Regulation of ARE transcript 3 end processing by the. yeast Cth2 mrna decay factor

Transcription

1 Regulation of ARE transcript 3 end processing by the yeast Cth2 mrna decay factor Manoël Prouteau, Marie-Claire Daugeron and Bertrand Séraphin Supplementary Information Material and Methods Plasmid construction details Plasmids expressing wild-type and mutant Cth2 An acceptor plasmid, pbs3125, was constructed by inserting simultaneously a Cth2 promoter/tap-tag fragment and a Cth2 3 UTR fragment flanked respectively by BamHI and HindIII or HindIII and XhoI restriction sites into BamHI- and XhoI-linearized pbs3122 (YCpLac111-SapI). The Cth2 promoter/tap-tag fragment was obtained by PCR fusion using Cth2 promoter and TAP-tag fragments as templates and primers OBS1876/OBS187. The Cth2 promoter and Cth2 3 UTR were obtained by PCR amplification using genomic DNA from the yeast strain BSY1233 as template and primers OBS1876/OBS1973, or OBS1974/OBS1877, respectively. The TAP-tag fragment was obtained by PCR amplification using pbs1761 as template and primers OBS1971/OBS1972. PCR reactions were used to amplify the coding sequences of Cth2; terminal truncations of Cth2 or Pop2 flanked by Sap1 sites and PCR fusion reactions were used to amplify the coding sequences of Cth2 internal deletions; Cth2 point mutant or Pop2-Cth2 fusions flanked by SapI sites using genomic DNA as template and the primer sets listed in supplementary table S4. The PCR products were then digested by SapI restriction enzyme and cloned into the SapIlinearized pbs3125. All constructions were verified by DNA sequencing.

2 SDH4 reporter plasmids The SDH4 coding sequence flanked by BamHI and HindIII restriction sites and the SDH4 3 UTR flanked by SapI and XhoI restrictions sites were amplified by PCR from genomic DNA using OBS1951/OBS2018 and OBS1969/OBS1952. The two digested PCR products were ligated to annealed OBS2019/OBS2018 primers. The resulting fragment was inserted under the control of the Gal1 promoteur in the YCplac33 plasmid yielding pbs3408 (psdh4-g18). The extended SDH4 3 UTR fragment flanked by SpeI and XhoI restriction sites was obtained by PCR amplification using genomic DNA as template and primers OBS2802/OBS2681. The SpeI-XhoI digested PCR product was then ligated into the SpeI-XhoI linearized pbs3408 yielding pbs3409 (psdh4-g18l). Plasmids were verified by DNA sequencing. Detailed description of the RNA analysis procedures Reverse ligation-mediated PCR (RL-PCR) Mapping of 5 and 3 ends of the SDH4 transcripts, as well as poly(a) tail size, was done by Reverse-ligation mediated PCR (Couttet et al., 1997). Briefly, 500 ng of total RNA were first treated with Calf intestinal phosphatase (Invitrogen), extracted with Phenol-Cloroform (PCI) and ethanol precipitated. RNA were then treated with Tobacco acid pyrophosphatase (Invitrogen), extracted again with PCI and precipitated again with ethanol. Highly diluted RNA samples (~1,0 ng of total RNA/μl) were incubated with T4 RNA ligase (GE Healthcare). The 3-5 junctions of circularized SDH4 mrnas were then specifically amplified by RT-PCR using the OBS2678/OBS1969 primer set and the resulting PCR products cloned into the TOPO blunt plasmid (Invitrogen). Insert sequences were obtained using the M13 forward and reverse primers. Extended transcripts analysis by quantitative RT-PCR

3 mrna species originating from the SDH4, SDH2, ACOI, CCP1 and ACT1 loci were quantified by RT-PCR using primer sets located upstream (OBS3028/OBS3029, OBS3016/OBS3017, OBS3020/OBS3021, OBS3024/OBS3025 and OBS3078/OBS3079) or downstream (OBS3030/OBS3031, OBS3018/OBS3019, OBS3022/OBS3023, OBS3026/OBS3027 and OBS3076/OBS3077) of the normal poly(a) site. These primers pairs were validated for the absence of dimer formation and good amplification efficiency using serial dilution of genomic DNA. RNA samples, treated with Turbo DNase I (Ambion), were reverse transcribed using mixtures of upstream (OBS3029, 3017, 3021, 3025 and 3079) or downstream (OBS3031, 3019, 3023, 3017 and 3077) primers. The resulting cdnas were used as template in quantitative PCR reactions in a LightCycler480 (Roche). Genomic DNA dilutions were used as references. Each measure, done in biological and technical triplicates, generated a Ct mean that was then converted in quantity using the corresponding reference. For each locus, the ratio of the downstream to upstream RNA quantity was expressed as a percentage of 3 extended molecules.

