Nucleus export reporter system
The present invention relates to reporter systems for RNA export, methods for searching for molecules which influence RNA export, and a method, based on these methods, for detecting a viral infection.
1. A method for detecting RNA export from the cell nucleus of a eukaryotic target cell, comprising
(a) providing a polynucleotide which codes for a reporter protein and which further comprises
(i) a cis-active RNA export signal which is in operative linkage with the polynucleotide
(ii) a functional 5′ splice donor without a 3′ splice acceptor,
(iii) an A/T content that is greater than 50%, which reduces the metabolic stability of a reporter RNA which encodes said reporter protein in the cell nucleus;
(b) introducing the polynucleotide into the cell nucleus of the target cell so that it is present therein in operative linkage with a transcription control sequence, and that upon transcription of the polynucleotide there is generation of a transcript in which the segment of the transcript coding for the reporter protein cannot be subjected to any efficient splicing process;
(c) providing a trans-active export factor;
(d) transcribing the polynucleotide; and
(e) determining whether the resulting transcript is exported from the cell nucleus.
2. The method according to claim 1 , wherein the polynucleotide additionally comprises a choice of codons which is at least partially adapted to the choice of codons of exported viral RNA, leading to a reduction of the metabolic stability of a reporter RNA in the cell nucleus.
3. The method according to claim 1 , wherein the polynucleotide additionally comprises a choice of which is at least partially adapted to the choice of codons of HIV-1 (human immunodeficiency virus 1), leading to a reduction of the metabolic stability of a reporter RNA in the cell nucleus.
4. The method according to claim 1 , wherein the polynucleotide which codes for the reporter protein is a synthetically produced polynucleotide.
5. The method according to claim 1 , wherein the reporter protein is a fluorescent protein.
6. The method according to claim 5 , wherein the fluorescent protein is GFP (green fluorescent protein), BFP (blue fluorescent protein), RFP (red fluorescent protein), YFP (yellow fluorescent protein), eGFP (enhanced green fluorescent protein), eBFP (enhanced blue fluorescent protein), eRFP (enhanced red fluorescent protein), eYFP (enhanced yellow fluorescent protein) or hrGFP.
7. The method according to claim 1 , wherein the reporter protein comprises a detectable enzymatic activity.
8. The method according to claim 7 , wherein the reporter protein is LUC (luciferase), AP (alkaline phosphatase), SEAP (secretory alkaline phosphatase) or CAT (chloramphenicol acetyltransferase).
9. The method according to claim 1 , wherein the reporter protein is an immunologically detectable protein.
10. The method according to claim 1 , wherein the reporter protein is a selection marker.
11. The method according to claim 1 , wherein the reporter protein is encoded by a regulatory gene which regulates the expression of other genes.
12. The method according to claim 1 , wherein the polynucleotide comprises a cis-active RNA export element.
13. The method according to claim 12 , wherein the cis-active RNA export element is a constitutive RNA export element.
14. The method according to claim 13 , wherein the cis-active RNA export element is MPMV CTE (Mason Pfizer monkey virus constitutive transport element), RSV CTE (Rous sarcoma virus constitutive transport element) or SRV CTE (simian retrovirus constitutive transport element).
15. The method according to claim 12 , wherein the cis-active RNA export element is an export element which is recognized by a viral export factor.
16. The method according to claim 15 , wherein the cis-active RNA export element is HIV-1 RRE (human immunodeficiency virus 1 Rev responsive element), HIV-2 RRE (human immunodeficiency virus 2 Rev responsive element), SIV RRE (simian immunodeficiency virus Rev responsive element), HTLV-I (human T-cell leukemia virus I) Rex responsive element or HTLV-II (human T-cell leukemia virus II) Rex responsive element.
17. The method according to claim 11 , wherein the regulatory gene is a transcription factor.
18. The method according to claim 1 , comprising employing a viral cis-active RNA export element.
19. The method according to claim 1 , wherein the A/T content is increased throughout a reporter construct.
20. The method according to claim 1 , wherein the eukaryotic cell is a mammalian cell.
21. A method for detecting RNA export from the cell nucleus of a eukaryotic target cell, comprising
(a) providing a polynucleotide which codes for a reporter protein and which further comprises
(i) a cis-active RNA export signal which is in operative linkage with the polynucleotide
(ii) a functional 5′ splice donor without a 3′ splice acceptor at 3′ from said cis-active RNA export signal
(iii) an A/T content that is greater than 50%, which reduces the metabolic stability of a reporter RNA which encodes said reporter protein in the cell nucleus;
(b) introducing the polynucleotide into the cell nucleus of the target cell so that it is present therein in operative linkage with a transcription control sequence, and that upon transcription of the polynucleotide there is generation of a transcript in which the segment of the transcript coding for the reporter protein cannot be subjected to any efficient splicing process;
(c) providing a trans-active export factor;
(d) transcribing the polynucleotide; and
(e) determining whether the resulting transcript is exported from the cell nucleus.