Difference between revisions of "Part:BBa K2403000"
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| − | + | ==Usage and Biology== | |
In eukaryotic cells, it is known that RNA is transcribed in the nucleus and then exported to the cytoplasm, a process mediated by nuclear export factors specific to each type of RNA. In the case of single stranded mRNA, the TAP / p15 complex binds and exports it to the cytoplasm. However, artificial RNAs designed for synthetic biology applications are often highly structured RNA which are not recognized by TAP / p15, and therefore a different pathway is required to export such target RNAs to the cytoplasm. | In eukaryotic cells, it is known that RNA is transcribed in the nucleus and then exported to the cytoplasm, a process mediated by nuclear export factors specific to each type of RNA. In the case of single stranded mRNA, the TAP / p15 complex binds and exports it to the cytoplasm. However, artificial RNAs designed for synthetic biology applications are often highly structured RNA which are not recognized by TAP / p15, and therefore a different pathway is required to export such target RNAs to the cytoplasm. | ||
In order to solve this problem, we used the Rev protein derived from HIV-1 which binds to and exports RNA containing the cis-acting RRE sequence (Rev Response Element). By fusing the RRE sequence to an RNA of interest and co-expressing Rev protein, complex RNA is exported. | In order to solve this problem, we used the Rev protein derived from HIV-1 which binds to and exports RNA containing the cis-acting RRE sequence (Rev Response Element). By fusing the RRE sequence to an RNA of interest and co-expressing Rev protein, complex RNA is exported. | ||
| − | + | ==Characterization== | |
In order to demonstrate the function of the Rev protein and RRE, we fused an RRE [1] to U6 snRNA, which has a complex structure and is not transported by the TAP / p15 pathway. In Xenopus oocytes, we ascertained whether this RNA was exported from the nucleus in a Rev-dependent manner. | In order to demonstrate the function of the Rev protein and RRE, we fused an RRE [1] to U6 snRNA, which has a complex structure and is not transported by the TAP / p15 pathway. In Xenopus oocytes, we ascertained whether this RNA was exported from the nucleus in a Rev-dependent manner. | ||
| − | As shown in the | + | As shown in the figure1 below, the U6-RRE RNA itself remains in the nucleus even 60 minutes after injection. On the other hand, the same RNA was exported to the cytoplasm when Rev was co-injected. From this data, we showed that a combination of RRE and Rev allowed target RNAs to be exported to the cytoplasm. |
<!-- (以下Rev アフリカツメガエルinjection result、and analysis fig) --> | <!-- (以下Rev アフリカツメガエルinjection result、and analysis fig) --> | ||
| − | [[File: | + | [[File:アフリカツメガエル結果injection.png|500px|thumb|center|'''Figure 1 : Result of the amount of expressing protein after injecting RNA to Xenopus oocytes. N is nucleus. C is cytosol.''']] |
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| + | ==Reference== | ||
| + | [1] Ichiro Taniguchi, Naoto Mabuchi and Mutsuhito Ohno (2014) HIV-1 Rev protein specifies the viral RNA export | ||
| + | pathway by suppressing TAP/NXF1 recruitment <i>Nucleic Acids Research</i>, 6645–665 | ||
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Latest revision as of 02:13, 2 November 2017
Rev protein
Rev protein binds to RRE(Rev Response Element) and CRM1. High molecular mRNA with Rev protein can be transported into cytoplasm.
Usage and Biology
In eukaryotic cells, it is known that RNA is transcribed in the nucleus and then exported to the cytoplasm, a process mediated by nuclear export factors specific to each type of RNA. In the case of single stranded mRNA, the TAP / p15 complex binds and exports it to the cytoplasm. However, artificial RNAs designed for synthetic biology applications are often highly structured RNA which are not recognized by TAP / p15, and therefore a different pathway is required to export such target RNAs to the cytoplasm.
In order to solve this problem, we used the Rev protein derived from HIV-1 which binds to and exports RNA containing the cis-acting RRE sequence (Rev Response Element). By fusing the RRE sequence to an RNA of interest and co-expressing Rev protein, complex RNA is exported.
Characterization
In order to demonstrate the function of the Rev protein and RRE, we fused an RRE [1] to U6 snRNA, which has a complex structure and is not transported by the TAP / p15 pathway. In Xenopus oocytes, we ascertained whether this RNA was exported from the nucleus in a Rev-dependent manner. As shown in the figure1 below, the U6-RRE RNA itself remains in the nucleus even 60 minutes after injection. On the other hand, the same RNA was exported to the cytoplasm when Rev was co-injected. From this data, we showed that a combination of RRE and Rev allowed target RNAs to be exported to the cytoplasm.
Reference
[1] Ichiro Taniguchi, Naoto Mabuchi and Mutsuhito Ohno (2014) HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment Nucleic Acids Research, 6645–665
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 64
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 97
Illegal BamHI site found at 281 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]