4 Tables Strain name Notes Genotype Reference BMA64 WT MATa ade2-1 his3-11,15 leu2-3 trp1 ura3-1 can1-100 BSY1424 cth1 MATa cth1::hismx6 ade2-1 his3-11,15 leu2-3 trp1 ura3-1 can1-100 This study BSY1517 cth1 cth2 MATa cth1::hismx6 cth2::hixmx6 ade2-1 his3-11,15 leu2-3 trp1 ura3-1 can1-100 This study BSY1429 CTH2-TAP MATα CTH2-t7-TAPtag-TRPKl ade2-1 his3-11,15 leu2-3 trp1 ura3-1 can1-100 This study BSY1235 TMA46-TAP MATa TMA46-TAPtag-TRPKl ade2-1 his3-11,15 leu2-3 trp1 ura3-1 can1-100 This study BSY1850 FAL1-TAP MATa FAL1-TAPtag-TRPKl ade2-1 his3-11,15 leu2-3 trp1 ura3-1 can1-100 This study BSY1606 TPI-CTH2-TAP MATα NatR-TPI-CTH2-t7-TAPtag-TRPKl ade2-1 his3-11,15 leu2-3 trp1 ura3-1 can1-100 This study FWY2-374 rna14-1 MATa rna14-1 ade2-1 his3-11,15 leu2-3 trp1 ura3-1 can1-100 FWY2-378 rna15-2 MATa rna14-2 ade2-1 his3-11,15 leu2-3 trp1 ura3-1 can1-100 FWY2-386 rna14-1 rna15-2 MATa rna14-1 rna14-2 ade2-1 his3-11,15 leu2-3 trp1 ura3-1 can1-100 YAO428-1B nab4-4 MATa leu2 2 ura3-52 nab4 2::LEU2 [pnab4.55] Minvielle-Sebastia et al. (1998) YAO431-1C nab4-7 MATa leu2 2 ura3-52 nab4 2::LEU2 [pnab4.58] Minvielle-Sebastia et al. (1998) Supplementary Table S1: Yeast strains used in this study Except for the nab4 mutants, all are derived from the BMA64 background.

5 Plasmid Notes Features References YCpLac111 - YCpLac111, LEU2, Amp R Gietz and Sugino (1988) pbs YcpLac11, Sap1 restriction site mutated This study pbs3148 Cth2 pbs3122-cth2 locus (without TAP tag) This study pbs pbs3122-cth2[prom]-taptag-(sap1) 2 -CTH2[3'UTR] This study pbs3133 TAP-Cth2 CTH2 fragment in SapI sites of pbs3125 This study pbs3137 (TAP-)Δ1-86 Δ1-86 fragment in SapI sites of pbs3125 This study pbs3138 (TAP-)Δ1-157 Δ1-157 fragment in SapI sites of pbs3125 This study pbs3139 (TAP-)Δ Δ fragment in SapI sites of pbs3125 This study pbs3142 (TAP-)Δ Δ fragment in SapI sites of pbs3125 This study pbs3146 (TAP-)Δ Δ fragment in SapI sites of pbs3125 This study pbs3147 (TAP-)Δ1-157/Δ Δ1-157/Δ fragment in SapI sites of pbs3125 This study pbs3397 (TAP-)Δ1-34 Δ1-34 fragment in SapI sites of pbs3125 This study pbs3398 (TAP-)Δ1-57 Δ1-57 fragment in SapI sites of pbs3125 This study pbs3399 (TAP-)Δ36-57 Δ36-57 fragment in SapI sites of pbs3125 This study pbs3400 (TAP-)Δ Δ fragment in SapI sites of pbs3125 This study pbs3401 (TAP-)Δ Δ fragment in SapI sites of pbs3125 This study pbs3402 (TAP-)Δ Δ fragment in SapI sites of pbs3125 This study pbs3403 (TAP-)Cth2(ACCH) 2 Cth2(ACCH)2 fragment in SapI sites of pbs3125 This study pbs3404 (TAP-)Δ1-86(ACCH) 2 Δ1-86(ACCH)2 fragment in SapI sites of pbs3125 This study pbs3405 (TAP-)POP2 POP2 fragment in SapI sites of pbs3125 This study pbs3406 (TAP-)POP2-Cth2 POP2-Cth2 fragment in SapI sites of pbs3125 This study pbs3407 (TAP-)POP2-Δ1-86 POP2-Δ1-86 fragment in SapI sites of pbs3125 This study YCpLac33 - YCpLac33, URA3, Amp R Reference pbs Insertion of 10Gal1[prom] in YCpLac33 This study pbs3408 SDH4-G 18 SDH4 fragment-g18-sdh4[3'utr] in pbs3152 upstream Gal1[prom] This study pbs3409 SDH4-G 18 -L SDH4 fragment-g18-sdh4[long 3'UTR] in pbs3152 upstream Gal1[prom] This study TopoBlunt II - Kan R Invitrogen Supplementary Table S2: Plasmids used in this study Plasmids were propagated in the MH1 strain.

