Structure and expression of the human p68 RNA helicase gene

Abstract

Nuclear DEAD box protein p68 is immunologically related to SV40 large tumour antigen and both proteins possess RNA helicase activity. In this report, we describe the structural organisation of the human p68 gene and aspects of the regulation of its expression. Northern blot and primer extension analyses indicate that, although its level is variable, the p68 RNA helicase appears to be expressed from a single transcription start site in all tissues tested. Sequence analysis revealed that the p68 promoter harbours a 'TATA', a 'CAAT' and an initiator element and contains high affinity binding sites for Sp1, AP-2, CRE and Myc. This and functional promoter analyses in transient expression assays suggest that transcriptional regulation of the p68 gene is complex. Furthermore, there are indications that p68 expression is also regulated post-transcriptionally. Steady-state pools of poly(A)(+)RNA from human cells contain completely spliced p68 mRNA and alternatively spliced forms that contain introns 8-11 or 8-12 (from a total of 12 introns) and are not translated. Analysis of a conditionally p68-overproducing HeLa cell line points to negative autoregulation at the level of splicing, which is confirmed by a recently reported association of p68 with spliceosomes in human cells.

The mRNA of DEAD Box Protein p72 Is Alternatively Translated into an 82-kDa RNA Helicase

“…This in turn requires tight control of at least some DEAD/DEXH box proteins like CsdA of Escherichia coli, which is regulated by an 11-nt "cold box" sequence in its 5Ј-UTR (6), or the Saccharomyces cerevisiae Dbp2, the gene of which contains an unusually large intron that is part of a post-transcriptional autoregulatory feedback loop (7). A similarly complex transcription regulation has been shown for nuclear p68 (8), a mammalian homologue of Dbp2. In addition, the 5Ј-UTR of p68 mRNA is long (170 nt) and GC-rich (57.6% as compared with a genome-wide average of 41%; see Ref.…”

An evolutionarily conserved, alternatively spliced, intron in the p68/DDX5 DEAD-box RNA helicase gene encodes a novel miRNA

“…1A). This observation, coupled with the fact that the p68 RNA, which includes this intron, is stable and differentially expressed between tissues (Stevenson et al 1998;Rossler et al 2000), suggests that sequences within this intron are important. However, to date, the function of this intron has remained unclear.…”

“…Taken together, these findings indicate that the conserved p68 intron encodes a novel miRNA. In this respect, the previous observations that expression of the intron-containing RNA appears to be differentially regulated (Stevenson et al 1998;Rossler et al 2000) would provide an additional mechanism by which alterations in p68 expression may impact on the cell.…”

“…The p68 gene contains a large intron (intron 11, 1.2 kb in the human gene), which has been conserved through evolution, being present even in yeast (Iggo et al 1991;Rossler et al 2000). Several studies have demonstrated the presence of two predominant p68 RNAs in cell lines and tissues, resulting from alternative splicing of this intron, and shown that their expression is differentially regulated, suggesting that the intron-containing RNA is not only stable but may also be functional (Stevenson et al 1998;Rossler et al 2000).…”

“…Several studies have demonstrated the presence of two predominant p68 RNAs in cell lines and tissues, resulting from alternative splicing of this intron, and shown that their expression is differentially regulated, suggesting that the intron-containing RNA is not only stable but may also be functional (Stevenson et al 1998;Rossler et al 2000). Inclusion of the intron introduces a stop codon, but, to date, there is no evidence for the presence of a truncated protein.…”