(A) Cells expressing GFP or GFP-FUS were induced by 2% galactose for 6 h. Cells were then fixed and viewed by fluorescence microscopy. DAPI was used to stain the nucleus. (B) Yeast with integrated GFP, 1XFUS (1 copy of untagged FUS integrated into the HIS3 locus in the genome), and 2XFUS (2 copies of untagged FUS integrated into the HIS3 locus and the TRP1 locus in the genome) were serially diluted (from left to right) and spotted onto plates containing either glucose (FUS expression “off”) or galactose (expression “on”). Picture was taken after 2 d growth at 30°C.

(A) A serial deletion of the full length FUS/TLS gene from c-terminus, labeled as 1–4, and from N-terminus, labeled as 6–8, was carried out. (B) The truncated genes (with GFP tag at N-terminus) were then placed under the control of GAL1 promoter on the yeast expression vector pYES2CT. Yeast with above constructs was serially diluted and spotted onto plate containing either glucose (expression “off”) or galactose (expression “on”). Pictures of the plates were taken after 2 d growth at 30°C. (C) Cells containing the above constructs were grown in the Ura-Raffinose medium to mid-log phase. Expression of the proteins was induced by 2% galactose for 6 h. Localization and aggregation of the proteins was visualized by fluorescence microscopy. (D) Constructs 3–5 as shown in (A) were also cloned into yeast expression vector pDEST52 without the GFP tag. Yeast containing the constructs was serially diluted and spotted onto glucose (expression “off”) or galactose plate (expression “on”). Picture of the plates was taken after 2 d growth at 30°C.

(A) GFP was fused with the nuclear localization signal of FUS/TLS (GFP_FUS) and Hrp1 (GFP_Hrp1) as shown in top part of (A). Expression of protein from yeast containing above constructs was induced by 2% galactose for 6 h. Localization and aggregation of protein was visualized by fluorescence microscopy. (B) Hrp1 NLS was added to the c-terminal end of FUS/TLS (FUS_plus) or was used to replace nuclear localization signal of FUS/TLS (FUS_switch), as shown on part top of (B). Protein expression from yeast containing above constructs was induced by 0.1% galactose for 6 h. Protein localization and aggregation was visualized by fluorescence microscopy. (C) The same yeast strains were grown in raffinose medium and 0.1% galactose medium. Cell growth was monitored using a Bioscreen machine for 2 d at 30°C. Typically, at least 10 replicates were done for each.

hUPF1 and hUPF2 were cloned into yeast expression vector pYES2CT. (A) The constructs were transformed into 1XFUS (one copy of FUS/TLS integrated at HIS locus). Spotting assay was performed to check the rescue of toxicity by hUPF1 and hUPF2. Empty vector and ECM32 construct from library screen were used as negative and positive controls. (B) The above constructs were transformed into 2XFUS (two copies of FUS/TLS integrated at HIS locus and TRP locus, respectively). Spotting assay was performed to check the rescue of toxicity by hUPF1 and hUPF2. (C) GFP tagged FUS/TLS (pYES2CT/GFP-FUS) and RFP tagged hUPF1 (pRSGal1hUPF1-DsRed) were transformed into yeast. Protein expression was induced by 2% galactose for 6 h and visualized by fluorescence microcopy.

Cells were grown in raffinose medium to early log phase. Protein expression was induced by 2% galactose for 6 h in 1XFUS strain expressing empty vector (1XFUS), human UPF1 (+hUPF1), or ECM32 (+ECM32). CYH2 pre-mRNA (A) and MER2 pre-mRNA level (B) were determined by qRT-PCR using 18sRNA as an internal control. Pre-mRNA in wild type yeast cell without integration of FUS (WT) was normalized to 1.

(A) Yeast cells containing GFP-tagged N-terminal huntingtin harboring pathogenic polyglutamine expansions (Htt103Q, stretch of 103 consecutive glutamines), normal huntingtin (Htt25Q, stretch of 25 consecutive glutamines), and GFP tagged FUS/TLS were induced with galactose for 6 h. Filter retardation assay was performed to characterize the aggregates. (B) GFP tagged FUS, Htt103Q, or Htt25Q was transformed into HSP104Δ, RNQ1Δ deletion strains and the isogenic wild type strain BY4743 (WT). Cells were serially diluted and spotted onto glucose (expression “off”) or galactose plate (expression “on”) to observe toxicity. Pictures were taken after 2 d growth at 30°C. GFP on the same vector (GFP) was used as control. (C) The same strains as above were visualized for localization and aggregation of the proteins by fluorescence microscopy. (D) pYES2CT/GFP-FUS (FUS) and empty vector (Vec) were transformed into wild type yeast, and yeast containing integrated Htt103Q. Freshly grown cells were then serially diluted and spotted onto glucose (expression “off”) or galactose plate (expression “on”) to observe toxicity. Pictures were taken after 2 d growth at 30°C.

(A) Empty vector and N-terminus GFP-tagged FUS/TLS on pYES2CT were transformed into yeast arginine methyl transferase deletion strain rmt1Δ, rmt2Δ, and its isogenic wild type BY4743 (WT). Spotting assay was performed to observe toxicity from the above yeast strains. (B) Expression of the proteins from above strains was induced by 2% galactose for 6 h. Localization and aggregation of the protein was visualized by fluorescence microscopy.

(A) ECM32, SBP1, SKO1, and VHR1 were individually transformed into 1X FUS strain. Spotting assay was performed to observe toxicity of yeast containing the above constructs. (B) Protein expression was induced by 2% galactose for 6 h from above yeast strains. Western blot analysis was performed using an antibody against FUS/TLS. PGK1 is shown as a control of protein loading.

(A) Protein expression was induced by 2% galactose for 6 h in 1XFUS strain expressing hUPF1, hUPF2, or ECM32. Western blot was performed using an antibody against FUS/TLS to check the expression of FUS/TLS. PGK1 is shown as a control of protein loading; (B) the same yeast cells were also subject to indirect immunofluorescence staining using primary antibody against FUS/TLS, and secondary antibody conjugated with fluorescein. Nuclear DNA was stained with DAPI. (C) Different domains of hUPF1 were cloned into a yeast expression vector and transformed into 1XFUS strain. Spotting assay was performed to check rescue of the toxicity. hUPF1-418, hUPF2 binding domain; hUPF419-1118, ATPase/Helicase domain; hUPF1R843C, inactivated ATPase/Helicase by arginine to cysteine mutation at residue of 843 in the full length FUS/TLS.