre-mRNA splicing takes place in spliceosomes, multicomponent nuclear complexes containing the small nuclear ribonucleoproteins (snRNPs) Ul, U2, U4, U5, and U6, and a large number of splicing factors (for reviews, see refs. 1 and 2). Evidence that splicing components are nonrandomly distributed in the nucleus was first provided by intranuclear localization studies, which showed that snRNPs are preferentially associated with nuclear structures designated as perichromatin fibrils, interchromatin granules, and coiled bodies (3-6). These associations can be visualized in the light microscope by immunofluorescent staining and by in situ hybridization methods. The interchromatin granule regions appear as 20-50 heavily stained regions referred to as nuclear "speckles" (refs. 7 and 8; see ref. 9 for review).The first indication that non-snRNP essential splicing factors are also associated with nuclear speckles was provided by immunofluorescent labeling studies with a monoclonal antibody (mAb) directed against the splicing factor SC35 (10). The SC35 protein (11) is a member of the SR family of general splicing factors (12), a group of proteins that also includes the splicing factor SF2/ASF (13)(14)(15)(16). All of the proteins in this family are recognized by MablO4, and they contain one or more RNP binding domains and a region rich in arginine/ serine dipeptides (RS domain; see ref. 12). Significantly, all of the SR family members examined to date colocalize with SC35 to nuclear speckles (10,12,17).RS domains are also present in the Drosophila alternative splicing factors suppressor-of-white-apricot [Su(wa)], Transformer (Tra), and Transformer 2 (Tra2), and these proteins localize to speckle domains in mammalian cells (ref. 17; this paper). In fact, the RS-rich domain is the only known sequence that is shared by all of the splicing factors that localize to speckles. Evidence that this domain may be involved in nuclear and/or subnuclear localization of splicing factors was provided by the observation that a 100-aa sequence of Tra, whichThe publication costs of this article were defrayed in part by page charge p...

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Purpose: The carcinogen activator cytochrome P450 1B1 (CYP1B1) is expressed on almost all human tumors with rare expression on normal tissues. Anti-CYP1B1–specific T cells kill CYP1B1-expressing tumors, providing the rationale to examine CYP1B1 as a target for immunotherapy. Experimental Design: ZYC300, a plasmid DNA of CYP1B1 encapsulated in biodegradable poly-dl-lactide-coglycolide microparticles, was used in a phase I clinical trial to treat 17 patients with advanced stage, progressive cancer. ZYC300 was administered i.m. at a fixed dose of 400 μg every other week for up to 12 doses. Results: Thirteen patients received six vaccinations and five received all 12 doses. No significant adverse events were observed. Six patients developed immunity to CYP1B1, three of whom developed disease stabilization. All but 1 of 11 patients who did not develop immunity to CYP1B1 progressed and did not respond to salvage therapy. Five patients who developed immunity to CYP1B1 required salvage therapy for progressive metastatic disease and showed marked response to their next treatment regimen, most of which lasted longer than 1 year. Conclusions: The association between immunity to CYP1B1 and response to next salvage therapy was not expected. Because six of the seven patients who had clinical benefit regardless of the nature of salvage therapy had developed immunity to CYP1B1, it seems highly unlikely that this occurred by chance alone. Regardless of the mechanism(s) that induced tumor regression, these findings force us to rethink how the generation of antitumor immunity might be integrated into the treatment of cancer.

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