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Results: 6

1.
Figure 1.

Figure 1. From: The Rpb7p subunit of yeast RNA polymerase II plays roles in the two major cytoplasmic mRNA decay mechanisms.

Rpb7p-GFP colocalizes with Dcp2p-RFP in PBs. Cells expressing RPB7-GFP and DCP2-RFP (yRL11) and cells expressing RPB7-GFP and RPB4-RFP (yMS8) were starved and then visualized microscopically, as described previously (Lotan et al., 2005). +CHX panels, cyclohexamide-treated cells (Lotan et al., 2005).

Rona Lotan, et al. J Cell Biol. 2007 September 24;178(7):1133-1143.
2.
Figure 5.

Figure 5. From: The Rpb7p subunit of yeast RNA polymerase II plays roles in the two major cytoplasmic mRNA decay mechanisms.

RPB7 affects the P bodies' number. Wild-type cells and their isogenic rpb7 ts derivatives, expressing DHH1-GFP (A and C) or PAT1-GFP (B) (see Table I), were allowed to proliferate at 30°C in a selective medium until late-log phase. Cultures were then shifted to 42°C for 45 min (A) or 60 min (B). Cells were washed 3× with water before they were visualized by fluorescent microscopy. (C) Average number of DHH1-GFP PBs per cell. Error bars indicate the SD from the mean values.

Rona Lotan, et al. J Cell Biol. 2007 September 24;178(7):1133-1143.
3.
Figure 3.

Figure 3. From: The Rpb7p subunit of yeast RNA polymerase II plays roles in the two major cytoplasmic mRNA decay mechanisms.

Rpb7p is required for efficient deadenylation and subsequent decay of both PBF and non-PBF mRNA. Cells were shifted rapidly to 42°C to block transcription and to inactivate the ts Rpb7-28p. RNA was extracted at the indicated time points post-shift and analyzed by the polyacrylamide Northern technique (Sachs and Davis, 1989). The same membrane was hybridized sequentially with the indicated probes. Actual lengths (relative to size marker) of the indicated mRNAs (without the poly(A) tail) are indicated in parentheses next to the gene name on the left. Lane “Δ(A)n” shows the position of fully deadenylated RNA. This RNA was obtained by hybridizing RNA sample from time point 0 with oligo(dT), followed by digestion of the poly(A) tail by RNase H. The star indicates the time point when deadenylation seems to be complete. We suspect that in some cases complete deadenylation occurred in between two time points. In these particular cases, the star is placed between lanes. SCR1 RNA (a Pol III transcript) serves to demonstrate equal loading.

Rona Lotan, et al. J Cell Biol. 2007 September 24;178(7):1133-1143.
4.
Figure 2.

Figure 2. From: The Rpb7p subunit of yeast RNA polymerase II plays roles in the two major cytoplasmic mRNA decay mechanisms.

PBF and non-PBF mRNAs decay abnormally slowly at the nonpermissive temperature in rpb7 ts mutants. (A) At the permissive temperature, rpb7-29 and WT exhibit similar mRNA decay kinetics. The indicated strains were proliferated at 30°C until mid-log phase. 1, 10, phenanthroline was added to block transcription (Grigull et al., 2004; Lotan et al., 2005). Decay kinetics was determined by monitoring mRNA levels at the indicated time points post-transcription block, using Northern analysis with the indicated probes. rRNA serves to demonstrate equal loading. Half-lives (designated T1/2) were determined as described previously (Lotan et al., 2005) and the ratios between T1/2 of the mutant and that of the wild-type [T1/2 (m)/T1/2 (WT)] are indicated on the right. Lengths of the open reading frames are indicated in parentheses next to the gene name on the left. (B) At the nonpermissive temperature, rpb7-29 cells exhibit defective mRNA decay. Cells were challenged simultaneously with 1, 10, phenanthroline and shifted to 42°C and mRNA decay was determined as in A. (C) rpb7-28 cells are defective in mRNA decay. Cells were proliferated at 30°C till mid-log phase and then shifted abruptly to 42°C (no drug was added). The high temperature led both to inactivation of Rpb7-28p ts and to transcription arrest (Lotan et al., 2005). mRNA decay was determined as in A. (D) Decay kinetics of YEF3 mRNA in WT and rpb7-28 cells was determined as described previously (Lotan et al., 2005).

Rona Lotan, et al. J Cell Biol. 2007 September 24;178(7):1133-1143.
5.
Figure 6.

Figure 6. From: The Rpb7p subunit of yeast RNA polymerase II plays roles in the two major cytoplasmic mRNA decay mechanisms.

