The physiological function of the prion protein (PrP) is unknown, but there is strong evidence that the infec ious agent of transmissible spongiform encephalopathies (the prion) consists at least in part of PrPsC, a mod)fied form of the normally occurring cellular PrP (PrPC)~. Both forms of PrP are encoded in a single exon of the PrP gene.
Goldgaber in his Scientific Correspondence noted a large open reading frame (ORF) on the antisense strand of the coding regions of mammalian PrP genes. Several genetic traits influencing spongiform encephalopathies in humans and animals have been linked to the PrP gene. If an 'anti-PrP' existed it could be responsible for the properties formerly ascribed to the prion protein. In support of this hypothesis, Hewinson and colleagues detected a 4.5-kilobase RNA in bovine brain which hybridized to PrP sense riboprobes, and which was thus expected to represent a PrP antisense transcript.
We have found a similar 4.5-kb mRNA in normal and scrapie infected hamsters and in mice; however, our results show that this presumed 'anti-prior protein' RNA is not derived from the antisense strand of the PrP gene. We detected a 4.5-kb 'antisense' RNA in brains of normal and scrapie infected hamsters at approximately the same level, as judged by northern analysis. The 4.5-kb RNA was polyadenylated and was not found in tissues other than brain. The fact that the 4.5-kb RNA was detected under stringent hybridization conditions initially suggested that the 'anti-PrP' RNA was transcribed from the opposite strand of the hamster PrP gene.
If indeed the 4.5-kb RNA were an antisense transcript of the PrP gene, then it should be absent or at least altered in mice homozygous for partially deleted PrP genes (Prn-p0~0)6. Using the above hamster probe A, which was shown to cross-hybridize to 'anti-PrP RNA from wild-type mouse brain, we detected the 4.5-kb RNA also in mice with Prp genes . Because probe A extended beyond the partial deletion in the PrP gene of PrP mice, we used an additional probe which lies entirely with in the deleted region. It de tected the same 4.5-kb RNA in both types of mice . These results unequivocally show that the 4.5-kb RNA is not derived from the opposite strand of the murine PrP gene; however, it appears to share considerable homology with the antisense strand of the PrP gene.
Our results confirm the presence of the 4.5-kb 'anti-PrP' RNA in two additional species, show that its concentration is not greatly altered after scrapie infection, and demonstrate that the RNA is not derived from the PrP gene. If the 4.5-kb mRNA, despite its being derived from a locus other than the PrP gene, encoded an 'anti-prior protein', this might have intriguing implications. It has recently been described for four independent proteins that peptides encoded by antisense sequences correspond structurally to binding proteins or receptors. Thus, a putative 'anti-prion protein' may be pertinent to the normal function of the prion protein.
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A host-encoded prion protein (PrP) is crucial in the pathogenesis of transmissible spongiform encephalopathies, such as scrapie, produce changes in complementary scrapie and scrapie-like diseases in man and PrP and anti-PrP mRNAs. The PrP gene is highly conserved in protein evolution and contains a single open reading. Several mutations in the human homologue prospective encoded protein, especially in PRNP gene were found in patients with view of its potential role in transmissible familial Gerstmann-Straussler-Scheinker's syndrome (GSS) and Creutzfeld-Jakob disease (CJD). While analysing PrP complementary DNA sequences with the DNA Strider computer program, I found a large overlapping ORF in the DNA strand opposite to the PrP transcriptional unit. The deduced amino acid sequence of the prospective encoded protein was unique. Its hydropathy plot is almost a mirror image of that of the PrP. For simplicity the name of anti-PrP is used in reference to this putative protein.
This ORF may be of biological importance first, because it is as large as the PrP ORF. Second, none of the differences in PrP sequences found in patients or animals resulted in additional stop codons in this ORF. It should be noted, however, that mutations found in GSS patients in codons 102 and 117 of the PRNP gene produced amino-acid changes in the anti-PrP, whereas mutations found in CJD patients in codon 178 and 200 did not. Third, there are ATG or CTG codons at the beginning of this ORF which could serve as translation initiation codons. Fourth, existing data on PrP gene expression do not exclude expression of the anti-PrP gene because only double-stranded DNA probes were used to detect PrP messenger RNA.
