Kim et al., Supplemental manuscript 1

A new MIF4G domain-containing protein CTIF directs nuclear cap-binding protein CBP80/20-dependent translation

Kyoung Mi Kim, Hana Cho, Kobong Choi, Jaedong Kim, Bong-Woo Kim, Young-Gyu Ko, Sung Key Jang*,and Yoon Ki Kim*

*Correspondences: S.K.J. () and Y.K.K. ()

Supplemental Materials and Methods

Plasmid constructions

CTIF cDNA (KIAA0427) was obtained by amplification from a human brain cDNA library (Clontech) using two oligonucleotides: 5’-AAGGATCCCCCATGGAAAACTCCTCTGC-3’(sense) and 5’-CCCTGCAGCCCTCAGGCTGTCAGTTTCTG-3’(antisense), where the underlined nucleotides specify the BamHI andPstI sites, respectively. Amplified CTIF cDNA was ligated to pSK(-) digested with BamHI and PstI to generate pSK(-)-CTIF. To confirm the fidelity of the clone, the DNA was sequenced using an ABI automated sequencer (ABI 3100).

To construct plasmid pcDNA3-FLAG-CTIF, a HindIII/Klenow-filled BamHI fragment from pSK(-)-CTIF was ligated to a HindIII/Klenow-filled BamHI fragment from pcDNA3-FLAG (a kind gift from Didier Poncet).

To construct pcDNA3-FLAG-CTIF(1-305), which expresses the C-terminally deleted form of CTIF, a BamHI/Klenow-filled HindIII fragment from pcDNA3-FLAG was ligated to a BamHI/T4 DNA polymerase-treated BglI fragment from a PCR product that contains the full-length CTIF cDNA sequence. pcDNA3-FLAG-CTIF(306-598), which expresses the N-terminally deleted form of CTIF, was constructed by ligating a HindIII/Klenow-filled BamHI fragment of pcDNA3-FLAG to HindIII/T4 DNA polymerase-treated BglI fragment from a PCR product that contains the full-length CTIF cDNA sequence. The full-length CTIF cDNAwas amplified using pSK(-)-CTIF and two oligonucleotides: 5’-AAGGATCCCATGGAAAACTCCTCTGCAG-3’ (sense) and 5’-TTAAGCTTTCAGGCTGTCAGTTTCT-3’ (antisense), where the underlined nucleotides specify the BamHI and HindIII sites, respectively.

Plasmid pEGFP-CTIF was constructed byligating a BamHI/EcoRI fragment from pSK(-)-CTIF to a BglII/EcoRIfragment from pEGFP-C1 (Clontech).

To construct plasmid pCMV-Myc-CTIF, a KpnI/Klenow-filled SpeI fragment from pSK(-)-CTIF was ligated to a KpnI/Klenow-filled SalI fragment from pCMV-Myc (Clontech).

To construct pcDNA3-FLAG-eIF4E, which encodes a FLAG-tagged, full-length human eIF4E cDNA,the BamHI/HindIII fragment from pcDNA3-FLAG was ligated to a PCR fragmentthat contained human eIF4E cDNA and was digested with BamHI and HindIII. eIF4E cDNA was amplified using total cDNAs obtained from HeLa cell and two oligonucleotides: 5’-CGGGATCCGATGGCGACTGTCGAACCGGAAACCACC-3’(sense) and 5’-CCCAAGCTTTTAAACAACAAACCTATTTTTAGTGGTG-3’(antisense), where the underlined nucleotides specify the BamHI and HindIII sites, respectively. PCR amplification was carried out using the Advantage-HF2 PCR Kit (Clontech).

To construct plasmid pCMV-Myc-CBP80, a Klenow-filled BamHI/HindIII fragment from pcDNA3-FLAG-CBP80 (Oh et al. 2007) was ligated to a Klenow-filled XhoI fragment from pCMV-Myc.

To generaterecombinant CTIF protein that harbors an N-terminal 6xHis tag, pQE31-CTIF was constructed by ligating a HindIII/Klenow-filled BamHI fragment from pSK(-)-CTIF to a HindIII/SmaI fragment from pQE31 (Qiagen).

