The Highly Conserved U Small Nuclear RNA 3′-End Formation Signal Is Quite Tolerant to Mutation

Abstract

Formation of the 3′ end of U1 and U2 small nuclear RNA (snRNA) precursors is directed by a conserved sequence called the 3′ box located 9 to 28 nucleotides downstream of all metazoan U1 to U4 snRNA genes sequenced so far. Deletion of part or all of the 3′ box from human U1 and U2 genes drastically reduces 3′-end formation. To define the essential nucleotides within this box that direct 3′-end formation, we constructed a set of point mutations in the conserved residues of the human U1 3′ box. The ability of the various mutations to direct 3′-end formation was tested by microinjection into Xenopus oocytes and transfection into HeLa cells. We found that the point mutations had diverse effects on 3′-end formation, ranging from no effect at all to severe inhibition; however, no single or double point mutation we tested completely eliminated 3′-end formation. We also showed that a rat U3 3′ flank can effectively substitute for the human U1 3′ flank, indicating that the 3′ boxes of the different U snRNA genes are functionally equivalent.