The 3 '-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in vivo

We describe a new approach to elucidate the role of 3'-end processing in pr e-mRNA splicing in vivo using the influenza virus NS1A protein. The effecto r domain of the NS1A protein, which inhibits the function of the CPSF and P ABII factors of the cellular 3'-end-processing machinery, is sufficient for the inhibition of not only 3'-end formation but also the splicing of singl e-intron pre-mRNAs in vivo. We demonstrate that inhibition of the splicing of single-intron pre-mRNAs results from inhibition of 3'-end processing, th ereby establishing that 3'-end processing is required for the splicing of a 3' terminal intron in vivo. Because the NS1A protein causes a global suppr ession of 3'-end processing in trans, we avoid the ambiguities caused by th e activation of cryptic poly(A) sites that occurs when mutations are introd uced into the AAUAAA sequence in the pre-mRNA. In addition, this strategy e nabled us to establish that the function of a particular 3'-end-processing factor, namely CPSF, is required for the splicing of single-intron pre-mRNA s in vivo: splicing is inhibited only when the effector domain of the NS1A protein binds and inhibits the function of the 30-kDa CPSF protein in 3'-en d formation. In contrast, the 3'-end processing factor PABII is not require d for splicing. We discuss the implications of these results for cellular a nd influenza viral mRNA splicing.