Role of RNA structure in splicing: excision of the intervening sequence in yeast tRNA3leu is dependent on the formation of a D stem.

A substitution mutant of the yeast tRNA3leu gene results in the sequence change of GCC to AAA at positions 10, 11, and 12 in the noncoding strand. The ability to form a D stem is lost. Transcription in the heterologous Xenopus germinal vesicle system is not drastically affected, but splicing of the tRNA precursor does not occur. To determine whether this effect is caused by the change in sequence or the change in conformation we constructed two new mutants. In one, mutation results in the sequence change of GGC to TTT at positions 24, 25, and 26. The ability to form a D stem is lost; transcription is unaffected, but excision of the intron does not occur. The other, a double mutant, is characterized by both substitutions described above, and the ability to form a D stem is retained. The precursor derived from the double mutant is accurately spliced in X. laevis germinal vesicle extracts, therefore excision of the intervening sequence appears to depend on the formation of a D stem.