Reconstituted mammalian U4/U6 snRNP complements splicing: a mutational analysis.

We have developed an in vitro complementation assay to analyse the functions of U6 small nuclear RNA (snRNA) in splicing and in the assembly of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. U6-specific, biotinylated 2'-OMe RNA oligonucleotides were used to deplete nuclear extract of the U4/U6 snRNP and to affinity purify functional U4 snRNP. The addition of affinity purified U4 snRNP together with U6 RNA efficiently restored splicing activity, spliceosome assembly and U4/U5/U6 multi-snRNP formation in the U4/U6-depleted extract. Through a mutational analysis we have obtained evidence for multiple sequence elements of U6 RNA functioning during U4/U5/U6 multi-snRNP formation, spliceosome assembly and splicing. Surprisingly, the entire 5' terminal domain of U6 RNA is dispensable for splicing function. In contrast, two regions in the central and 3' terminal domain are required for the assembly of a functional U4/U5/U6 multi-snRNP. Another sequence in the 3' terminal domain plays an essential role in spliceosome assembly; a model is strongly supported whereby base pairing between this sequence and U2 RNA plays an important role during assembly of a functional spliceosome.