(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.


Figure 6


Fig. 6. Ccr4 regulates CRT1 mRNA poly(A) tail length. (A) Poly(A) tail lengths were determined by LM-PAT analysis of total cellular RNA isolated from isogenic ccr4{Delta} (MT3768) and wild-type (BY4741), and from isogenic ccr4-1 (Y359) and wild-type (Y136) strain pairs, each either untreated or treated with 100 mM HU for 90 minutes. The estimated number of adenosine residues in the poly(A) tails from ccr4{Delta} strains are noted for specific transcripts. Notice that a segment of 12 adenosines is introduced by the linker primer into all cDNA produced, and thus this length represents the lowest limit of PCR product size from short tailed mRNAs. (B) CRT1 mRNA levels were determined by quantitative Real Time PCR of total RNA isolated from wild-type (BY4741), ccr4{Delta} (MT3768), dun1{Delta} (MT3769) or ccr4{Delta} dun1{Delta} (MT3772) strains that were either untreated or treated with 100 mM HU for 90 minutes. CRT1 mRNA levels were normalized to ACT1 mRNA levels and reported as the fraction of untreated wild-type CRT1 mRNA abundance. (C) Alteration of CRT1 mRNA poly(A) tail length in wild-type (Y136), ccr4-1 (Y359), ccr4{Delta} (Y294), caf1{Delta} (Y297), pan2{Delta} (YTP1), ccr4-1 pan2{Delta} (YTP2), ccr4{Delta} (MT3768) and wild-type (BY4741) strains by LM-PAT analysis of total RNA. (D) Crt1 protein abundance was determined in wild-type (BY4741), ccr4{Delta} (MT3768), dun1{Delta} (MT3769) and ccr4{Delta} dun1{Delta} (MT3772) strains that were either untreated or treated with 100 mM HU for 90 minutes. Crt1 was detected with polyclonal anti-Crt1 antibody. Strains crt1{Delta} (MT3770) and ccr4{Delta} dun1{Delta} crt1{Delta} (MT3773) served as negative controls for antibody specificity; equivalent protein loading was assessed with a monoclonal anti-Pgk1 antibody. (E) CRT1 mRNA poly(A) tail lengths were determined in wild-type (BY4741), ccr4{Delta} (MT3768), dun1{Delta} (MT3769), ccr4{Delta} dun1{Delta} (MT3772), pbp1{Delta} (MT3776), ccr4{Delta} pbp1{Delta} (MT3777), dun1{Delta} pbp1{Delta} (MT3842), ccr4{Delta} dun1{Delta} pbp1{Delta} (MT3843), chk1{Delta} (MT3784) and ccr4{Delta} chk1{Delta} (MT3785) strains by LM-PAT analysis of total RNA. The number of adenosine residues for major poly(A) tail species for specific transcripts are indicated. (F) The effect of CRT1 overexpression on tolerance to replication stress was determined with an integrated GAL-CRT1 allele in strains GAL1-CRT1 (MT3806), ccr4{Delta} GAL1-CRT1 (MT3807) and dun1{Delta} GAL1-CRT1 (MT3808). Sensitivity to HU was determined by serial dilution and spotting on indicated concentrations of HU under conditions of GAL1-CRT1 repression (+GLU) or induction (+GAL). Wild-type (BY4741), ccr4{Delta} (MT3768), dun1{Delta} (MT3769) and ccr4{Delta} dun1{Delta} (MT3772) strains were used as controls.