Poly(A)⁺ RNA export is inhibited in sem1Δ but is not impaired in mutants of the RP. (A) The depicted yeast strains were grown in YPD at 30°C; the localization of poly(A)⁺ RNA in the strains was assessed by in situ hybridization using Cy3-oligo (dT)³⁰, and DNA was stained with DAPI. The mex67-5 strain was grown at 25°C and subsequently shifted to 37°C for 1 h. (B) The cim3-1 and cim5-1 were grown to logarithmic phase at 25°C and subsequently shifted to 37°C for 4 h. Other details were as in A.

Sem1-TAP is not functional in the mRNA export pathway. (A) Wild-type and Sem1-TAP strains were grown in YPD at 30°C; the localization of poly(A)⁺ RNA in the strains was assessed by in situ hybridization using Cy3-oligo (dT)³⁰, and DNA was stained with DAPI. (B) sl of the Sem1-TAP strain when combined with strains that contain mutant alleles mex67-5 and mtr2-21. The strains carrying the indicated wild-type and mutant alleles were spotted in serial 10-fold dilutions onto synthetic dextrose complete medium + 5-FOA plates, and incubated at 30°C for 4 d.

Sac3-GANP and PAM domain protein pairs in budding yeast. Sac3-GANP and PAM domain–containing proteins are part of three large multisubunit complexes: RP, TREX-2, and the CSN. The schematic of the proteins shows their domain organization. Sequence analysis was performed using the online program SMART (http://smart.embl-heidelberg.de/).

sem1Δ and Sem1-TAP, but not mutants of the RP, are impaired in transcription of long, GC-rich genes. Gene expression analysis of the pCM184-LAUR and pCM189-LEU2 constructs, as shown by analysis of the capacity of sem1Δ and the isogenic wild-type (W303-1A) strains carrying the tetpr∷lacZ-URA3 fusion to accumulate β-galactosidase and to form colonies on SD-Trp-Ura after 3 d at 30°C, as well as by Northern analysis. RNA was isolated from mid-log phase cultures. As a P³²-labeled DNA probe, we used the 3-kb BamHI–lacZ fragment and an internal 589-bp 25S rDNA fragment obtained by PCR. RNA levels in arbitrary units (A.U.) were normalized with respect to rRNA levels of each sample.

Spontaneous and AID-induced mutation frequencies in sem1Δ. Analysis of the genetic instability (mutation and recombination) phenotype in W303-1A and sem1Δ isogenic strains, using the tetpr∷lacZ-URA3 fusion construct. Ura⁻ mutants were selected on 5-FOA. The human AID gene, present in the p413-GALAID plasmid, was either overexpressed in 2% galactose (+AID) or repressed in 2% glucose (−AID). Median and SD of three independent experiments are shown.

SEM1 is genetically linked to essential components of the mRNA export machinery. (A and B) sl or se of the sem1Δ strain when combined with mutant alleles mex67-5, mtr2-21, yra1ΔRBD, yra1-1, and sub2-85. The rpn9Δ strain was used as a control. The strains carrying the indicated wild-type and mutant alleles were spotted in serial 10-fold dilutions onto 5-FOA plates (when sl) or YPD (when se) and incubated at 25°C and 30°C for 3–5 d. (C) The effect of SUB2 overexpression on sem1Δ. Viability of wild-type (W303-1A) and sem1Δ isogenic strains transformed with the plasmid Ptet-SUB2, containing the SUB2 gene under the control of a tet promoter, which is expressed in the absence of doxycyclin (dox). Transformants were spotted as 10-fold serial dilutions on selective medium with and without doxycyclin. Growth was analyzed after 4 d at 30°C. (D) Schematic representation of the genetic network between Sem1 and factors involved in transcription-coupled mRNA export. Arrows indicate sl/se; an absence of arrows indicates the lack of genetic links with Sem1.