To generaterecombinant CTIF protein that harbors an N-terminal GST tag, pGEX-CTIF was constructed by ligating the BamHI/EcoRI fragment from pGEX-4T3 (Amersham-Pharmacia Biotech) to a PCR-amplified fragment of CTIF digested with BamHI and EcoRI. The PCR fragment of CTIF was amplified using pSK(-)-CTIF and two oligonucleotides: 5’-AAAGGATCCATGGAAAACTCCTCTGCAG-3’ (sense) and 5’-TTTGAATTCTAGGCTGTCAGTTTCTGGA-3’ (antisense), where the underlined nucleotides specify the BamHI and EcoRI sites, respectively.

For bacterial production of human CBP80 that harbors an N-terminal GST tag, pGEX-CBP80 was constructed by ligating the BamHI/EcoRI fragment from pGEX-6p-1+NdeI(Kim et al. 2005) to a PCR-amplified fragment digested with BamHI and EcoRI. The PCR fragment was amplified using the human CBP80 cDNA expression vector pMS2-HA-CBP80 and two oligonucleotides: 5’-CGGGATCCATGTCGCGGCGGCGGCACAGCGACGAG-3’(sense) and 5’-CGGAATTCTTAGGCCTGCAGGGCACAGAACTGCTG-3’(antisense), where the underlined nucleotides specify the BamHI and EcoRI sites, respectively.

For bacterial production of human CBP20, pGEX-CBP20 was constructed by ligating the BamHI/EcoRI fragment from pGEX-6p-1+NdeI to a PCR-amplified fragment digested with BamHI and EcoRI. The PCR fragment was amplified using the human CBP20 cDNA expression vector pCNS-D2-CBP20 and two oligonucleotides: 5’-CGGGATCCATGTCGGGTGGCCTCCTGAAGGCGCTG-3’(sense) and 5’-CGGAATTCTCACTGGTTCTGTGCCAGTTTTCCATAG-3’(antisense), where the underlined nucleotides specify the BamHI and EcoRI sites, respectively. pCNS-D2-CBP20 waspurchased from 21C Human Gene Bank, GenomeResearchCenter, KRIBB, Korea.

Immunoprecipitation using Cos-7 cells

Cos-7 cells (4 x 107) were transfected by calcium phosphate precipitation (Wigler et al. 1979) with the indicated plasmids. Two days after transfection, Cos-7 cellswere washed with ice-cold phosphate-buffered saline (PBS), harvested by centrifugation at 3,000 x g for 10 min at 4°C. After centrifugation, the pellet was resuspended in 500 l of NET-2 buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM PMSF, 2 mM benzamidine, 0.05% NP-40] containing 100 U ofRNaseOUT (Invitrogen). Cells were sonicated on ice with 2x30 bursts of 1 sec each (Branson Sonifier 250, output control 3, duty cycle 30%). Aftercentrifugation at 13,000 x g for 10 min at 4°C,the supernatant was cleared by incubation with 50 l of protein G-agarose beads (GE Healthcare) for more than 1 hr at 4°C. Pre-cleared supernatant was incubated with an appropriate antibody for 2hr at 4°C. A 50l aliquot of protein G-agarose beads was added to the mixture of supernatant/antibodyand further incubated for 90 min at 4°C. The beads were washed five times with ice-cold NET-2 buffer, suspended in 100l of 2 X SDS sample buffer [0.125 M Tris-HCl (pH 6.8), 4% SDS, 10% -mercaptoethanol,18% glycerol, and 0.1% Bromophenol Blue].

