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

Abstract

p68 and p72 are two highly related DEAD box proteins with similar biochemical activities in the nucleus of vertebrate cells; it is unknown whether they have redundant or differential in vivo functions. We report on a third member of this subfamily that is alternatively expressed from p72 mRNA. A detailed analysis of HeLa p72 mRNA was performed. It has an overall length of more than 5 kb and contains a 0.75-kb 5-untranslated region and a 3-untranslated region of 2.5 kb. Its open reading frame extends to nucleotide ؊243 upstream of the first in-frame AUG (A in the AUG triplet is ؉1) which serves as the p72 translation initiator codon. We provide evidence that alternative translation at a non-AUG within the extra coding region of this mRNA yields an 82-kDa protein (p82). Immunological studies substantiate that p82 is a naturally existing p72 variant and that both proteins are expressed at similar concentrations. p82 purified from HeLa cells is an ATP-dependent RNA helicase with biochemical properties almost identical to those of p72.Control of transcription is generally considered the main regulation level of mammalian gene expression, although mRNA maturation and modulation of mRNA stability or translational efficiency also contribute to expression control (1). Generation of protein variants by alternative pre-mRNA splicing or alternative initiation of mRNA translation enlarges the coding capacity and may explain the relatively low number of genes actually found in the human genome. According to recent estimations, on average one gene encodes three proteins (2).Although the 5Ј-untranslated leader sequences (UTRs) 1 of most vertebrate mRNAs are less than 100 nucleotides (nt) long (3), those of tightly controlled genes usually are longer and are GC-rich. Such structure-prone 5Ј-UTRs can modulate translation by the presence of upstream (up) open reading frames (ORFs) in addition to the main ORF, by formation of secondary structures, and/or by RNA-protein interactions (4). Indeed, they appear particularly characteristic of mRNAs encoding growth factors, receptor proteins, transcription factors, signal transduction components, proto-oncogenes, tumor suppressor proteins, and even proteins that mostly are constitutively expressed at a low level ("housekeeping" proteins).DEAD/DEXH box proteins belonging to superfamily II of RNA helicases play a universal role in RNA metabolism of proand eukaryotes. Not surprisingly, they are involved in gene expression control, for instance during germ cell and embryonic development or in cell proliferation and differentiation (5). 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,...

A Novel Domain within the DEAD-Box Protein DP103 Is Essential for Transcriptional Repression and Helicase Activity

“…Whereas the biological activity of putative RNA helicases has been demonstrated (12,16,28,36,54), ATP-dependent RNA helicase activity has been characterized for only some of these proteins (13,14,45,46,62,63). Our data clearly indicate that, similar to other RNA helicases (15,45,63), DP103 exhibits RNA helicase activity in an ATP-dependent fashion and in a 5Ј to 3Ј direction with a 5Ј single-strand overhang and is thus distinguished from activity of DNA helicases (51,54,61).…”

Involvement of RNA Helicases p68 and p72 in Colon Cancer

“…1A, p68 was overexpressed in all four tumor cell lines studied compared with the three nontumor cell lines. Similarly, p72 and its isoform p82, both of which are generated from the same transcript but use different translation start codons (40), were overexpressed in tumor cells. Interestingly, the degree of p68/p72 overexpression was at least as high as the known overexpression of h-catenin in colon cancer cells (Fig.…”

Concerted activation of the Mdm2 promoter by p72 RNA helicase and the coactivators p300 and P/CAF

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