RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection.
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.
Kleinschmidt
A M
AM
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Pederson
T
T
D001483:Base Sequence; D002460:Cell Line; D006801:Humans; D008722:Methods; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping; D012315:RNA Caps / Q000235:genetics; D012322:RNA Precursors / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional; D012342:RNA, Small Nuclear / Q000235:genetics*; D012261:Ribonucleoproteins / Q000235:genetics*; D017411:Ribonucleoproteins, Small Nuclear; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D012315:RNA Caps; D012322:RNA Precursors; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear