Abstract:
:We present a method for studying RNA processing and ribonucleoprotein assembly in vivo, by using RNA synthesized in vitro. SP6-transcribed 32P-labeled U2 small nuclear RNA precursor molecules were introduced into cultured human 293 cells by calcium phosphate-mediated uptake, as in standard DNA transfection experiments. RNase protection mapping demonstrated that the introduced pre-U2 RNA underwent accurate 3' end processing. The introduced U2 RNA was assembled into ribonucleoprotein particles that reacted with an antibody specific for proteins known to be associated with the U2 small nuclear ribonucleoprotein particle. The 3' end-processed, ribonucleoprotein-assembled U2 RNA accumulated in the nuclear fraction. When pre-U2 RNA with a 7-methylguanosine group at the 5' end was introduced into cells, it underwent conversion to a 2,2,7-trimethylguanosine cap structure, a characteristic feature of the U-small nuclear RNAs. Pre-U2 RNA introduced with an adenosine cap (Ap-ppG) also underwent processing, small nuclear ribonucleoprotein assembly, and nuclear accumulation, establishing that a methylated guanosine cap structure is not required for these steps in U2 small nuclear ribonucleoprotein biosynthesis. Beyond its demonstrated usefulness in the study of small nuclear ribonucleoprotein biosynthesis, RNA transfection may be of general applicability to the investigation of eukaryotic RNA processing in vivo and may also offer opportunities for introducing therapeutically targeted RNAs (ribozymes or antisense RNA) into cells.
journal_name
Proc Natl Acad Sci U S Aauthors
Kleinschmidt AM,Pederson Tdoi
10.1073/pnas.87.4.1283subject
Has Abstractpub_date
1990-02-01 00:00:00pages
1283-7issue
4eissn
0027-8424issn
1091-6490journal_volume
87pub_type
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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更新日期:2016-02-09 00:00:00