For experiments to analyze the RNA dependency of protein interactions,thecell pellet was resuspended in 500 l of NET-2 buffer without RNaseOUT. After sonication as described above, 10 g of RNase A (Sigma) or 10 g of BSA (New England Biolabs), as a control,was added to the supernatant before pre-clearing, and incubated for 30 min at 37°C. Before treatment with RNase A or after treatment with RNase Afollowed by centrifugation at 13,000 x g for 10 min at 4°C, one-tenth of the supernatant was extracted with phenol, chloroform, and isoamyl alcohol, and precipitated using ethanol to purify total RNAs for semi-quantitative RT-PCR. The remaining supernatant was used as a source of protein for IP, which was analyzed by SDS-PAGE and Western blotting.

For experiments to analyze the co-immunopurified mRNAs (Supplemental Fig. 4), IP was performed as described above, except that the supernatant obtained after sonication was added to tRNA-saturated FLAG-conjugated agarose beads (Sigma) and incubated for 3 hr at 4°C. The beads were washed ten times with ice-cold NET-2 buffer to minimize nonspecific RNA binding, and suspended in 100l of 2 X SDS sample buffer. After a quick spin-down, two-thirdsof the supernatant was extracted with phenol, chloroform, and isoamyl alcohol, and precipitated using ethanol to purify total RNAs. The remaining supernatant was analyzed by SDS-PAGE and Western blotting.

Immunoprecipitationof endogenous CTIF

HeLa cells (6x106) were lysed using Triton lysis buffer [0.5% Triton X-100, 25 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 0.1 mM EDTA, 2 mM sodium orthovanadate, 1 μg aprotinin/ml, 1 μg/ml antipain, 1 μg/ml bestatin, 1 μg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride]. Extracts were clarified by centrifugation at 14,000 x g at 4°C for 15 min. The supernatant was treated with 20 l of proteinAagarose at 4°C for 3 hr with or without 5 g/ml RNase A (Sigma), and then incubated at 4°C for 6 hr with anti-CTIF antibodies conjugated with proteinAagarose(Amersham-Pharmacia Biotech). Immunoprecipitates were washed four times with lysis buffer and analyzed by SDS-PAGE, followed by Western blotting with the indicated antibodies.

Supplemental Figure Legends

Supplemental Figure 1. The expression levels of CTIF in various mouse tissues and human cell lines. (A) Sequence alignment of the predicted MIF4G domain of human CTIF and human eIF4GI. The numbers on either side of the alignment represent the position of the first and last residue of the domain in each protein.The alignment was performed using the program BLAST 2 SEQUENCES available through the NCBI (NationalCenter for Biotechnology Information). Each amino acid is indicated in a different color depending on its properties: D and E, acidic residues are red; A, G, I, L, and V, hydrophobic residues are white; N and Q, amido residues are sky-blue; F, W, and Y, aromatic residues are orange; R, H, and K, basic residues are blue; S and T, hydroxyl residues are pink; P, proline residues are green; and C and M, sulfur residues are yellow. (B,C) Western blotting of endogenous CTIF in various mouse tissues (B) and human cell lines (C).

Supplemental Figure2. CTIF interacts with CBP80. (A) IP of endogenous CTIF. As in Fig. 1B, except that HeLa-cell extracts were analyzed either before or after IP using -CTIF antibodyby Western blotting for the indicated proteins. -actin served as a negative control. Additional details for IP of endogenous CTIF are provided in the Supplemental Materials and Methods. (B)IP of Myc-CBP80. As in Fig. 1B, except that extracts of Cos-7 cells transientlytransfected with 15 g ofpCMV-Myc-CBP80 were analyzed after IP using-Mycantibody ornormal mouse serum (mIgG), as a control for nonspecific IP. GAPDH served as a negative control. (C)IP of endogenous eIF4E. As in Fig. 1B, except that whole cell-extracts of untransfected Cos-7 cells were analyzed after IP using-eIF4E antibody ormIgG without RNase treatment.

Supplemental Figure3. Purified recombinant proteins used for GST pull-down assays in Fig. 1D. Purified proteins fromE.coli were analyzed by SDS-PAGE and stainedwithCoomassie blue.Asterisk indicates a contaminant in purified GST protein.

Supplemental Figure4.CTIF associates with newly synthesized pre-mRNA and spliced mRNA. As in Fig. 1B, except that cells were cotransfected with 10 gof pmCMV-Gl,5 gof phCMV-MUP, and10 gof pcDNA3-FLAG, pcDNA3-FLAG-CBP80, pcDNA3-FLAG-eIF4E, or pcDNA3-FLAG-CTIF. After IP, the co-immunopurified proteins and RNAs were analyzed by Western blotting (upper) and semi-quantitative RT-PCR (lower). Western blotting using -FLAG antibody and --actin antibody showed the specificity of IP (upper). Semi-quantitative RT-PCR was performed to detect intron-containing Gl pre-mRNA, Gl mRNA, and MUP mRNA (lower).Each panel of results is representative of at leastthree independently performed experiments.

Supplemental Figure 5.Preparations of FLAG-CBP80-bound or FLAG-eIF4E-bound RNP-complex. (A) Cos-7 cells were transiently transfected with 10 gof a reporter plasmid pRL-CMV to express Renilla luciferase (RLuc)and 10 gof pcDNA3-FLAG, pcDNA3-FLAG-CBP80, or pcDNA3-FLAG-eIF4E. Two days after transfection, total-cell extractswere analyzed by Western blotting before or after IP using-FLAG antibody followed by the elution with FLAG peptides, as described in the Materials and Methods. -actin served as a negative control. (B,C) Total RNAs were purified from total-cell extractsbefore IP or after IP followed by the elution with FLAG peptides. The levels of RLuc mRNAs before IP or after IP followed by elution were analyzed by semi-quantitative RT-PCR (B) and quantitative real-time PCR (C). The level of each RLuc mRNA in the eluted RNP-complex was normalized to that in the extract before IP. The normalized level of RLuc mRNA in the eluted RNP-complex after IP of FLAG-CBP80 was defined as 100%. (D) The RLuc activities were monitored before or after IP followed by elution with FLAG peptides. RLuc activities obtained from cell extracts expressing FLAG tag only before IP were defined as 1.

Supplemental Figure 6. Downregulation of CTIF abolishes NMD. As in Fig. 5A-C, except that CTIF-1 siRNA [5’-r(GAAGUGGAGAUCGCACACA)d(TT)-3’], which targets a distinct sequence of CTIF mRNA, was used. (A) Western blotting results indicate that the levels of endogenous Upf1 and CTIF were downregulated to 2% and 1% of normal, respectively, where normal is defined as the level in the presence of nonspecific Control siRNA. (B,D) Semi-quantitative RT-PCR showed that downregulation of Upf1 and CTIF abrogated the NMD of PTC-containing Gl mRNA by 3.5-foldand3.0-fold (B), respectively, andthe NMD of PTC-containing GPx1 mRNA by 3.3-foldand2.5-fold (D),respectively. RT-PCR results obtained in at least three independently performed experiments varied by less than 6%. (C,E) Quantitative real-time PCR of Gl mRNAs (C) and GPx1 mRNAs (E). The same samples obtained from (B) and (D) were analyzed by quantitative real-time PCR. The statistical differences in the results were evaluated bytwo-tailed, equal-sample variance Student’s t-test. Each P-value is indicated abovethe bar.

Supplemental Figure 7. CTIF localizes to the perinuclear region. (A) As in Fig.6, except that Cos-7 cells were transiently transfected with plasmid expressing FLAG-CTIF. (B) Immunostaining of Myc-CTIF in HeLa cells.

Supplemental References

Kim, Y.K., Furic, L., Desgroseillers, L., and Maquat, L.E. 2005. Mammalian Staufen1 recruits Upf1 to specific mRNA 3'UTRs so as to elicit mRNA decay. Cell120(2): 195-208.

Oh, N., Kim, K.M., Cho, H., Choe, J., and Kim, Y.K. 2007. Pioneer round of translation occurs during serum starvation. Biochem. Biophys. Res. Commun.362(1): 145-151.

Wigler, M., Sweet, R., Sim, G.K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S., and Axel, R. 1979. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell16(4): 777-785.