Maintenance and regulation of mRNA stability of the Saccharomyces cerevisiae OLE1 gene requires multiple elements within the transcript that act through translation-independent mechanisms.
The Saccharomyces cerevisiae OLE1 gene encodes a membrane-bound Delta-9 fatty acid desaturase, whose expression is regulated by unsaturated fatty acids through both transcriptional and mRNA stability controls. In fatty acid-free medium, the mRNA has a half-life of 10 +/- 1.5 min (basal stability) that drops to 2 +/- 1.5 min when cells are exposed to unsaturated fatty acids (regulated stability). A deletion analysis of elements within the transcript revealed that the sequences within the protein-coding region that encode transmembrane sequences and a part of the cytochrome b5 domain are essential for the basal stability of the transcript. Deletion of any of the three essential elements produced unstable transcripts and loss of regulated instability. By contrast, substitution of the 3'-untranslated region with that of the stable PGK1 gene did not affect the basal stability of the transcript and did not block regulated decay. Given that Ole1p is a membrane-bound protein whose activities are a major determinant of membrane fluidity, we asked whether membrane-associated translation of the protein was essential for basal and regulated stability. Insertion of stop codons within the transcript that blocked either translation of the entire protein or parts of the protein required for co-translation insertion of Ole1p had no effect. We conclude that the basal and regulated stability of the OLE1 transcript is resistant to the nonsense-mediated decay pathway and that the essential protein-encoding elements for basal stability act cooperatively as stabilizing sequences through RNA-protein interactions via a translation-independent mechanism.
Vemula
Muralikrishna
M
Division of Life Sciences, Department of Cell Biology and Neuroscience, Rutgers University, Nelson Laboratories, Piscataway, New Jersey 08854, USA.
Kandasamy
Pitchaimani
P
Oh
Chan-Seok
CS
Chellappa
Ramesh
R
Gonzalez
Carlos I
CI
Martin
Charles E
CE
D020413:3' Untranslated Regions; D018388:Codon, Terminator; D015786:Cytochromes b5 / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000502:physiology*; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013230:Stearoyl-CoA Desaturase; D013997:Time Factors; D014158:Transcription, Genetic
D020413:3' Untranslated Regions; D018388:Codon, Terminator; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D012313:RNA; D004247:DNA; D015786:Cytochromes b5; D044943:Fatty Acid Desaturases; D013230:Stearoyl-CoA Desaturase; C029627:delta-9 fatty acid desaturase