U1 snRNA promotes the selection of nearby 5' splice sites by U6 snRNA in mammalian cells

Communication between exon boundaries is a central feature of the exon definition model of pre-mRNA splice-site selection and an exon-bridging interaction involving U1 small nuclear RNA (snRNA) paired with the 5' splice site (5'ss) has been identified previously. It has become increasingly clear, however, that the 5'ss is not defined relative to the base-pairing interaction with U1, suggesting that a connection in the proposed line of communication between exon boundaries is missing. To explore this issue, we have first sought to characterize the role in mammalian 5'ss selection of a previously suggested base-pairing interaction with U6 snRNA. Using transfection experiments, we show that mutations at positions 5 and 6 of a 5'ss associated with an internal exon can be suppressed by compensatory changes in the first two positions of a conserved hexanucleotide of U6 RNA. The specificity of the effect was established by covariation experiments as well as by experiments with two splice sites arranged in tandem. Suppression of 5'ss mutations by U6 was more efficient when U1 could pair nearby than when pairing was restored further away and individual U1 RNAs stimulated U6-defined proximal sites more efficiently than distal sites. These results are interpreted to suggest that U1 acts to direct 5'ss choice by U6 to matching sequences nearby. Our work supports a central role for base-pairing with U6 snRNA in mammalian 5'ss selection and suggests how the interaction may be established properly despite the limited complementarity involved.