Obviously, only direct experimental analysis of infected and uninfected cells and tissues with single-stranded probes will show whether the anti-PrP gene is expressed.The expression of the anti-PrP gene would introduce a new player, present a different view of scrapie infection, and raise new and important questions. Do PrP and anti-PrP proteins interact in infected cells? Do RNA unwindases capable of modifying double-stranded RNA... Do GSS mutations, which, unlike CJD mutations, result in amino-acid changes in both proteins, explain the differences between the two? In conclusion, the existence of a long anti frame (ORF) located within a large exon. PrP ORF clearly warrants a search for the
1 Prusiner s. B. A Rev Microbiol 43 345-374 (1989). 2 . Marck c. Nucleic Acids Res. 18 1829 - 1836 (1988). 3 Kyte J. ; Doolittle R. F. J. molec. Bio 157 105-132(1982). 4 Hsiao c. Science250, 1587-1590 (1990). 5 Doh-ura K. Biochem. biophys Res. Commun.163 974-979 (1989). 6. Goldfarb L. G. Lancet t337 425 (1991). 7. Goldgaber D. Exp Neuro 106 204-206 ( 1990). 8. Prats Proc.natn. Acad sci. U.S.A. 86 1836-1840 (198g). 9. Srentani R. R. J. theor Bio 135 495 - 499 (1988). 10 Lamb. R. A, Creyfuss G. Nature 337 19 20 ( 1989).
The normal function of the pathogenicity-related protein, PrP (or prion protein), is unknown. We have characterized a candidate marsupial PrP gene.A particular feature of the marsupial[and mink] gene is the lack of a continuous ORF on the antisense strand, as is found in most eutherian PrP. We propose that antisense ORFs found in other species are artefactual.
[The authors argue in the body of the paper that 5% of eukcaryotic genes have these open reading frames in the anti-gene, that this is a consequence of low useage of anti-codons to the stop codons to speed synthesis, that the hydrophobicity reversal is a simple property of the genetic code, and that all known long ORFs are in phase. -- webmaster]
MVKSHIGSWILVLFVAMWSDVGLCKKRPKPGGGWNTGGSRYPGQ GSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGGWG QGGTHGQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGSDYEDRY YRENMHRYPNQVYYRPVDQYSNQNNFVHDCVNITVKEHTVTTTTKGENFTETDIKMME RVVEQMCITQYQRESQAYYQRGASVILFSSPPVILLISFLIFLIVG
Base composition 197 a 193 c 244 g 161 t ORIGIN 1 atggtgaaaa gccacatagg cagttggatc ctggttctct ttgtggccat gtggagtgac 61 gtgggcctct gcaagaagcg accaaaacct ggaggaggat ggaacactgg ggggagccga 121 tacccaggac agggcagtcc tggaggcaac cgttatccac ctcagggagg gggtggctgg 181 ggtcagcccc atggaggtgg ctggggccag cctcatggag gtggctgggg ccagcctcat 241 ggaggtggct ggggtcagcc ccatggtggt ggctggggac agccacatgg tggtggaggc 301 tggggtcaag gtggtaccca cggtcaatgg aacaaaccca gtaagccaaa aaccaacatg 361 aagcatgtgg caggagctgc tgcagctgga gcagtggtag ggggccttgg tggctacatg 421 ctgggaagtg ccatgagcag gcctcttata cattttggca gtgactatga ggaccgttac 481 tatcgtgaaa acatgcaccg ttaccccaac caagtgtact acaggccagt ggatcagtat 541 agtaaccaga acaactttgt gcatgactgt gtcaacatca cagtcaagga acacacagtc 601 accaccacca ccaaggggga gaacttcacc gaaactgaca tcaagatgat ggagcgagtg 661 gtggagcaaa tgtgcattac ccagtaccag agagaatccc aggcttatta ccaacgaggg 721 gcaagtgtga tcctcttctc ttcccctcct gtgatcctcc tcatctcttt cctcattttt 781 ctcatagtag gatag
