The sub2-201 mutation is synthetically lethal with deletion of any one of the HPR1, THO2, and MFT1 genes of the THO complex. Δsub2, Δsub2/Δhpr1, Δsub2/Δtho2, and Δsub2/Δmft1 cells, expressing Sub2-201p from a TRP1-marked plasmid and Sub2p from a URA3-marked plasmid, were grown at 24°C on medium containing 5-FOA (to rid the cells of Sub2p) or on URA dropout plates (CSM-URA), as indicated.

Deletion of HPR1 or MFT1 leads to Rrp6p-dependent transcription site focus accumulation of HSP104 RNA. (A) HSP104 and poly(A)⁺ RNA FISH analysis of Δhpr1 cultures grown at 25°C or temperature shifted to 37°C for 15 or 60 min and HSP104 RNA FISH analysis of Δhpr1/Δrrp6 cultures temperature shifted to 37°C for 15 min, as indicated. (B) RNA FISH analysis of Δmft1 and Δmft1/Δrrp6 cultures, as described for panel A. For the Δhpr1/Δrrp6 strain, HSP104 RNA FISH analysis was also performed on a culture grown for 60 min at 37°C. DNA was stained with DAPI (4′,6′-diamidino-2-phenylindole).

Rrp6p-dependent generation of 3′-end-truncated transcripts in Δhpr1, sub2-201, and Δmft1 mutant backgrounds. (A) Northern analysis of HSP104 RNA (or U4 snRNA as a control) isolated from W303, Δhpr1, and Δhpr1/Δrrp6 cultures harvested at the indicated time points after a 37°C temperature shift. Prior to gel electrophoresis, all samples were treated with RNase H and the DL163 oligonucleotide complementary to nt 2583 to 2606 of the HSP104 gene. The row above the upper gel (dT) indicates whether the RNA sample was also treated with oligo(dT) to remove the poly(A) tail. HSP104 RNA was visualized using a radiolabeled DNA oligonucleotide (DL164) complementary to nt 2631 to 2690 of the HSP104 gene. (B) Schematic representation of the HSP104 RNA, indicating positions cleaved by RNase H and the DNA oligonucleotides DL195, DL163, and oligo(dT). Positions recognized by the HSP104 RNA FISH probes (THJ203 to THJ206) are also shown. (C) Top row, Northern analysis of HSP104 RNA isolated from W303, Δhpr1, and Δhpr1/Δrrp6; middle row, Northern analysis of HSP104 RNA isolated from W303, sub2-201, and sub2-201/Δrrp6; bottom row, Northern analysis of HSP104 RNA isolated from W303, Δmft1, and Δmft1/Δrrp6. All cultures were harvested at the indicated time points after a 37°C temperature shift. To measure 5′-end levels, oligonucleotide DL195 complementary to nt 94 to 118 of the HSP104 gene was used in the RNase H cleavage reaction and a radiolabeled DNA oligonucleotide spanning the first 57 nt of HSP104 was used for hybridization. HSP104 3′-end abundance was measured as described for panel A. All samples were treated with oligo(dT) in the RNase H reaction to facilitate quantitation analysis. To quantitate relative HSP104 5′- and 3′-end levels, signals were normalized to the amount of U4 RNA and expressed in comparison to the percentage of RNA produced in a wild-type strain (set to a value of 100) at the same time point.

HSP104 transcripts harboring extended 3′ untranslated regions accumulate in Rrp6p-dependent transcription site foci. (A) HSP104 mRNA FISH analysis of rna14-3, rna14-3/Δrrp6, rna15-2, and rna15-2/Δrrp6 cultures temperature shifted to 37°C for 15 or 60 min. DNAs were stained with DAPI. (B) Northern analysis of HSP104 mRNA (or U4 snRNA as a control) isolated from rna14-3 and rna14-3/Δrrp6 cultures harvested at the indicated time points after a 37°C temperature shift. Prior to gel electrophoresis, samples were treated with the DL163 oligonucleotide complementary to nt 2583 to 2606 of the HSP104 gene and RNase H. Approximate size markers are indicated. (C) Northern analysis of HSP104 RNA isolated from W303 and rna14-3/Δrrp6 cultures 15 min after a 37°C temperature shift. Prior to gel electrophoresis, samples were treated with RNase H and the indicated oligonucleotide [none (−), oligo(dT), D1, or D2] complementary to the region of the HSP104 gene (as schematized above the gel) for read-through as well as polyadenylated transcripts. Migration of bands corresponding to oligo(dT), D1, and D2 cleavage is shown. (D) Northern analysis of read-through as well as polyadenylated HSP104 RNA content in rna14-3 and rna14-3/Δrrp6 strains at 15- and 30-min time points after a 37°C temperature shift. To facilitate concomitant detection of read-through and polyadenylated HSP104 RNA, samples were treated with RNase H and a mix of oligonucleotide D1 and oligo(dT) prior to gel electrophoresis. (E) Quantitation of the result described for panel D. Signals were normalized to the amount of U4 RNA and expressed in comparison to the percentage of RNA produced in the rna14-3/Δrrp6 strain (set to a value of 100) at the same time point.

Transcripts whose 3′ end is generated by a self-cleaving hammerhead ribozyme accumulate in Rrp6p-dependent transcription site foci. (A) GFP RNA FISH analysis of wild-type (top row) and Δrrp6 (middle row) strains transformed with a 2μm high-copy-number plasmid construct expressing GFP RNA terminated by a self-cleaving hammerhead ribozyme (polII-GFP-RZ) and wild-type cells transformed with a 2μm plasmid expressing GFP RNA terminated by a conventional 3′-end processing signal (polII-GFP) (bottom row). Δrrp6 cells were cotransformed with either a plasmid expressing the Rrp6p protein (top and bottom rows) or empty vector (middle row). Log-phase cultures growing at 30°C were fixed and subjected to RNA FISH utilizing probes specific for the GFP ORF. DNA was stained with DAPI. dT, oligo(dT). (B) Northern analysis of GFP RNA from the cultures described above. WT, wild type.