6 Name Sequence OBS482 5'-GTAAAACGACGGCCAG-3' OBS483 5'-CAGGAAACAGCTATGAC-3' OBS1876 5'-GAGGGATCCGCTGCCGTAACCATTCTCTC-3' OBS1877 5'-GAGCTCGAGTCGACGAAATGACCATCAGA-3' OBS1884 5'-GTGGGATGGGATACGTTGAG-3' OBS1885 5'-ATGGTTCAGGACACACTCCA-3' OBS1951 5'-GCCGGATCCACGCTTTCGACTTTCTTCCT-3' OBS1952 5'-GCCCTCGAGTTAAAAATCCGCGAGTGACG-3' OBS1969 5'-GCGGCTCTTCTTGTAACATGCACATCTATAGCAAC-3' OBS1971 5'-TGCAAACTCAATACGTAAAAAATAACGCAGTTATCCATGGCAGGCCT-3' OBS1972 5'-CGCAAGCTTCGCGCTCTTCTAAGTTTATCGTCATCATCAAGTG-3' OBS1973 5'-GCGTTATTTTTTACGTATTGAGTTTGCA-3' OBS1974 5'-CGCAAGCTTCGCGCTCTTCTTGAAACAATTCCTGGCCTG-3' OBS1979 5'-CGCGCTCTTCTCTTATGTGGGCTCAATTATCATATACTAGA-3' OBS1980 5'-CGCGCTCTTCTTCACCAGGTCATTCTCTGCA-3' OBS2018 5'-CGCAAGCTTGACCGAACAAATGATTCGTG-3' OBS2019 5'-AGCTTGGGGGGGGGGGGGGGGGG-3' OBS2020 5'-ACACCCCCCCCCCCCCCCCCCA-3' OBS2104 5'-AGAGACGTTACAGTAAGTACCAGCCACCTTAGGTTGAGCTGAGGA-3' OBS2105 5'-GCTGGTACTTACTGTAACGTCTCT-3' OBS2106 5'-GGCGCTCTTCATCAACCATTGCTTCTCTTTTCGTC-3' OBS2211 5'-GGCGCTCTTCACTTCAGTTAATGAACCCTTTTCTCCCA-3' OBS2212 5'-GGCGCTCTTCACTTGTGAAAAGCCAAGTGCAGGAAACT-3' OBS2213 5'-GGCGCTCTTCATCAAGAGACGTTACAGTAAGTACCAGC-3' OBS2417 5'-GGCGCTCTTCACTTCAGATCAATATTAGGGAACTGGAG-3' OBS2418 5'-GGCGCTCTTCACTTGAAACAAGCAGTGAAATATCTTCG-3' OBS2515 5'-CGAAGATATTTCACTGCTTGTTTCCTGGTAGTCATTTAATGGATTTTGC-3' OBS2516 5'-GAAACAAGCAGTGAAATATCTTCG-3' OBS2517 5'-CGGATTTATTTGTACCTCTAGTTTCTTTTCCTGTATGTTATCCTCGTTTAA-3' OBS2518 5'-AAGAAACTAGAGGTACAAATAAATCCG-3' OBS2519 5'-AGAAGCATCATTTTTGGGCTTAGTAGTAGGTGCCGTGCTATTCAG-3' OBS2520 5'-AAGCCCAAAAATGATGCTTCT-3' OBS2521 5'-TTCCTGCACTTGGCTTTTCACTTGAGACAACTGTTGCAAATTCTG-3' OBS2522 5'-GTGAAAAGCCAAGTGCAGGAA-3' OBS2605 5'-GGCGCTCTTCACTTCAATCTATGAATGTACAACCGA-3' OBS2678 5'-TTCTTAGCGGTAGACTGGAAGG-3' OBS2681 5'-TCAAAAGATTGAAGCCAAGAAGT-3' OBS2797 5'-GATATTCAGAAAAAAGCATAAGCC-3' OBS2802 5'-GGCCTCGAGACTAAAGATCCCGATGAACAACA-3' OBS2813 5'-GACGCAAACACACACGTATATCT-3' OBS2887 5'-TTGGTCCCCATCAATACCGTA-3'