gataggatgatactctttttactcctttctctactcctcctagtgtcctccccttctcttctcctagtgtgaacggggagcaaccattattcggaccctaa gagagaccatgacccattacgtgtaaacgaggtggtgagcgaggtagtagaactacagtcaaagccacttcaagagggggaaccaccaccaccactg acacacaaggaactgacactacaactgtgtcagtacgtgtttcaacaagaccaatgatatgactaggtgaccggacatcatgtgaaccaaccccattgc cacgtacaaaagtgctatcattgccaggagtatcagtgacggttttacatattctccggacgagtaccgtgaagggtcgtacatcggtggttccggggga tggtgacgaggtcgacgtcgtcgaggacggtgtacgaagtacaaccaaaaaccgaatgacccaaacaaggtaactggcacccatggtggaactggggt cggaggtggtggtacaccgacaggggtcggtggtggtaccccgactggggtcggtggaggtactccgaccggggtcggtggaggtactccgaccgggg tcggtggaggtaccccgactggggtcggtgggggagggactccacctattgccaacggaggtcctgacgggacaggacccatagccgaggggggtcac aaggtaggaggaggtccaaaaccagcgaagaacgtctccgggtgcagtgaggtgtaccggtgtttctcttggtcctaggttgacggatacaccgaaaagtggta
ctatcctactatgagaaaaatgaggaaagagatgaggaggatcacaggaggggaagagaagaggatcacacttgcccctcgttggtaataagcctgggattc tctctggtactgggtaatgcacatttgctccaccactcgctccatcatcttgatgtcagtttcggtgaagttctcccccttggtggtggtggtgactgtgtgttcc ttgactgtgatgttgacacagtcatgcacaaagttgttctggttactatactgatccactggcctgtagtacacttggttggggtaacggtgcatgttttcacgat agtaacggtcctcatagtcactgccaaaatgtataagaggcctgctcatggcacttcccagcatgtagccaccaaggccccctaccactgctccagctgcagc agctcctgccacatgcttcatgttggtttttggcttactgggtttgttccattgaccgtgggtaccaccttgaccccagcctccaccaccatgtggctgtccccag ccaccaccatggggctgaccccagccacctccatgaggctggccccagccacctccatgaggctggccccagccacctccatggggctgaccccagccaccc cctccctgaggtggataacggttgcctccaggactgccctgtcctgggtatcggctccccccagtgttccatcctcctccaggttttggtcgcttcttgcagaggc ccacgtcactccacatggccacaaagagaaccaggatccaactgcctatgtggcttttcaccat
Translation of anti-sense strand: note total absence of stop codons:
LSYYEKNEERDEEDHRRGREEDHTCPSLVISLGFSLVLGNAHLLHHSLHHLDVSFGEVLPLGGGGDCVFL DCDVDTVMHKVVLVTILIHWPVVHLVGVTVHVFTIVTVLIVTAKMYKRPAHGTSQHVATKAPYHCSSCSS SCHMLHVGFWLTGFVPLTVGTTLTPASTTMWLSPATTMGLTPATSMRLAPATSMRLAPATSMGLTPATPS LRWITVASRTALSWVSAPPSVPSSSRFWSLLAEAHVTPHGHKENQDPTAYVAFHH
W-KAT-AVGSWFSLWPCGVTWASARSDQNLEEDGTLGGADTQDRAVLEATVIHLREGVAGVSPMEVAGAS LMEVAGASLMEVAGVSPMVVAGDSHMVVEAGVKVVPTVNGTNPVSQKPT-SMWQELLQLEQW-GALVATC WEVP-AGLLYILAVTMRTVTIVKTCTVTPTKCTTGQWISIVTRTTLCMTVSTSQSRNTQSPPPPRGRTSP KLTSR-WSEWWSKCALPSTRENPRLITNEGQV-SSSLPLL-SSSSLSSFFS--D
5'3' Frame 3: sense strand, frameshifted by two
GEKPHRQLDPGSLCGHVE-RGPLQEATKTWRRMEHWGEPIPRTGQSWRQPLSTSGRGWLGSAPWRWLGPA SWRWLGPASWRWLGSAPWWWLGTATWWWRLGSRWYPRSMEQTQ-AKNQHEACGRSCCSWSSGRGPWWLHA GKCHEQASYTFWQ-L-GPLLS-KHAPLPQPSVLQASGSV--PEQLCA-LCQHHSQGTHSHHHHQGGELHR N-HQDDGASGGANVHYPVPERIPGLLPTRGKCDPLLFPSCDPPHLFPHFSHSRI
YPTMRKMRKEMRRITGGEEKRITLAPRW--AWDSLWYWVMHICSTTRSIILMSVSVKFSPLVVVVTVCSL TVMLTQSCTKLFWLLY-STGL-YTWLG-RCMFSR--RSS-SLPKCIRGLLMALPSM-PPRPPTTAPAAAA PATCFMLVFGLLGLFH-PWVPP-PQPPPPCGCPQPPPWG-PQPPP-GWPQPPP-GWPQPPPWG-PQPPPP -GG-RLPPGLPCPGYRLPPVFHPPPGFGRFLQRPTSLHMATKRTRIQLPMWLFT
3'5' Frame 3: anti-sense strand, frameshifted by two
ILL-EK-GKR-GGSQEGKRRGSHLPLVGNKPGILSGTG-CTFAPPLAPSS-CQFR-SSPPWWWW-LCVP- L-C-HSHAQSCSGYYTDPLACSTLGWGNGACFHDSNGPHSHCQNV-EACSWHFPACSHQGPLPLLQLQQL LPHASCWFLAYWVCSIDRGYHLDPSLHHHVAVPSHHHGADPSHLHEAGPSHLHEAGPSHLHGADPSHPLP EVDNGCLQDCPVLGIGSPQCSILLQVLVASCRGPRHSTWPQREPGSNCLCGFSP
Taken at face value, this leaves prions emerging more or less with the appearance of the vertebrates and not being part of any protein super-family. Normally, proteins appear from diverging gene duplications or domain assembly, but if so here, the orginal gene cannot yet be found. So we are left without the usual clues as to the biological function of normal prion protein or its domain.