A SPONTANEOUS DUPLICATION IN U6 SPLICEOSOMAL RNA UNCOUPLES THE EARLY AND LATE FUNCTIONS OF THE ACAGA ELEMENT IN-VIVO

U6 RNA enters the spliceosome base paired with U4 RNA, but dissociates from U4 RNA before the catalytic steps of splicing. We have identifie d a cold-sensitive lethal mutation in U4 RNA (U4-cs1) that blocks the splicing pathway after U4/U6 complex formation, but before the first c atalytic step of splicing. Remarkably, selection for suppressors of th e cold-sensitive growth of the U4-cs1 strain yielded a tandem duplicat ion of the highly conserved ACAGA sequence of U6 RNA (U6-Dup). The ACA GA sequence plays an essential role in spliceosome assembly and in the second catalytic step of pre-mRNA splicing; one or both of these role s involves direct base pairing to the pre-mRNA 5' splice site. In a U4 -cs1/UG-Dup double-mutant strain grown at low temperature, the upstrea m ACAGA sequence of U6 RNA is required for suppression of the U4 mutat ion, whereas the downstream ACAGA sequence is required for other essen tial functions. Based on the sequence requirements for function of the upstream ACAGA element of U6-Dup, we propose that it pairs with the p re-mRNA 5' splice site during incorporation of the U4/U6 complex into the spliceosome and that the subsequent dissociation of U4 RNA exposes the downstream ACAGA sequence, which functions in the catalytic steps . The properties of this mutant U4/U6 complex provide compelling in vi vo evidence that U6 RNA normally base pairs with the 5' splice site be fore disruption of its pairing with U4 RNA.