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The mRNA encoding the yeast ARE-binding protein Cth2 is generated by a novel 3' processing pathway.

Ciais D, Bohnsack MT, Tollervey D - Nucleic Acids Res. (2008)

Bottom Line: CTH2 carries a consensus ARE element and levels of the pre-mRNA and mRNA were elevated by mutation of the ARE or inactivation of the nuclear 5'-exonuclease Rat1.We propose that CTH2 mRNA is processed from a 3'-extended primary transcript by the exosome, TRAMP and Nrd1/Nab3/Sen1 complexes.This unusual pathway may allow time for nuclear, ARE-mediated regulation of CTH2 levels involving Rat1.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, EH9 3JR, UK.

ABSTRACT
Microarray analyses of mRNAs over-expressed in strains lacking the nuclear exosome component Rrp6 identified the transcript encoding the ARE-binding protein Cth2, which functions in cytoplasmic mRNA stability. Subsequent northern analyses revealed that exosome mutants accumulate a 3'-extended transcript at the expense of the mature CTH2 mRNA. The 3' ends of the CTH2 mRNA were mapped to a [GU(3-5)](5) repeat, unlike any previously characterized polyadenylation site. CTH2 mRNA accumulation was not inhibited by mutations in 3'-cleavage and polyadenylation factors, Rna14, Rna15 and Pap1, which block accumulation of other mRNAs. The 3'-extended CTH2 pre-mRNA strongly accumulated in strains with mutations in the TRAMP4 polyadenylation complex or the Nrd1/Nab3/Sen1 complex, and contains multiple Nrd1 and Nab3 binding sites. CTH2 carries a consensus ARE element and levels of the pre-mRNA and mRNA were elevated by mutation of the ARE or inactivation of the nuclear 5'-exonuclease Rat1. We propose that CTH2 mRNA is processed from a 3'-extended primary transcript by the exosome, TRAMP and Nrd1/Nab3/Sen1 complexes. This unusual pathway may allow time for nuclear, ARE-mediated regulation of CTH2 levels involving Rat1.

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Extended Cth2 transcripts are detected in wild type and trf4 strains. (A) Locations of PCR primers for detection of 3′ extended CTH2 transcripts. (B) Some CTH2 mRNA is not cleaved co-transcriptionally. RT–PCR reactions were performed on RNA samples from wild-type, trf4Δ and cth2Δ strains with PCR primers located on either side of the CTH2 poly(A) site, and separated on an agarose gel.
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Figure 2: Extended Cth2 transcripts are detected in wild type and trf4 strains. (A) Locations of PCR primers for detection of 3′ extended CTH2 transcripts. (B) Some CTH2 mRNA is not cleaved co-transcriptionally. RT–PCR reactions were performed on RNA samples from wild-type, trf4Δ and cth2Δ strains with PCR primers located on either side of the CTH2 poly(A) site, and separated on an agarose gel.

Mentions: To map the CTH2 poly(A) site, 5 µg of wild-type mRNA were reverse transcribed with superscript II RT for 50 min at 44°C in presence of the oligo V(T)25-Adaptor. cDNA was then amplified with a reverse adaptor oligonucleotide and a forward oligonucleotide at the end of the coding sequence (Cth2-CDS-F-750). A second round of PCR amplification was performed with a forward oligonucleotide located 30-nt downstream (Cth2-CDS-F-780). After gel purification, PCR products were cloned into pGEM-T and 19 clones were sequenced. The same procedure was used to characterize the end of the 3′-extended transcript, using tfr4Δ mRNA, V(T)25-Adaptor oligonucleotide for the reverse transcription and forward primers Cth2-3′-UTR-F-1300 and Cth2-3′-UTR-F-1350, with the reverse adaptor oligonucleotide for the nested-PCR. To characterize the presence of an extended transcript (Figure 2), we reverse transcribed 5 µg of wild-type mRNA or tfr4Δ mRNA in presence of random hexanucleotides. Primers Cth2-CDS-F-750 and Cth2-3′-UTR-R-1340, located on either side of the predicted poly(A) site were used for the PCR reaction. Oligonucleotide sequences are listed in Supplementary Table S2.