7 OBS2888 OBS2889 OBS3018 OBS3019 OBS3020 OBS3021 OBS3022 OBS3023 OBS3024 OBS3025 OBS3026 OBS3027 OBS3028 OBS3029 OBS3030 OBS3031 OBS3076 OBS3077 OBS3078 OBS3079 OBS3172 OBS3173 5'-TACGGTATTGATGGGGACCAAATGTGGGCTCAATTATCATATACTAGA-3' 5'-TACGGTATTGATGGGGACCAACTGAATAGCACGGCACCTACT-3' 5'-ATTTTCGTTGTGAAATTTCTGTTAAG-3' 5'-CATGGTGTTCTCTTCTCATTGC-3' 5'-GGCCGATGAGAAGAAATAATG-3' 5'-CAAGGGTACGGTGTACAGAAGA-3' 5'-GTTGCTGATTATGTACTCAGTTTAACG-3' 5'-TCTGACAGACGGTGCACTTATT-3' 5'-GGAGAACGGTATCACTTTCCCTA-3' 5'-TTTCCTCAGTGACCTATAAACCTTG-3' 5'-TGAAAAATGGAAAGCCATACAA-3' 5'-AAGAGGCGGACAATGTTTTCT-3' 5'-ACGAATCATTTGTTCGGTCAT-3' 5'-AATATACCGGCGGAAAACAAG-3' 5'-CCAGCTCCGAAGATTTTGAA-3' 5'-TGTACTGTCATGAAAAGGAACTGAC-3' 5'-ACGAAAATTTCAAAAATTGACCA-3' 5'-ACACGGTCCAATGGATAAACA-3' 5'-TCCATCTATCGTTCACCACAAG-3' 5'-ACATAAACATACGCGCACAAA-3' 5'-TCGCCGAGTCCGTGAGCAAACTGACACTTACTGCCATAAGGGCAACTTCCTTTTAAAGTGAATGATTCGGCCAGCTCTGTCTTGTATAATTGTTTG-3' 5'-GTCAGTTTGCTCACGGACTCGGCGAATTAAAGGTTAAGAAATCGTGCAAGAATTTCAGGACGAAGCCCGCGGTGAATTGGGAGAAACTAGGTTATT-3' Supplementary Table S3: Oligonucleotides used in this study

