Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2010 Aug 23;285(44):33589–33601. doi: 10.1074/jbc.M110.117150

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

PMC Copyright notice

FIGURE 3. — Stabilization of No-AUG-FLAG-MS2 mRNA by tethered Pab1p does not require an interaction between Pab1p and eIF4G. a, tethering of Pab1p without the RRM1–2 domains stabilizes non-translated mRNA containing no AUG codon. W303 cells harboring pIT2071 (No-AUG-FLAG-MS2, left) or pIT2070 (No-AUG-FLAG, right) were transformed with a control p415TEF1p-FLAG-MS2 plasmid or with plasmids expressing the indicated FLAG-MS2-Pab1p fusion proteins. The cells were grown in SC-UraLeu medium containing 2% galactose (SC-Gal UraLeu medium). The cells were re-suspended in medium containing 2% glucose to inhibit transcription from the GAL1 promoter and harvested at the indicated times. RNA samples were prepared, and the levels of the remaining mRNA were determined using Northern blot analysis with DIG-labeled MPT4 probe. The relative mRNA levels of the samples were determined by comparison with a series of 2-fold dilutions of samples time 0 (first through third lanes). These dilutions were used as standard curves as described previously (25). b, tethering of Pab1p stabilizes non-translated mRNA. W303 cells harboring pIT2071 (No-AUG-FLAG-MS2) or pIT2070 (No-AUG-FLAG) were transformed with the control p415TEF1p-FLAG-MS2 plasmid or with plasmids expressing the indicated FLAG-MS2-Pab1p fusion proteins. The cells were grown in SC-Gal UraLeu medium, and mRNA stability was measured as described in a. Relative quantities are shown as the mean values of three independent experiments. The half-lives of mRNAs are shown as the mean values of three independent experiments with S.D. c, tethering of Pab1p stabilizes translated mRNA. W303 cells harboring pIT2069 (AUG-FLAG-MS2) or pIT2068 (AUG-FLAG) were transformed with the control p415TEF1p-FLAG-MS2 plasmid or with plasmids expressing the indicated FLAG-MS2-Pab1p fusion proteins. The cells were grown in SC-Gal UraLeu medium, and mRNA stability was measured as described in a. d, tethering of Pab1–34Cp stabilizes non-translated mRNA in eIF4G1-ΔN300 mutant YAS2071 (W303tif4631⁻tif4632⁻ ptif4631-ΔN300TRPCEN) cells. YAS2071 cells, harboring pIT2071 (No-AUG-FLAG-MS2) or pIT2070 (No-AUG-FLAG), were transformed with a plasmid containing a FLAG-MS2-Pab1–34C fusion gene or with a control p415TEF1p-FLAG-MS2 plasmid. The cells were grown in SC-Gal UraLeu medium, and mRNA stability was measured as in a. e, tethering of Pab1–34Cp stabilizes translated mRNA in eIF4G1-ΔN300 mutant. YAS2071 (W303tif4631⁻tif4632⁻ ptif4631-ΔN300TRPCEN) cells harboring pIT2069 (AUG-FLAG-MS2) or pIT2068 (AUG-FLAG) plasmids were transformed with plasmids encoding the FLAG-MS2-Pab1–34C fusion gene or with the control p415TEF1p-FLAG-MS2 plasmid. The cells were grown in SC-Gal UraLeu medium, and mRNA stability was measured as described in a.