DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80). We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-2) and clusterin. KUB2 is Ku80. KUB3 and KUB4 are unknown, >10 kb trans-cripts. Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively. The interaction of apoJ/XIP8 with Ku70 was confirmed by far-western analyses. Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation.

RNA processing is carried out in close proximity to the site of transcription, suggesting a regulatory link between transcription and pre-mRNA splicing. Using an in vitro transcription/splicing assay, we demonstrate that an association of RNA polymerase II (Pol II) transcription and pre-mRNA splicing is required for efficient gene expression. Pol II-synthesized RNAs containing functional splice sites are protected from nuclear degradation, presumably because the local concentration of the splicing machinery is sufficiently high to ensure its association over interactions with nucleases. Furthermore, the process of transcription influences alternative splicing of newly synthesized pre-mRNAs. Because other RNA polymerases do not provide similar protection from nucleases, and their RNA products display altered splicing patterns, the link between transcription and RNA processing is RNA Pol II-specific. We propose that the connection between transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs.

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