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. 2000 Feb 15;28(4):932-9.
doi: 10.1093/nar/28.4.932.

Structure and expression of the human p68 RNA helicase gene

O G Rössler  1 , P Hloch, N Schütz, T Weitzenegger, H Stahl
Affiliations

Structure and expression of the human p68 RNA helicase gene

O G Rössler et al. Nucleic Acids Res. .

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.

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Figures

Figure 1
Figure 1
Structure of the human p68 gene (EMBL accession no. AF015812). (A) Relative distribution of exons and introns with an allocation of the conserved DEAD box protein motifs of p68 among exons. Filled boxes represent exons. Introns and flanking regions are depicted by thin lines (bar = 500 nt). Restriction sites used for subcloning are indicated. Amino acid consensus sequences of DEAD box proteins are given in single letter code. (B) Detailed structure of exon–intron boundaries. Exons are indicated by Roman numbers on the right and left side, with their sizes in parentheses. Nucleotide sequences at the exon and intron boundaries and around the putative branch sites are shown in comparison to the respective consensus sequences (M = A or C; R = A or G; Y = C or T; N = any nucleotide). The size of exon I is numbered from the transcription start site (see below). The size of exon XIII is 706 bp up to the polyadenylation site. Underneath the donor and acceptor sites, the amino acids are given in the three letter amino acid code. Note that some codons are split by intron sequences.
Figure 2
Figure 2
Identification of the transcription initiation site in the p68 gene. Primer extension reactions were performed with a p68-specific 32P-labelled primer (see Materials and Methods) and 5 µg of poly(A)+ RNA from HeLa-S3 cells or the indicated human tissues. tRNA was used in a control reaction. Products were analysed on a sequencing gel side-by-side with DNA sequencing reactions (lanes 1–4). The nucleotide sequence around the p68 transcription start site (indicated by an arrow) is shown on the left in comparison to the consensus sequence of TATA box-containing promoters (31).
Figure 3
Figure 3
Analysis of the p68 gene promoter. (A) Sequence of the upstream p68 genomic DNA. The complete nucleotide sequence of the human p68 gene was determined, and the sequence of the 5′-flanking region is depicted (EMBL accession no. AF015812). The transcription initiation site is marked +1, and nucleotide numbers relative to the transcription start site are given on the left. Consensus sequences for transcription factor binding sites are underlined. (B) Activities of p68 promoter–CAT fusion genes in HeLa cell transfection assays. p68 promoter–CAT gene constructs are schematically shown on the left side of the figure. Numbers refer to p68 coordinates in (A). CAT activity was normalised for variations in transfection efficiency by dividing it by β-galactosidase activity and expressed in relation to the Herpes simplex virus thymidine kinase promoter activity obtained in parallel experiments. All results are mean values (±SD) from four independent transfection experiments.
Figure 4
Figure 4
Expression of the p68 gene in human cells. (A) Northern blot analysis of different human tissues. A Human Multiple Tissue Northern Blot [with 2 µg poly(A)+ RNA/lane; Clontech no. 7760-1] was hybridized with a p68 full-length 32P-labelled cDNA probe and analysed by autoradiography (left, exon DNA probe). The stripped membrane was rehybridized with an intron 11-specific (StuI–MunI 1017 bp fragment) 32P-labelled DNA probe (right, intron DNA probe). RNA markers, run in parallel, are indicated on the right, and estimated sizes of p68 RNAs are shown on the left. (B) p68 RNA (upper) and protein (lower) analysis of different human culture cells. Northern blot analysis with 2 µg poly(A)+ RNA from the indicated cell lines was performed as in (A) except that the blot was hybridized wih the p68 exon- and intron-specific and a β-actin probe at the same time. For western blot analysis, each lane was loaded with 20 µg protein extract from the respective cells and separated by 10% SDS–PAGE. After transfer to nitrocellulose, the blot was probed with the p68-specific monoclonal antibody C10. Only the p68 area of the membrane is shown.
Figure 5
Figure 5
Structure and cellular localisation of the alternatively spliced human p68 RNA. (A) The structure of two alternatively spliced p68 cDNAs. Two p68-specific clones with inserts of ~4.4 kb were isolated by screening of a λgt11 HeLa cDNA library with a StuI–MunI p68 intron 11-specific probe. Determination of their sequence revealed that they are copies of alternatively spliced p68 poly(A)+ RNAs, the structures of which are schematically shown with open and filled boxes representing introns and exons, respectively. (A)n is the poly(A) tail. (B) RT–PCR analysis of HeLa-S3 poly(A)+ RNA. Cytoplasmic (lanes 2 and 5) and nuclear (lanes 3 and 6) poly(A)+ RNAs were analysed with the indicated probes. Their positions in the genomic DNA are shown in the scheme on top of this part (pr 1, intron 11-specific primer; pr 2, exon 12-specific primer; rev pr, reverse primer; arrows indicate direction of DNA synthesis). PCR reactions were performed with the same primers but with p68 cDNA (lane 7) or cloned genomic p68 DNA (lanes 4 and 8) used as controls. RT–PCR, with PCR products analysed by agarose gel electrophoresis and ethidium bromide staining. Sizes of λ DNA markers, run in parallel (lane 1), are indicated on the left side.
Figure 6
Figure 6
High level expression of exogenous p68 suppresses maturation of endogenous p68 mRNA. (A and B) Analysis of conditionally p68-overproducing HeLa cells for p68-specific transcripts at different times after induction. Poly(A)+ RNA was prepared from a conditionally p68-overproducing HeLa cell clone at indicated times after withdrawal (–tet) from or in the presence (control) of tetracycline (2 µg/ml) in the medium and analysed by northern blotting with DIG-labelled antisence RNA probes representing either (A) nucleotides 1–1300 (p68 exon probe) or (B) nucleotides 5336–6352 (p68 intron probe) of the human p68 cDNA or the human β-actin coding sequence (β-actin mRNA, bottom). Sizes of RNA markers are indicated on the left. (C) Western blot analysis of the same cell clone. Each lane was loaded with 20 µg protein extract from the respective cells and separated by 10% SDS–PAGE. After transfer to nitrocellulose, the blots were probed with the p68-specific monoclonal antibody Pab 204 (1). p68 signals were quantitated with a Molecular Dynamics densitometer and expressed as a multiple of the control.
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