Difference between revisions of "Part:BBa K2403000"

 
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Rev protein binds to RRE(Rev Response Element) and CRM1. High molecular mRNA with Rev protein can be transported into cytoplasm.  
 
Rev protein binds to RRE(Rev Response Element) and CRM1. High molecular mRNA with Rev protein can be transported into cytoplasm.  
  
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===Usage and Biology===
 
===Usage and Biology===
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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.
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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.
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===Characterization===
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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.
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As shown in the figure, 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.
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===Reference===
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[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041468/pdf/gku304.pdf
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Revision as of 17:58, 31 October 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 figure, 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]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041468/pdf/gku304.pdf


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 64
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 97
    Illegal BamHI site found at 281
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]