As a control to demonstrate that we could isolate PCR products that result from ligating

Transcription

1 SUPPLEMENTAL INFORMATION (Mullen and Marzluff) RESULTS Oligo(dA) Clones and Oligo(dT) RT-PCR controls. As a control to demonstrate that we could isolate PCR products that result from ligating the authentic 3 ends of the mrnas, and to eliminate the possibility of nuclease contamination in our crt-pcr reagents, we analyzed the 3 end of histone H2a mrna formed in an in vitro processing reaction. Since the in vitro transcribed substrate histone pre-mrna contains a 5 triphosphate, it is also necessary to treat these RNAs with TAP prior to ligation. At time 0 we cloned only products corresponding to the synthetic pre-mrna substrate. After processing for 120 minutes, when most of the substrate has been cleaved, we recovered only histone RNA that ended with the conserved ACCCA nts following the base of the stemloop, precisely where we and others (Scharl and Steitz, 1994;Dominski et al., 2005) have mapped the cleavage site in vitro (Suppl. Figure S3A). These results demonstrate that we were able to recover RNA molecules that had their 3 and 5 ends intact and hence nuclease activity in our reagents was minimal. 1

2 EXPERIMENTAL PROCEDURES Supplemental Table 1. sirna sequences Target Sequence Reference C2 5 -GGUCCGGCUCCCCCAAAUG-3 (Wagner and Garcia- Blanco, 2002) PTB 5 -GCCUCUUUAUUCUUCUCGG-3 (Wagner and Garcia- Blanco, 2002) 3 hexo 5 -UUACGAAUGGCUGUAUUAA-3 This paper Lsm CAAACUUAGUGCUACAUCA-3 (Stoecklin et al., 2006) Lsm CCAGCAAGUAUCCAUUGAA-3 (Stoecklin et al., 2006) Upf1 5 -GAUGCAGUUCCGCUCCAUU-3 (Kaygun and Marzluff, 2005) Dcp2 5 -GUGGCAUGUAAUGGACAUUGC-3 (Wang et al., 2002) Xrn1 5 -UGAUGAUGUUCACUUUAGA-3 (Stoecklin et al., 2006) PM/Scl GCUGCAGCAGAACAGGCCA- 3 (Lejeune et al., 2003) Rrp CUAUGCAGCUUGUGUGAAUUU-3 This paper Rrp CGGACAGGGCCCUAGUGAAUU-3 This paper TUTase-1 5 -CGAGCACAUUCACUAACAA-3 This paper TUTase-2 5 -CGUUAGUGCUGGUGAUUAA-3 This paper TUTase-3 5 -GGACGACACUUCAAUUAUU-3 This paper TUTase-4 5 -UGAUAGUGCUUCAGGAAUU-3 This paper TUTase-5 5 -CUACGGUACCAAUAAUAAA-3 This paper U6 TUTase 5 -GCAGCCAAUUACUGCCGAA-3 (Trippe et al., 2006) TUTase-7 5 -GAAAAGAGGCACAAGAAAA-3 This paper 2

3 Supplemental Table 2. Primers for RT-PCR of endogenous TUTase mrna Target Oligo sequence TUTase-1 sense: 5 -GAAGATCCTGTCTGTGTTAGGAG-3, antisense: 5 -GGAAGGTCGATCTGTATCTTCC-3 TUTase-2 sense: 5 -GCAGACTTGTCTAGAGCTGTG-3, antisense: 5 -CTCGAATCAGCTGAGGTCTCTC-3 TUTase-3 sense: 5 -GAGTAACAGATGAAGTTGCCAC-3 ), antisense: 5 -GTTTGAGTTGTACCTTGGAAGC-3 TUTase-4 sense: 5 -GGCAACCAGTTTACAGGCAATAGC-3 antisense: 5 -CGTCCGATACGTCTTCAATTCC-3 TUTase-5 sense: 5 -GGTCCTATCCAAACAGAGACG-3 antisense: 5 -GTTCCTTCTACACCAGCTTGTC-3 TUTase-7 sense: 5 -GGTTATAGGTGGCAAGACACAAG-3 antisense: 5 -CAGCCTCTGTTCCAAGTTCTC-3 FIGURE LEGENDS Suppl. Figure 1. Cell cycle analysis of knockdowns. (A-F) HeLa cells were treated with sirnas targeting the indicated genes. Cells were fixed in 70% ethanol, stained with propidium iodide, and analyzed by FACS. Populations of G1 (2N DNA content), S phase (2N < S < 4N DNA content), and G2/M (4N DNA content) were quantified and are represented as a percentage of the total population. All experiments are paired samples that correspond to the RNA samples analyzed by Northern and/or S1 nuclease protection assays shown in the text. 3

4 Suppl. Figure 2. Knockdown of PTB does not affect histone mrna degradation (A) Dilution series Western blot estimating degree of PTB knockdown. C2 protein lysate was serial diluted as indicated. A cross-reacting band with a larger molecular weight than PTB is used as a control (B) Northern blot simultaneously detecting human histone H2a mrna and 7SK snrna levels in response to 5 mm HU treatment for 0, 20 and 45 minutes. (C) Summary of 3 independent knockdowns of PTB compared to C2 control (, C2;, PTB). Stabilization of histone mrna in response to RNAi is represented as a percent of histone mrna remaining. Quantification of hybridized radiolabelled probe was performed using a PhosphorImager. 4

