5'-flanking sequences can dramatically influence 4.5SH RNA gene transcription by RNA-polymerase III.

4.5SH RNA is a 94 nt small nuclear RNA with an unknown function. Hundreds of its genes are present in the genomes of rodents of six families including Muridae. 4.5SH RNA genes contain an internal RNA-polymerase III promoter consisting of A and B boxes. Here we studied the influence of 5'-flanking sequences on the transcription of a mouse 4.5SH RNA gene. We found that replacement of the upstream sequence can dramatically change the 4.5SH RNA gene transcription efficiency. Various DNA fragments inserted immediately upstream from 4.5SH RNA gene completely inhibited its in vitro transcription, whereas others promoted it. The shortening of the native mouse 5'-flanking sequence of 4.5SH RNA gene to 42 bp resulted in the activation of an additional illegal transcription start site in upstream region. Transcription of the 4.5SH RNA gene with various upstream sequences in transfected HeLa cells revealed the differences between the tests performed in vivo and in vitro: in whole cells, only the construct with 5'-flanking native sequence could be transcribed. Apparently, at least some regions of the native 5'-flanking sequence of 4.5SH RNA genes have been selected during evolution for high transcription activity.