Transcription of a U6 Small Nuclear RNA Gene in Vitro

U6 small nuclear RNA (snRNA), an essential component of the eukaryotic spliceosomes, is unique in that it is ynthesized by RNA polymerase 111, while all other U-snRNAs are synthesized by RNA polymerase 11. U6 genes are notable for functional upstream regulatory elements which resemble RNA polymerase I1 regulatory sequence motifs. In this study, the optimal conditions for transcription of the U6 snRNA gene in vitro were found to be similar to conditions optimal for transcription of 5 s RNA genes. To purify the trans-acting factors necessary for the transcription of the U6 RNA gene, HeLa cell extracts were fractionated on a DEAE-Sephadex column, and three fractions, designated DE-50, DE-175, and DE-500, were obtained by stepwise elution with 50, 175, and 500 mM ammonium sulfate, respectively. DE175 fraction transcribed tRNA and 55 RNA genes but not a mouse U6 RNA gene. Complementation of the DE-175 fraction with the DE-50 fraction resulted in the transcription of the U6 RNA gene. Experiments in which the transcription factor (TFIIIA) was selectively inactivated indicated that TFIIIA is not required for the transcription of the U6 RNA gene. These results show that the U6 snRNA gene, although transcribed by RNA polymerase 111, differs from tRNA and 55 RNA genes in that factors other than TFIIIA, -IIIB, and -1IIC are required for U6 gene transcription in vitro.