8 PCR fragments Primers set(s) Cth2 locus OBS1876/OBS1973 Cth2 OBS1979/OBS1980 Δ1-86 OBS2211/OBS1980 Δ1-157 OBS2212/OBS1980 Δ OBS1979/OBS2104, OBS2105/OBS1980, BS1979/OBS1980 Δ OBS1979/OBS2106 Δ OBS1979/OBS2213 Δ1-157/Δ OBS2212/OBS2213 Δ1-34 OBS2417/OBS1980 Δ1-57 OBS2418/OBS1980 Δ36-57 OBS1979/OBS2515, OBS2516/OBS1980, OBS1979/OBS1980 Δ OBS1979/OBS2517, OBS2518/OBS1980, OBS1979/OBS1980 Δ OBS1979/OBS2519, OBS2520/OBS1980, OBS1979/OBS1980 Δ OBS1979/OBS2521, OBS2522/OBS1980, OBS1979/OBS1980 Cth2(ACCH)2 OBS1979/OBS3172, OBS3173/OBS1980, OBS1979/OBS1980 Δ1-86(ACCH)2 OBS2211/OBS3172, OBS3173/OBS1980, OBS1979/OBS1980 POP2 OBS2505/OBS2506 POP2-Cth2 OBS2605/OBS2887, OBS2888/OBS1980, OBS2605/OBS1980 POP2-Δ1-86 OBS2605/OBS2887, OBS2889/OBS1980, OBS2605/OBS1980 SDH4 probe OBS1951/OBS1952 CCP1 probe OBS2813/OBS2797 SCR1 probe OBS1884/OBS1885 Supplementary Table S4: Primer sets used for PCR amplification of fragments used for cloning or as probes

9 Loci Upstream Poly(A) site (PCR5') Downstream Poly(A) site (PCR3') SDH4 OBS3028/OBS3029 OBS3030/OBS3031 SDH2 OBS3016/OBS3017 OBS3018/OBS3019 ACO1 OBS3020/OBS3021 OBS3022/OBS3023 CCP1 OBS3024/OBS3025 OBS3026/OBS3027 ACT1 OBS3078/OBS3079 OBS3076/OBS3077 Supplementary Table S5: Primer sets used for qrt-pcr

10 Supplementary Figure 1 Cth2-dependent mediated SDH4 mrna downregulation results from faster RNA decay A schematic representation of the SDH4-G18 reporter is shown at the top of the figure. The SDH4 coding sequence followed by SDH4 3 UTR (300nt downstream from the ORF) containing a stretch of 18 G just after the stop codon is under the control of the galactose inducible and glucose repressible GAL1 promoter on a yeast centromeric plasmid. The lower part depicts the results of northern analysis of SDH4-G18 mrna degradation after transcriptional shut-off. cth1 cth2 cells co-transformed by SDH4-G18 reporter (psdh4- G18) and an empty vector or a vector expressing TAP-Cth2 were grown 4 hours in presence of galactose to express reporter mrna and 100 μm BPS to express TAP-Cth2. Glucose was added to stop the reporter transcription and samples were collected at various time points. Reporter mrna and decay intermediates were detected using a 5 end-labeled oligonucleotide probe complementary to the G18 sequence and SCR1 (loading control) with a random labeled DNA probe. Reporter mrna half-live were calculated as the average of two independent experiments.

11 Supplementary Figure 2 All Cth2 mutants lacking CR1 accumulate extended SDH4 transcripts. Northern blot analysis of SDH4 mrna and SCR1 (loading control) from cells expressing the various cth2 mutants, or wild-type control, was performed and analyzed as described in Fig. 1 except that cells were grown for 5 hours in low iron conditions. The extended SDH4 transcripts that accumulate in these conditions are then clearly visible in all Cth2 mutants lacking CR1.

12 Supplementary Figure 3 High levels of Cth2 are insufficient to promote the formation of extended transcripts The upper part presents a northern blot analysis of SDH4 mrna and SCR1 (loading control) from cells expressing either the wild-type TAP tagged Cth2 under the control of its natural promoter, a similar construct expressing Cth or the wild type tagged protein under the control of the strong constitutive TPI promoter. Cells were grown in iron replete or low iron conditions for 5 hours and analyzed as described in Fig. 1. The lower part of the Figure presents a western blot analysis to assay the levels of these three proteins detected through the TAP tag. Three different dilutions of each sample were loaded to ensure that the signal was proportional to the input material and allow for better comparison. Equal loading was controlled by monitoring the level of the constitutively expressed Stm1 factor.

13 References Couttet, P., Fromont-Racine, M., Steel, D., Pictet, R. and Grange, T. (1997) Messenger RNA deadenylylation precedes decapping in mammalian cells. Proc Natl Acad Sci U S A, 94, Gietz, R.D. and Sugino, A. (1988) New yeast-escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene, 74, Minvielle-Sebastia, L., Beyer, K., Krecic, A.M., Hector, R.E., Swanson, M.S. and Keller, W. (1998) Control of cleavage site selection during mrna 3' end formation by a yeast hnrnp. EMBO J, 17,