Heterologous HIV-nef mRNA trans-splicing: a new principle how mammalian cells generate hybrid mRNA and protein molecules.
Heterologous trans-splicing is a messenger RNA (mRNA) processing mechanism, that joins RNA segments from separate transcripts to generate functional mRNA molecules. We present here for the first time experimental evidence that the proximal segment of the HIV-nef RNA segment can be trans-spliced to both viral (e.g. SV40 T-antigen) and cellular transcripts. Following either microinjection of in vitro synthesized HIV-nef and SV40 T-antigen pre-mRNA or transfection of the HIV-nef DNA into T-antigen positive cells (CV1-B3; Cos7), it was found that recipient cells synthesized HIV-nef/T-antigen hybrid mRNA and protein molecules. To generate the hybrid mRNA, the cells utilized the 5' cryptic splice sites of the HIV-nef (5'cry 66 and 5'cry 74) and the SV40 T/t-antigen 3' splice site. To demonstrate that heterologous trans-splicing also occurs between the HIV-nef RNA and cellular transcripts, a cDNA library was established from HIV-nef positive CV1-B3 cells (CV1-B3/13 cells) and screened for hybrid mRNA molecules. Reverse transcription-PCR and Northern blot analysis revealed that a significant portion of the HIV-nef transcript is involved in heterologous trans-splicing. To date, eight independent HIV-nef/cellular hybrid mRNA molecules have been identified. Five of these isolates contain segments from known cellular genes (KIAA1454, PTPkappa, Alu and transposon gene families), while three hybrid segments contain sequences of not yet known cellular genes (genes 1-3).
Caudevilla
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Institut für Molekularbiologie und Biochemie, Freie Universität Berlin, Arnimallee 22, D-14195, Berlin, Germany.
Da Silva-Azevedo
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L
Berg
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Guhl
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E
Graessmann
M
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Graessmann
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A
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D002478:Cells, Cultured / Q000821:virology; D002678:Chimera; D015693:Gene Products, nef / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D008322:Mammals / Q000821:virology*; D008845:Microinjections; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012326:RNA Splicing; D012333:RNA, Messenger; D054311:nef Gene Products, Human Immunodeficiency Virus
D000952:Antigens, Polyomavirus Transforming; D015693:Gene Products, nef; D012333:RNA, Messenger; D054311:nef Gene Products, Human Immunodeficiency Virus