Inhibition of pre-mRNA splicing by synthetic branched nucleic acids.
The cellular transformation of a precursor mRNA (pre-mRNA) into its mature or functional form proceeds by way of a splicing reaction, in which the exons are ligated to form the mature linear RNA and the introns are excised as branched or lariat RNAs. We have prepared a series of branched compounds (bRNA and bDNA), and studied the effects of such molecules on the efficiency of mammalian pre-mRNA splicing in vitro. Y-shaped RNAs containing an unnatural L-2'-deoxycytidine unit (L-dC) at the 3' termini are highly stabilized against exonuclease hydrolysis in HeLa nuclear extracts, and are potent inhibitors of the splicing pathway. A bRNA containing internal 2'-O-methyl ribopyrimidine units and L-dC at the 3' ends was at least twice as potent as the most potent of the bRNAs containing no 2' modifications, with an IC50 of approximately 5 micro M. Inhibitory activity was maintained in a branched molecule containing an arabino-adenosine branchpoint which, unlike the native bRNAs, resisted cleavage by the lariat- debranching enzyme. The data obtained suggest that binding and sequestering of a branch recognition factor by the branched nucleic acids is an early event, which occurs prior to the first chemical step of splicing. Probably, an early recognition element preferentially binds to the synthetic branched molecules over the native pre-mRNA. As such, synthetic bRNAs may prove to be invaluable tools for the purification and identification of the putative branchpoint recognition factor.
Department of Chemistry, Otto Maass Chemistry Building, McGill University, 801 Sherbrooke St West, Montreal, QC, H3A 2K6, Canada.
Sandra
S
Carriero
Masad J
MJ
Damha
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D002457:Cell Extracts; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012326:RNA Splicing / Q000187:drug effects*; D020871:RNA Stability; D013379:Substrate Specificity
D002457:Cell Extracts; D009696:Nucleic Acids; D009841:Oligonucleotides; D012322:RNA Precursors; D000241:Adenosine