Rpb7p interacts with Pat1p; the extent of interaction correlates with the capacity of Rpb7p to stimulate mRNA decay. (A) High copy effect of the indicated genes on the proliferation capacity of pat1Δ cells or their WT counterparts. Two strain backgrounds were examined. WT strain (yMC229) and its isogenic pat1Δ strain (yMC363) are designated “Strain A”; WT (yMC269) or its pat1Δ derivative yMC272 are designated “Strain B”. Cells carrying high copy plasmid expressing the indicated genes were spotted in fivefold serial dilutions onto selective plates. Cells were allowed to grow at the nonpermissive temperature for pat1Δ cells, 36°C. (B) Two-hybrid interactions between Rpb7p as the bait and genes whose products are involved in mRNA decay as prey were performed as described in Materials and methods. Only 6 of 20 genes tested are shown as indicated around the plate. The other genes that did not exhibit interactions with the Rpb7p-DBD and are not shown here are Lsm1p, Lsm3p–Lsm7p, Ccr4p, Not1p, Caf1p, Pan2p, Pan3p, Pop2p, Edc1p, Edc2p, Edc3p, and Pab1p. Lsm2p showed weak interaction (not depicted). Equal amount of cells, carrying the indicated prey plasmids, were streaked onto an indicator plate as described in Materials and methods. We then verified that the growth on the indicator plates was dependent on both plasmids by evicting one plasmid at a time from the positive clones (not depicted). (C) Two-hybrid interaction between various mutant forms of Rpb7p-DBD and Pat1p-AD. Cells were spotted in threefold serial dilutions, starting with 106 cells/spot, on an indicator plate as in B (selective conditions). Plates were incubated for 3 d at the indicated temperatures. To demonstrate spotting of equal amounts of cells, cells were spotted on a nonselective plate that allowed growth of cells that carry both the bait and prey plasmids independently of the two-hybrid interactions (using medium lacking only leucine and tryptophane). β-galactosidase (β-Gal) values, quantitative means to determine interactions (Uetz et al., 2000), are indicated on the right. They reflect the average values determined from two independent experiments (variations were <15%). Background values obtained with cells expressing only the bait plasmid were subtracted. Levels of the various mutant forms of Rpb7p-DBD were found to be comparable to that of the WT Rpb7-DBD (not depicted).

Rona Lotan, et al. J Cell Biol. 2007 September 24;178(7):1133-1143.
6.
Figure 4.

Figure 4. From: The Rpb7p subunit of yeast RNA polymerase II plays roles in the two major cytoplasmic mRNA decay mechanisms.

Rpb7p is required for efficient execution of the 3′ to 5′ degradation of MFA2pG mRNAs. (A) rpb7-28 cells contain high level of MFA2pG degradation intermediate. WT and rpb7-28 cells expressing Tet-Off-MFA2pG (yVB32 and yVB34) were harvested in mid-log phase. MFA2pG mRNA (designated F.L.) and its degradation intermediate fragment (designated Frag.) were examined using PAGE Northern technique as in Fig. 3, except that electrophoresis was shorter to allow detection of the fragment. MFA2pG-specific probe was used (see Materials and methods). As a control for the probe specificity, RNA from cells lacking the plasmid was also analyzed (“No plasmid” lane). SCR1 RNA (a Pol III transcript) serves to demonstrate equal loading. The ratio between the fragment radioactivity and that of the full length was obtained by PhosphorImager technology and is indicated at the bottom. (B) Deletion of XRN1 displays synthetic lethality in combination with rpb7-29 and rpb7-34, synthetic sickness in combination with rpb7-28, but not with rpb7-26. A strain lacking XRN1 and carrying pRPB7∷URA3 in lieu of RPB7 (yMC414) was transformed with HIS3 plasmid carrying RPB7 or rpb7 mutants as indicated on the left. Synthetic lethality assay was performed as described in Materials and methods. In parallel, cells were spotted on a plate lacking 5-FOA (SC lacking histidine), designated “non-selective medium”, to demonstrate spotting of equal number of cells. (C) rpb7-29 cells are defective in 3′ to 5′ exonuclease activity. WT and rpb7-29 cells were grown to mid-log phase at 30°C. 2 μg/ml doxycycline was added to block transcription of Tet-Off-MFA2pG mRNA. Immediately thereafter, 100 μg/ml cyclohexamide was added in order to block decapping and consequently block Xrn1p activity. Immediately thereafter, cells were shifted to 37°C and samples were harvested at the indicated time points and their RNAs were analyzed by PAGE Northern as in A. Lane designated “No plasmid” used as a control as in A. SCR1 RNA was used for normalization. Short exposure of the membrane is shown in the top autoradiogram. Longer exposure of the fragment is shown in the bottom autoradiogram, for better detection. (D) 3′ to 5′ decay kinetics of the fragment. Fragment level was monitored by PhosphorImager, normalized to that of SCR1 RNA, and plotted as a function of time post-transcription and decapping block. A band intensity at time 0 was defined as 100%, and intensities at other time points were calculated relative to time 0. Similar results were obtained by four hybridization analyses. Variations were <15%.

Rona Lotan, et al. J Cell Biol. 2007 September 24;178(7):1133-1143.

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