The mRNA encoding the yeast ARE-binding protein Cth2 is generated by a novel 3' processing pathway.

Ciais D, Bohnsack MT, Tollervey D - Nucleic Acids Res. (2008)

Extended Cth2 transcripts are detected in wild type and trf4 strains. (A) Locations of PCR primers for detection of 3′ extended CTH2 transcripts. (B) Some CTH2 mRNA is not cleaved co-transcriptionally. RT–PCR reactions were performed on RNA samples from wild-type, trf4Δ and cth2Δ strains with PCR primers located on either side of the CTH2 poly(A) site, and separated on an agarose gel.
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Related In: Results  -  Collection

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Figure 2: Extended Cth2 transcripts are detected in wild type and trf4 strains. (A) Locations of PCR primers for detection of 3′ extended CTH2 transcripts. (B) Some CTH2 mRNA is not cleaved co-transcriptionally. RT–PCR reactions were performed on RNA samples from wild-type, trf4Δ and cth2Δ strains with PCR primers located on either side of the CTH2 poly(A) site, and separated on an agarose gel.
Mentions: To map the CTH2 poly(A) site, 5 µg of wild-type mRNA were reverse transcribed with superscript II RT for 50 min at 44°C in presence of the oligo V(T)25-Adaptor. cDNA was then amplified with a reverse adaptor oligonucleotide and a forward oligonucleotide at the end of the coding sequence (Cth2-CDS-F-750). A second round of PCR amplification was performed with a forward oligonucleotide located 30-nt downstream (Cth2-CDS-F-780). After gel purification, PCR products were cloned into pGEM-T and 19 clones were sequenced. The same procedure was used to characterize the end of the 3′-extended transcript, using tfr4Δ mRNA, V(T)25-Adaptor oligonucleotide for the reverse transcription and forward primers Cth2-3′-UTR-F-1300 and Cth2-3′-UTR-F-1350, with the reverse adaptor oligonucleotide for the nested-PCR. To characterize the presence of an extended transcript (Figure 2), we reverse transcribed 5 µg of wild-type mRNA or tfr4Δ mRNA in presence of random hexanucleotides. Primers Cth2-CDS-F-750 and Cth2-3′-UTR-R-1340, located on either side of the predicted poly(A) site were used for the PCR reaction. Oligonucleotide sequences are listed in Supplementary Table S2.

Bottom Line: CTH2 carries a consensus ARE element and levels of the pre-mRNA and mRNA were elevated by mutation of the ARE or inactivation of the nuclear 5'-exonuclease Rat1.We propose that CTH2 mRNA is processed from a 3'-extended primary transcript by the exosome, TRAMP and Nrd1/Nab3/Sen1 complexes.This unusual pathway may allow time for nuclear, ARE-mediated regulation of CTH2 levels involving Rat1.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, EH9 3JR, UK.

ABSTRACT
Microarray analyses of mRNAs over-expressed in strains lacking the nuclear exosome component Rrp6 identified the transcript encoding the ARE-binding protein Cth2, which functions in cytoplasmic mRNA stability. Subsequent northern analyses revealed that exosome mutants accumulate a 3'-extended transcript at the expense of the mature CTH2 mRNA. The 3' ends of the CTH2 mRNA were mapped to a [GU(3-5)](5) repeat, unlike any previously characterized polyadenylation site. CTH2 mRNA accumulation was not inhibited by mutations in 3'-cleavage and polyadenylation factors, Rna14, Rna15 and Pap1, which block accumulation of other mRNAs. The 3'-extended CTH2 pre-mRNA strongly accumulated in strains with mutations in the TRAMP4 polyadenylation complex or the Nrd1/Nab3/Sen1 complex, and contains multiple Nrd1 and Nab3 binding sites. CTH2 carries a consensus ARE element and levels of the pre-mRNA and mRNA were elevated by mutation of the ARE or inactivation of the nuclear 5'-exonuclease Rat1. We propose that CTH2 mRNA is processed from a 3'-extended primary transcript by the exosome, TRAMP and Nrd1/Nab3/Sen1 complexes. This unusual pathway may allow time for nuclear, ARE-mediated regulation of CTH2 levels involving Rat1.

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