Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.

Abstract

Promoter elements in the 5' flanking regions of vertebrate U6 RNA genes have been shown to be both necessary and sufficient for transcription by RNA polymerase III. We have recently isolated and characterized a variant human U6 gene (87U6) that can be transcribed by RNA polymerase III in vitro in the absence of any natural 5' or 3' flanking sequences. This gene contains 10 nucleotide differences from the previously characterized human U6 gene (wtU6) within the coding region but has no homology to wtU6 in the upstream promoter region. By constructing chimeras between these two genes, we have shown that mutation of as few as two nucleotides in the coding region of the human U6 RNA gene is sufficient to create an internal promoter that is functional in vitro. A T-to-C transition at position 57 and a single T deletion at position 52 produce an internal U6 promoter that is nearly as active in vitro as the external U6 polymerase III promoter utilized by wtU6. Neither of these residues is absolutely conserved during evolution, and both of these nucleotide changes occur within the previously noted A box homology. Deletion and linker scanning mutations within the coding region of this variant U6 gene suggest that, in addition to the central region including bp 52 and 57, sequences at the extreme 5' end of the gene are critical for efficient transcription. In contrast, flanking sequences have a minor effect on transcriptional efficiency. This arrangement is unique among internal RNA polymerase III promoters and may indicate unique regulation of this gene.The transcription of the small nuclear RNA U6 has been well characterized in both vertebrate (2,8,17,21,22,28) and yeast (6, 7, 9) species. In the three vertebrate species in which a U6 gene has been cloned (humans, mice, and Xenopus tropicalis), transcription has been shown to be entirely dependent on 5' flanking sequences, including a classical TATA box and the U small nuclear RNA specific proximal sequence element (for review, see references 19 and 38). Both of these elements are normally found in RNA polymerase II promoters but have been shown to function in the RNA polymerase III promoter of U6. Indeed, the TATA box has been shown to be the element responsible for conferring RNA polymerase III specificity to the U6 promoter (23,24). In yeast species, the promoter structure is more complex, consisting of both external and internal elements. The U6 gene in Saccharomyces cerevisiae requires a 5' flanking TATA box, but transcription in vivo requires the presence of an internal A block and a B block located 120 nucleotides downstream (4, 6, 7, 9). The fission yeast Schizosaccharomyces pombe also contains a potentially functional B block homology within the intron of its U6 gene (11,32). However, in all cases studied thus far, the transcription of U6 relies wholly or in part on sequences upstream of the coding region.The human genome contains approximately 200 copies of U6-related sequences (14), only five of which have been sequenced and only three of which have been stud...

The life of U6 small nuclear RNA, from cradle to grave

“…At least four human U6 genes encoding identical RNAs are transcriptionally active to various degrees (Domitrovich and Kunkel 2003). Additionally, a variant of human U6 snRNA with nine substitutions and one nucleotide deletion is expressed under the control of an internal promoter, unlike other transcriptionally active human U6 genes (Tichelaar et al 1994(Tichelaar et al , 1998. The presence of multiple U6 genes of varying transcriptional activity has complicated their individual study, and whether paralogous but divergent U6 snRNAs exhibit differences in modification, localization or function is poorly understood.…”

The Potential of U6 and Its Copies in the Regulation of the Human Genome

“…However, the presence of these sequences along the human evolution has shaped the genome and new functional elements have been generated in most cases by a unique mechanism called retrotransposition [39]. Retrotransposition is the greatest remodeling force of the human genome, and it is currently represented by the Long Interspersed Nuclear Element 1 (LINE 1) [40][41][42][43]. These are 6-7kb elements that have all the machinery to be copied and mobilized into the genome.…”

In Vivo Expression of a Variant Human U6 RNA from a Unique, Internal Promoter

“…Assay. The minimal promoter constructs were made by annealing mutually priming, overlapping oligonucleotides spanning the entire length of the coding region and filling in the overhangs with T4 DNA polymerase as described previously (21). Analysis of the transcription of these constructs was carried out in HeLa S100 extract as described previously (20).…”

“…We have previously described a naturally occurring sequence variant of the human U6 gene (20) and shown that it is controlled by a unique promoter (21). This RNA differs from the consensus mammalian U6 RNA at 10 of 107 residues, with none of the changes occurring at positions that are perfectly conserved throughout evolution (22).…”

“…We also show that 87U6 RNA is capable of assembling into Sm-precipitable snRNP complexes with an efficiency similar to the consensus U6 RNA. In addition, both naturally occurring alleles of this RNA (21) are capable of being capped with the unique γ-monomethyl phosphate cap of U6. These results suggest that 87U6 may participate in the splicing reaction in a manner similar to the consensus U6 RNA.…”