Characterization and Organization of the U6 SnRNA Gene in Zebrafish and Usage of Their Promoters to Express Short Hairpin RNA

We have characterized three U6 snRNA genes in zebrafish and randomly designated them as U6-1, U6-2, and U6-3. The U6-1 gene is closely related to the mammal U6 snRNA genes and that the U6-2 and U6-3 genes are more closely related to the Drosophila and Xenopus U6 snRNA genes. The upstream regulatory sequences were located based on their conserved position relative to the transcription start site. Furthermore, we speculate that the "CCAAT box" functions as the distal sequence element in the zebrafish U6 snRNA genes. Genomic BLASTn analysis revealed that at least 555 copies of the U6-1 gene are dispersed throughout the zebrafish genome, whereas the U6-2 and U6-3 genes are each present as a single copy. Three U6 snRNA genes are functionally expressed in various tissues. All three putative promoters were able to transcribe short hairpin RNA (shRNA) in zebrafish cell extracts. Our findings demonstrate that these putative promoters have the potential to be used for vector-based RNA interference (RNAi) in zebrafish. Another U6 snRNA was found from the genomic BLASTn search and designated as U6-4, demonstrating that there are four different types of zebrafish U6 snRNA genes.