Sequence of the rat Factor VIII cDNA M. Watzka, C. Geisen, E. Seifried

Sequence of the rat Factor VIII cDNA

M. Watzka, C. Geisen, E. Seifried and J. Oldenburg

Factor VIII functions as an essential cofactor in the blood coagulation cascade for the factor IXa-mediated activation of factor X. Here we report the cDNA corre- sponding to the rat homologue of the human factor VIII gene.

Materials and Methods

To find sequences containing the rat FVIII gene, DNA data of the rat genome pro- ject were aligned with mouse and human FVIII cDNAs. Alignment of the most homologue contig with mouse cDNA revealed several exon-intron boundaries.

These data were used to design primers to span the complete rat FVIII cDNA. PCR was performed using expand long template PCR System (Roche) under standard conditions including 1 µl human or rat liver cDNA (corresponding to 100 ng total RNA). Column or gel purified fragments of appropriate length were sequenced.

Results

Sequencing of rat FVIII cDNA with primers derived from the rat genome project resulted in 6777 bp of the complete rat FVIII cDNA (NM_183331). Overall similari- ty of this cDNA compared to human and mouse cDNA is 61% and 68 %, respectively (Table 1). Most posttranslational modification sites of the human factor VIII are

I. Scharrer/W. Schramm (Ed.)

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Hemophilia Symposium Hamburg 2003

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Table 1. Nucleotide and Amino Acid Homology

nucleotide homology amino acid homology domain rat/mouse rat/human rat/mouse rat/human

A1 70 % 68 % 70 % 67 %

a1 53 % 57 % 56 % 57 %

A2 74 % 73 % 76 % 71 %

a2 66 % 58 % 45 % 41 %

B6 2 % 49 % 41 % 26 %

a3 40 % 34 % 29 % 22 %

A3 70 % 68 % 69 % 69 %

C1 78 % 72 % 76 % 76 %

C2 79 % 72 % 73 % 66 %

overall 68 % 61 % 59 % 51 %

conserved in the rat factor VIII. A consensus polyadenylation signal was identified at nt 8142. Screening 203 bp 5´ of the ATG start codon revealed a potential tran- scription initiation point at nt -29 with a basic promoter consisting of a TATA box (26 bp), CAAT box (78 bp), and GC box (115 bp) situated upstream of this point.

While investigating rat liver FVIII cDNA, extra bands appeared when ampli- fying exons 16-22. Sequencing these bands, we found exon skipping of exon 17 and 328 M. Watzka et al.

Fig. 1. Characterization of rat and human Factor VIII alternative mRNAs. Lanes 1-3 rat liver cDNA, lane 4, 6 negative controls, lane 5 human liver cDNA. In human liver, neither exon skip- ping of exon 17, nor alternative splicing of exon 20 is observed.

A

B

600 500

400

300

200

700 600 500

400

300

200

bp 1 2 3 4 5 6 7 8

an alternative spliced exon 20 containing 26 additional bp (Fig. 1). While sequencing FVIII cDNA 13 polymorphisms in 5 alleles of 3 rats could be detected (Table 2).

Discussion

The rat FVIII nucleotide and the resulting amino acid sequence show significant similarity to human and mouse FVIII through the A and C domains, but not in the B and a domains. The low degree of conservation and high frequency of detected polymorphisms in the B domain supports the current view that the B domain does not significantly contribute to procoagulant activity.

The alternative transcript lacking exon 17 is formed due to a weak wt acceptor splice site flanking this exon. This is underlined by the relatively high expression of this transcript. The transcript results from alternative splicing of exon 20 because of a second acceptor splice site situated 26 bp upstream. Since the alternative splice site is weaker, expression of wt mRNA is clearly higher.

Protein variants derived from stop mutations in the human FVIII gene are not secreted in general [1]. Thus also the predicted alternative rat FVIII proteins are not expected to be secreted due to the phenomenon of nonsense mediated mRNA decay, a mechanism described for human FVIII and other genes [2].

This mechanism selectively degrades nonsense mRNAs with premature stop codons. Recently, regulated unproductive splicing and translation (RUST), a mecha- nism of alternative splicing coupled with NMD and nonsense associated altered splicing was discussed to be an ubiquitous mechanism in regulating protein expres- sion [3–4]. However, the biological relevance of the alternative rat FVIII RNA vari- ants with subsequent downregulation of the wt FVIII protein expression is not known. The diversity of rat FVIII is also reflected by 13 polymorphisms found in only 5 alleles of three animals. Four polymorphisms are found in the functionally important C1 and C2 domains, even one with an aa exchange from the sterically demanding Prolin to Leucin. The majority of changes represent C to T transitions Sequence of the rat Factor VIII cDNA 329

Table 2. Variations of the Rat FVIII cDNA

Exon nt exchange aa exchange domain

14 AAC>AAT Asn>Asn B

14 CCC>CCT Pro>Pro B

14 GAA>GAG Glu>Glu B

14 GCC>GCT Ala>Ala B

14 GAA>AAA Glu>Lys B

14 ATG>ATC Met>Ile B

14 AGA>AGG Arg>Arg B

20 CTC>CTT Leu>Leu C1

23 CTG>CTT Leu>Leu C1

24 CCG>CTG Pro>Leu C2

25 GGC>GGT Gly>Gly C2

3´UTR G>C – –

3´UTR ins A –

in CpG dinucleotides (7 of 13).With the discovery of the rat FVIII cDNA, the rat now becomes available as an animal model for further studies on the function of the FVIII protein. Through its highly divergent amino acid sequence, rat FVIII might provide new insights into the importance and function of particular amino acids, cleavage sites and sites of posttranslational modification, as the FVIII molecules from other species can do.

References

1. David D, Santos IMA, Johnson K, Tuddenham EGD, McVey JH. Analysis of the consequences of premature termination codons within the factor VIII coding sequences. J Thromb Haemost 2003; 1(1):139-146.

2. Culbertson MR, Leeds PF. Looking at mRNA decay pathways through the window of mole- cular evolution. Curr Opin Genet Dev 2003; 13(2):207-214.

3. Lewis BP, Green RE, Brenner SE. Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc Natl Acad Sci U S A 2003; 100(1):189- 192.

4. Wang J, Chang YF, Hamilton JI, Wilkinson MF. Nonsense-associated altered splicing: a frame-dependent response distinct from nonsense-mediated decay. Mol Cell 2002;

10(4):951-957.

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