Three novel components of the human exosome

The yeast exosome is a complex of 3' --> 5' exoribonucleases. Sequence anal ysis identified putative human homologues for exosome components, although several were found only as expressed sequence tags. Here we report the clon ing of full-length cDNAs, which encode putative human homologues of the Rrp 40p, Rrp4lp, and Rrp46p components of the exosome. Recombinant proteins wer e expressed and used to raise rabbit antisera. In Western blotting experime nts, these decorated HeLa cell proteins of the predicted sizes. All three h uman proteins were enriched in the HeLa cells nucleus and nucleolus, but we re also clearly detected in the cytoplasm. Size exclusion chromatography re vealed that hRrp40p, hRrp41p, and hRrp46p were present in a large complex. This cofractionated with the human homologues of other exosome components, hRrp4p and PM/ Scl-100. Anti-PM/Scl-positive patient sera coimmunoprecipita ted hRrp40p, hRrp41p, and hRrp46p demonstrating their physical association. The immunoprecipitated complex exhibited 3' --> 5' exoribonuclease activit y in vitro. hRrp41p was expressed in yeast and shown to suppress the lethal ity of genetic depletion of yeast Rrp4lp. We conclude that hRrp40p, hRrp41p , and hRrp46p represent novel components of the human exosome complex.