Sem1 is a bona fide subunit of TREX-2. (A and B) The indicated TAP- or protein A–tagged bait proteins were isolated from yeast lysates by affinity purification using an IgG-Sepharose. TEV eluates of purified bait proteins were separated on a 4–12% SDS–polyacrylamide gradient gel and subjected to silver staining. Western blot analysis of the purified protein complexes was performed with indicated antibodies against CBP, Sem1, and Rpt6. Asterisks indicate the positions of bait proteins. (C) In order to show that Sem1 is a stoichiometric component of TREX-2, sequential affinity purifications were performed. In the first step, protein A–Sac3 was purified from lysates derived from strains expressing Thp1-GFP and Thp1 (negative control) to isolate TREX-2. The bound material was released by TEV cleavage, and a part of the eluates was separated on a 4–12% SDS–polyacrylamide gradient gel, then analyzed by Western blotting using antibodies against GFP and Sem1 (top). In the second step, the rest of the TEV eluates were incubated with Sepharose coupled to anti-GFP antibodies. The bound material was eluted and separated on a 4–12% SDS–polyacrylamide gradient gel and analyzed by Western blotting using antibodies against GFP and Sem1 (IP anti-GFP, bottom). (D) Protein A–Sac3 and Thp1-TAP were isolated from yeast lysates from thp1Δ and sac3Δ strains by affinity purification using an IgG-Sepharose. Lysates derived from wild-type strains were used as controls. The bound material was released by TEV cleavage. TEV eluates of purified bait proteins were separated on a 4–12% SDS–polyacrylamide gradient gel and subjected to silver staining. Western blot analysis of the purified protein complexes was performed with indicated antibodies against CBP and Sem1. (E) Thp1-TAP was isolated from yeast lysates in which Sac3 was tagged with GFP by affinity purification using an IgG-Sepharose. The bound material was incubated with and without 20 µg/ml RNase A for 1 h. The IgG-Sepharose resin with the bound material was washed with 5 bed volumes of wash buffer. The bound TREX-2 was released by TEV cleavage, and the TEV eluates were separated on a 4–12% SDS–polyacrylamide gradient gel; Western analyses were performed using antibodies against GFP, CBP, and Sem1 to detect components of TREX-2.

Thp1 is inefficiently recruited to the NPC in sem1Δ cells. (A) The depicted yeast strains were grown to logarithmic phase at 30°C. The localization of Thp1-GFP and Sac3-GFP in sem1Δ cells and the isogenic wild-type strain was visualized by fluorescence microscopy. (B) Whole cell extracts were prepared from Thp1-GFP in wild-type and sem1Δ strains. The extracts were analyzed by SDS-PAGE and subjected to Western analysis using anti-GFP antibodies. Ribosomal protein L1 was used as loading control. (C) Thp1-TAP purification was performed from SEM1 and sem1Δ strains in which Sac3 was tagged with GFP. TEV eluates were separated on a 4–12% SDS–polyacrylamide gradient gel, and Western blot analysis was performed with indicated antibodies against GFP (Sac3-GFP), CBP (Thp1-CBP), and Sem1.

sem1Δ and Sem1-TAP, but not mutants of the RP, induce TAR. (A and B) Recombination frequency of sem1Δ, sac3Δ, thp1Δ, Sem1-TAP, and RP mutants (rpn10Δ and pre9Δ), and an isogenic wild-type strain (BY4741) in the plasmid-borne recombination systems. A small diagram of the systems (not drawn to scale) is shown. Repeats are shown as gray boxes, and gray arrows indicate relevant transcripts produced from the construct. Recombinants were selected as leu⁺. Median and SD of three independent experiments are shown. (C) Recombination analysis of sem1Δ and isogenic wild type in the plasmid-borne systems L-lacZ and GL-lacZ. Recombination frequencies, plotted as a function of the transcription levels, are shown. “Low” transcription refers to the GL-lacZ system, in which the first leu2 repeat is under the control of the GAL1 promoter, in strains cultured in 2% glucose; “medium” refers to L-lacZ, in which the leu2 repeat is under its own promoter, in 2% glucose; and “high” refers to GL-lacZ in 2% galactose. Other details were as in A. (D) RNAPII occupancy at the GAL1pr-YLR454w gene in sem1Δ mutant. ChIP analyses (using N20 or 8WG16 anti-RNAPII antibodies) in wild-type (W303-1A) and sem1Δ isogenic strains carrying the GAL1pr∷YLR454w fusion construct located at the endogenous YLR454w chromosomal locus are shown. The scheme of the gene and the PCR-amplified fragments are shown. The DNA ratios in the 5′ (1), middle (2), and 3′(3) regions, were calculated from the DNA amount of these regions relative to the intergenic region. The recruitment data shown are first referred to the value of the 5′ region taken as 100% and then relative to the wild-type levels for each region. Median and SD of three independent experiments are shown. WT, isogenic wild-type strain.