Substitution of pre-mRNA with phosphorothioate linkages reveals a new splicing-related reaction.
In vitro transcription in the presence of a nucleoside 5'-O-(1-thiotriphosphate) has been used to prepare pre-mRNA analogues of the small intron of a rabbit beta-globin gene and flanking exon sequences. Incubation of transcripts prepared with adenosine 5'-O-(1-thiotriphosphate) in a HeLa cell nuclear extract showed that the presence of the thionucleotide in a transcript inhibited splicing, but a novel product was formed by cleavage three nucleotides upstream of the 3' splice site. This product was formed with the same kinetics as the intermediates of a normal splicing reaction, and its formation depended on the presence of intact small nuclear RNAs U1, U2, and U6. We conclude that activation of 3' splice site-proximal sequences need not be linked to exon ligation.
Griffiths
A D
AD
Department of Biochemistry, University of Leicester, United Kingdom.
Potter
B V
BV
Eperon
I C
IC
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000378:metabolism; D005091:Exons; D005092:Exonucleases / Q000378:metabolism; D005914:Globins / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D011817:Rabbits; D013604:T-Phages / Q000201:enzymology; D013873:Thionucleotides / Q000378:metabolism*; D014158:Transcription, Genetic
D000345:Affinity Labels; D012322:RNA Precursors; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D013873:Thionucleotides; C022570:adenosine 5'-O-(2-thiotriphosphate); D000255:Adenosine Triphosphate; D005914:Globins; D012321:DNA-Directed RNA Polymerases; D004722:Endoribonucleases; D005092:Exonucleases; C022650:spleen exonuclease; C022543:ribonuclease T(2)