Direct introduction and transient expression of capped and non-capped RNA in Saccharomyces cerevisiae.
We report the introduction of functional RNA molecules into yeast spheroplasts. Plasmids containing the firefly luciferase coding region were transcribed to yield RNAs suitable for introduction into yeast cells and direct assay of their translation products. The 5' noncoding regions of the RNAs were derived either from the 5' noncoding regions of firefly luciferase, poliovirus, or yeast virus-like-particle (VLP) L-A or M1 RNAs. Capped and non-capped mRNAs were made by T7 RNA polymerase-directed transcription and introduced into yeast spheroplasts. The peak time of luciferase transient expression from introduced RNAs was 2-4 h after their introduction. In contrast, transient expression of luciferase from a non-replicative, luciferase-encoding plasmid introduced into the cells was maximal at 16 h. For capped mRNAs, luciferase activity increased linearly with transcript amount for both yeast and human (HeLa) cells. Although non-capped luciferase mRNAs were expressed more efficiently following introduction into yeast than into HeLa cells, the 5' noncoding sequences from yeast double-stranded (ds)RNA VLP RNAs conferred no greater apparent cap-independence than non-VLP RNA sequences in this transient expression assay. The RNA transient expression system will allow the study of translation of capped and non-capped RNAs in yeast cells and of the replicative cycle of yeast virus-like RNA genomes.
Russell
P J
PJ
Department of Molecular, Cellular and Developmental Biology, Howard Hughes Medical Institute, University of Colorado, Boulder 80309.
Hambidge
S J
SJ
Kirkegaard
K
K
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000235:genetics; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D017955:Poliovirus / Q000235:genetics; D012315:RNA Caps / Q000235:genetics*; D012331:RNA, Fungal / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013604:T-Phages / Q000201:enzymology; D014158:Transcription, Genetic; D014162:Transfection
D015226:Dinucleoside Phosphates; D012315:RNA Caps; D012331:RNA, Fungal; D012333:RNA, Messenger; D012367:RNA, Viral; C062229:7-methyl-diguanosine triphosphate; D008156:Luciferases; D012321:DNA-Directed RNA Polymerases