The human Ul snRNA promoter correctly initiates transcription in vitro and is activated by PSEl

A DNA-dependent in vitro transcription system for the human Ul small nuclear RNA (snRNA) promoter has been developed. This in vitro transcription system uses extracts of tissue culture cells to drive transcription of an RNA polymerase Il-transcribed snRNA gene. A Ul promoter (-393 to +192) template was constructed in which the sequences from +10 to +171 were replaced with a 179-bp sequence from a G-less cassette. This DNA template thus retained all of the known Ul promoter elements, including the Ul 3'-end box (positions +175 to +191), which is responsible for snRNA 3'-end formation. HeLa cell nuclear extracts were shown to drive specific transcription of this promoter by RNA polymerase II. This transcription system has many of the properties observed for wild-type snRNA promoters in vivo. Transcription was shown to initiate at +1 (and 2) relative to the Ul promoter and to efficiently (>90%) form a 3' end corresponding to the 3' end found in the primary transcript of Ul in vivo. The transcription signal is responsive to either deletion or replacement of the Ul distal sequence (enhancer-like) and proximal sequence (TATA-like) elements, as well as the 3'-end box. Additionally, the signal was shown by depletion/repletion experiments to be responsive to a protein called PSEl (related to Ku), which has recently been shown to specifically bind sequences in the Ul promoter. This in vitro snRNA transcription system should facilitate the biochemical analysis of the human Ul snRNA promoter and lead to a better understanding of the differences between snRNA and mRNA promoters.