Limits...
Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast.

Gnanasundram SV, Koš M - Mol. Biol. Cell (2014)

Bottom Line: Various approaches using different repressible promoters, inducible degrons, or their combinations were developed.The target protein does not require a tag, and its elimination is several fold faster compared with standard promoter shutoff systems.A depletion time of <40 min was sufficient to achieve a robust phenotype.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.

Show MeSH
Analysis of RNA processing and splicing during depletion of Has1p and Prp19p using different cassettes. Total RNA was isolated at the indicated time points following the addition of doxycycline. (A) Analysis of 20S pre-rRNA synthesis in TetO7-Has1, TetO7-Ubi-Leu-Has1, and TetO7-tc-Ubi-Leu-Has1 strains. Total RNA was separated in a 1% glyoxal agarose gel and blotted, and the membrane was hybridized with oligonucleotides complementary to 20S pre-rRNA and SCR1 RNA, which was used as a loading control. (B) Analysis of U3 snoRNA splicing in TetO7-Prp19 and TetO7-Ubi-Leu-Prp19 strains. The total RNA was separated in 6% denaturing polyacrylamide gel and blotted, and the membranes were hybridized with oligonucleotides complementary to the U3 snoRNA intron and 5S rRNA (loading control).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4325845&req=5

Figure 5: Analysis of RNA processing and splicing during depletion of Has1p and Prp19p using different cassettes. Total RNA was isolated at the indicated time points following the addition of doxycycline. (A) Analysis of 20S pre-rRNA synthesis in TetO7-Has1, TetO7-Ubi-Leu-Has1, and TetO7-tc-Ubi-Leu-Has1 strains. Total RNA was separated in a 1% glyoxal agarose gel and blotted, and the membrane was hybridized with oligonucleotides complementary to 20S pre-rRNA and SCR1 RNA, which was used as a loading control. (B) Analysis of U3 snoRNA splicing in TetO7-Prp19 and TetO7-Ubi-Leu-Prp19 strains. The total RNA was separated in 6% denaturing polyacrylamide gel and blotted, and the membranes were hybridized with oligonucleotides complementary to the U3 snoRNA intron and 5S rRNA (loading control).

Mentions: The results presented so far show that growth in the strains carrying the TetO7-Ubi-Leu cassette slows several hours before growth in the strains carrying the standard TetO7 promoter. While convenient, monitoring of cell growth allows only a gross measure of the defects resulting from the reduced levels of the protein studied. The change in the growth rate cannot be used to reliably time the first appearance of a phenotype resulting from the target protein depletion. Therefore we took advantage of the easily detectable RNA-processing phenotypes of Has1p and Prp19p and determined the time of appearance of first RNA-processing defects in the depleted strains. The Has1p protein is essential for rRNA processing, in particular for production of the 20S pre-rRNA intermediate (Emery et al., 2004). Therefore we used Northern blotting to determine the shortest depletion time required to abolish 20S pre-rRNA synthesis. Surprisingly, only 40 min after the start of depletion, the 20S pre-rRNA levels were clearly reduced and synthesis of 20S was barely detectable after 60 min in the TetO7-Ubi-Leu-Has1 strain (Figure 5A). The inclusion of the tc aptamer had only a mild positive effect on the depletion (Figure 5A, bottom panel). In comparison, in the standard TetO7-Has1 strain, the reduction in the 20S pre-rRNA levels was noticeable after only 240 min, with the 20S pre-rRNA signal comparable with the 40-min time point in the TetO7-Ubi-Leu-Has1 strain. Therefore the depletion time required to achieve a robust measurable phenotype was reduced sixfold in the TetO7-Ubi-Leu-Has1 strain. The Prp19p is an essential splicing factor. Northern blotting was used to detect unspliced, intron-containing U3 small nucleolar RNA (snoRNA) at different time points during depletion (Figure 5B). It is important to note that the unspliced U3 snoRNA (similar to unspliced pre-mRNAs) can also be detected in normally growing wild-type cells, explaining the weak signal present in all the analyzed time points. Increased levels of the unspliced U3 were detected after 1–2 h in the TetO7-Ubi-Leu-Prp19 strain compared with 8 h in TetO7-Prp19 strain. Thus, also in the case of Prp19p, the appearance of the first processing defects resulting from the protein depletion is largely accelerated.


Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast.

Gnanasundram SV, Koš M - Mol. Biol. Cell (2014)

Analysis of RNA processing and splicing during depletion of Has1p and Prp19p using different cassettes. Total RNA was isolated at the indicated time points following the addition of doxycycline. (A) Analysis of 20S pre-rRNA synthesis in TetO7-Has1, TetO7-Ubi-Leu-Has1, and TetO7-tc-Ubi-Leu-Has1 strains. Total RNA was separated in a 1% glyoxal agarose gel and blotted, and the membrane was hybridized with oligonucleotides complementary to 20S pre-rRNA and SCR1 RNA, which was used as a loading control. (B) Analysis of U3 snoRNA splicing in TetO7-Prp19 and TetO7-Ubi-Leu-Prp19 strains. The total RNA was separated in 6% denaturing polyacrylamide gel and blotted, and the membranes were hybridized with oligonucleotides complementary to the U3 snoRNA intron and 5S rRNA (loading control).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4325845&req=5

Figure 5: Analysis of RNA processing and splicing during depletion of Has1p and Prp19p using different cassettes. Total RNA was isolated at the indicated time points following the addition of doxycycline. (A) Analysis of 20S pre-rRNA synthesis in TetO7-Has1, TetO7-Ubi-Leu-Has1, and TetO7-tc-Ubi-Leu-Has1 strains. Total RNA was separated in a 1% glyoxal agarose gel and blotted, and the membrane was hybridized with oligonucleotides complementary to 20S pre-rRNA and SCR1 RNA, which was used as a loading control. (B) Analysis of U3 snoRNA splicing in TetO7-Prp19 and TetO7-Ubi-Leu-Prp19 strains. The total RNA was separated in 6% denaturing polyacrylamide gel and blotted, and the membranes were hybridized with oligonucleotides complementary to the U3 snoRNA intron and 5S rRNA (loading control).
Mentions: The results presented so far show that growth in the strains carrying the TetO7-Ubi-Leu cassette slows several hours before growth in the strains carrying the standard TetO7 promoter. While convenient, monitoring of cell growth allows only a gross measure of the defects resulting from the reduced levels of the protein studied. The change in the growth rate cannot be used to reliably time the first appearance of a phenotype resulting from the target protein depletion. Therefore we took advantage of the easily detectable RNA-processing phenotypes of Has1p and Prp19p and determined the time of appearance of first RNA-processing defects in the depleted strains. The Has1p protein is essential for rRNA processing, in particular for production of the 20S pre-rRNA intermediate (Emery et al., 2004). Therefore we used Northern blotting to determine the shortest depletion time required to abolish 20S pre-rRNA synthesis. Surprisingly, only 40 min after the start of depletion, the 20S pre-rRNA levels were clearly reduced and synthesis of 20S was barely detectable after 60 min in the TetO7-Ubi-Leu-Has1 strain (Figure 5A). The inclusion of the tc aptamer had only a mild positive effect on the depletion (Figure 5A, bottom panel). In comparison, in the standard TetO7-Has1 strain, the reduction in the 20S pre-rRNA levels was noticeable after only 240 min, with the 20S pre-rRNA signal comparable with the 40-min time point in the TetO7-Ubi-Leu-Has1 strain. Therefore the depletion time required to achieve a robust measurable phenotype was reduced sixfold in the TetO7-Ubi-Leu-Has1 strain. The Prp19p is an essential splicing factor. Northern blotting was used to detect unspliced, intron-containing U3 small nucleolar RNA (snoRNA) at different time points during depletion (Figure 5B). It is important to note that the unspliced U3 snoRNA (similar to unspliced pre-mRNAs) can also be detected in normally growing wild-type cells, explaining the weak signal present in all the analyzed time points. Increased levels of the unspliced U3 were detected after 1–2 h in the TetO7-Ubi-Leu-Prp19 strain compared with 8 h in TetO7-Prp19 strain. Thus, also in the case of Prp19p, the appearance of the first processing defects resulting from the protein depletion is largely accelerated.

Bottom Line: Various approaches using different repressible promoters, inducible degrons, or their combinations were developed.The target protein does not require a tag, and its elimination is several fold faster compared with standard promoter shutoff systems.A depletion time of <40 min was sufficient to achieve a robust phenotype.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.

Show MeSH