5 Suppl. Figure 3. Oligo(dA) Clones and Oligo(dT) RT-PCR controls. (A) HIST2H2AA clones from nested oligo(da) RT-PCR reactions. 2 μl of PCR product (0 minutes HU, 15 minutes HU) from the second round of PCR (+Taq) were TA cloned, transformed and plated onto kanamycin-containing LB agar plates. (B, top Panel) Positive control for oligo(dt) RT-PCR. Knockdown of SLBP results in polyadenylated HIST2H2AA mrna due to misprocessing of the pre-mrna. Total RNA was subjected to oligo(dt) cellulose selection and 500 ng of the elution was random primed for reverse transcription. 2 μl of RT reaction was used for PCR similarly as in Figure 5A. (B, bottom panel) Oligo(dT) control for HIST2H2AA mrna from cells treated with hydroxyurea. Total RNA from the experiments performed in Figure 5B were random primed and subjected MMLV reverse transcriptase. PCR was performed identically to that in Figure 5A and 5B to detect HIST2H2AA mrna. The 110 bp product is too small to be HIST2H2AA mrna and does not change in response to HU treatment. (C) Oligo(dT) control for HIST2H3D (top panel) and HIST2H3A/C (bottom panel) mrna from cells treated with hydroxyurea. RT- PCR was performed as in panel B, bottom. 5

6 Suppl. Figure 4. Detection of the 5 and 3 ends of histone H3 by crt-pcr (A) Synthetic histone pre-mrna was processed in a nuclear extract, and the unprocessed and processed RNAs were treated with TAP and subjected to crt-pcr, and the products cloned and sequenced. All 4 clones of processed RNA sequenced ended at ACCCA. The arrow indicates the site cleaved in vitro. (B) The 5 (top) and 3 (bottom) ends of capped (+TAP) HIST2H3A/C mrnas. The genomic sequence of HIST2H3A/C is indicated on the top line. The sequences of the TA cloned crt-pcr products are shown. (C) The 5 (top) and 3 (bottom) of the capped histone HIST2H3D mrna. The genomic sequence of HIST2H3D is shown in the top lines. The number of times a particular clone was obtained is in parentheses. Suppl. Figure 5. Enlargement of sequences from decapped histone H3 mrna degradation intermediates. (A and B) The degradation intermediates from Figure 4D and 4E are enlarged. HIST2H3A/C (panel A) or HIST2H3D (panel B) mrnas that were not capped were cloned by crt-pcr. Primer 2 and 3 represent sites where we targeted amplification towards the 5 and 3 ends. ORF sequences between these 2 oligonucleotides are missing. Numbers in parentheses are the number of times each clone was obtained. (C) Summary of the various species of decapped histone H3 degradation intermediates recovered by cloning uncapped (-TAP) crt-pcr reactions. The sequences from (A) and (B) were analyzed based on the criteria depicted on the left column. The frequency of recovery is represented as a percentage of the total number of clones recovered. 6

7 Suppl. Figure 6. RT-PCR to endogenous TUTase mrna. TUTase mrna levels were measured by RT-PCR after treatment of the cells with RNAi (Fig. 6). 2 μg of total RNA was random primed and subjected to MMLV reverse transcriptase followed by PCR. In a parallel reaction 7SK RNA levels were determined as a control. Lanes 1 and 2: TUTase-2; Lanes 3 and 4: TUTase-4; Lanes 5 and 6: TUTase-7. 7

8 Supplemental Reference List Dominski, Z., Yang, X.C., and Marzluff, W.F. (2005). The polyadenylation factor CPSF- 73 is involved in histone pre-mrna processing. Cell. 123: Kaygun, H. and Marzluff, W.F. (2005). Regulated degradation of replication-dependent histone mrnas requires both ATR and Upf1. Nat. Struct. Mol. Biol. 12: Lejeune, F., Li, X., and Maquat, L.E. (2003). Nonsense-mediated mrna decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities. Mol. Cell. 12: Scharl, E.C. and Steitz, J.A. (1994). The site of 3' end formation of histone messenger RNA is a fixed distance from the downstream element recognized by the U7 snrnp. EMBO J. 13: Stoecklin, G., Mayo, T., and Anderson, P. (2006). ARE-mRNA degradation requires the 5'-3' decay pathway. EMBO Rep. 7: Trippe, R., Guschina, E., Hossbach, M., Urlaub, H., Luhrmann, R., and Benecke, B.J. (2006). Identification, cloning, and functional analysis of the human U6 snrna-specific terminal uridylyl transferase. RNA. 12: Wagner, E.J. and Garcia-Blanco, M.A. (2002). RNAi-Mediated PTB Depletion Leads to Enhanced Exon Definition. Mol. Cell. 10: Wang, Z., Jiao, X., Carr-Schmid, A., and Kiledjian, M. (2002). The hdcp2 protein is a mammalian mrna decapping enzyme. Proc. Natl. Acad. Sci. U. S. A. 99:

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