The Cis-Acting Elements Involved in Endonucleolytic Cleavage of the 3′ UTR of Human IGF-II mRNAs Bind a 50 kDa Protein

Wiep Scheper 0 P. Elly Holthuizen 0 John S. Sussenbach 0 0 Laboratory for Physiological Chemistry, Graduate School of Developmental Biology, Utrecht University , PO Box 80042, 3508 TAUtrecht, The Netherlands - cooperate in the binding of trans-acting factors involved in cleavage of IGF-II mRNAs. The identification of proteins binding to the cis-acting elements required for cleavage may provide critical insight into the mechanism underlying the specific endonucleolytic cleavage of IGF-II mRNAs. In the present study we have initiated the identification of trans-acting factors that interact with the IGF-II cleavage unit. MATERIALS AND METHODS Plasmid pBluescript II (KS+) was obtained from Stratagene (La Jolla, CA). Enzymes were purchased from Boehringer Mannheim (Germany), with the exception of Pfu DNA polymerase (Stratagene, La Jolla, CA), RNase-free DNase (Promega, Madison, WI) and RNase T1 (CalBiochem, La Jolla, CA). Enzymes were used according to the manufacturers instructions. NTPs and dNTPs were obtained from Kabi-Pharmacia (Uppsala, Sweden). G418 was purchased from Sigma (St Louis, MO). A random priming DNA labeling kit was purchased from Boehringer Mannheim (Germany) and a DNA sequencing kit from Kabi-Pharmacia (Uppsala, Sweden). Guanidinium thiocyanate was obtained from Fluka (Buchs, Switzerland) and GeneScreen membranes from Du Pont de Nemours (Dreiech, Germany). [a -32P]dCTP (3000 Ci/mmol) and [a -32P]CTP (760 Ci/mmol) were purchased from Amersham (Buckinghamshire, UK). The cell lines HeLa and Ltk were grown in Dulbeccos Modified Eagles Medium (DMEM), the stable Ltk cell line EP7-9 (22) was grown in the DMEM in the presence of 300 m g/ml G418 and Hep3B cells were grown in a -Minimal Essential Medium (a -MEM). All media were supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 m g/m l streptomycin and 300 m g/ml glutamine. Cytoplasmic and nuclear cell fractions were prepared employing a method adapted from Vakalopoulou et al. (12) with slight modifications. Briefly, the cells were lysed in 10 mM Tris pH 8.0, 10 mM NaCl, 3 mM MgCl2, 1 mM DTT, 1 mM phenylmethylsulphonic acid, 0.5% NP-40 and 0.5% sodium deoxycholate on ice for 10 min. Subsequently, the lysate was spun in a microfuge for 5 min at 4 C and the supernatant was collected and used for cytoplasmic RNA isolation or stored at 80 C in 10% glycerol (cytoplasmic extract). The pellet was used to isolate nuclear RNA or to prepare nuclear extracts as described in (23). Protein concentrations were determined by the Bradford protein assay (BioRad, Mnchen, Germany). In vivo cross-linking experiments were performed essentially as in (24). Briefly, cells were grown on 100 mm plates, washed twice with cold PBS and exposed to UV light (254 nm) at 1.9 J/cm2 (Stratalinker, Stratagene, La Jolla, CA) in 5 ml PBS on ice prior to preparation of the extracts. Cellular RNA isolation and analysis RNA was isolated from the cytoplasmic, nuclear and total cell fractions by the single-step guanidinium thiocyanate method (25). RNA (10 m g) was glyoxalated and size-separated on a 1% agarose 10 mM sodium phosphate gel and transferred to a GeneScreen membrane. The RNA was fixed on the membrane by irradiation with long-wavelength UV light for 2.5 min and baking at 80 C for 2 h. Northern blots were hybridized in the presence of 50% formamide according to GeneScreen protocols in glass cylinders with continuous rotation at 42 C. DNA fragments were labeled by random priming with [a -32P]dCTP and added after 3 h prehybridization at a final concentration of 106 c.p.m./ml. After overnight hybridization, blots were washed to a final stringency of 0.5 or 0.1 SSC, 1% SDS at 65 C (1 SSC is 0.15 M NaCl, 0.015 M sodium citrate) and exposed on Fuji RX X-ray film. Two human IGF-II exon 9 probes were used: a 532 bp EcoRVAvaI fragment encompassing the region between positions 557 and 26 (5-specific probe) and a 1.0 kb SmaI fragment (positions +81/+1094; 3-specific probe). A 26 nt-long oligonucleotide was used as a 28S ribosomal probe: 5-AACGATCAGAGTAGTGGTATTTCACC-3. Hybridization conditions for this probe were identical to the IGF-II probe except for the formamide concentration (25%). Blots were washed for 30 min in 2 SSC, 1% SDS and 30 min in 1 SSC, 1% SDS at 30 C. The autoradiographs were analyzed by densitometric scannning. For the preparation of BS-S/S, BS-AS/S and BS-AS/AS (Fig. 6A), oligonucleotides 5 BamHI 2340/2334 3 and 5 EcoRI +169/+151 3 (numbers indicate positions in exon 9) were used as primers in PCR reactions on plasmids S/S, AS/S and AS/AS respectively. The latter constructs are IGF-II expression plasmids containing elements I/II in the sense (S) or antisense (AS) orientation as indicated and are described in detail in (21). The PCR products were subcloned in the BamHI and EcoRI sites of pBluescript KS+, resulting in the plasmids BS-S/S, BS-AS/S and BS-AS/AS. BS-II was prepared by PCR with primers 5 BamH1 173/155 3 and 5 EcoRI +169/+151 3 on S/S. BS-AS/AS was prepared from BS-AS/AS by replacing the BglII (110)EcoRI (+170) fragment with a PCR product made with primers 5 BglII 14/+4 3 and 5 EcoRI +169/+151 3 on template BS-AS/AS, resulting in a deletion of nucleotides 104/15 from BS-AS/AS. All constructs were checked by restriction enzyme analysis or sequencing if necessary. Synthesis of RNA probes and competitors Radiolabeled RNA probes and unlabeled competitor RNAs were synthesized using T7 RNA polymerase on linearized DNA templates according to instructions of the manufacturer in the presence of 1 mM ATP, GTP and UTP each and 20 m Ci [a -32P]CTP and 0.1 mM CTP (radiolabeled) or 1 mM CTP (unlabeled). Templates used for synthesis of the RNAs: I: BS-S/S linearized with BglII (110), II: BS-II linearized with EcoRI, I/II: BS-S/S linearized with EcoRI, AS/S: BS-AS/S linearized with EcoRI, AS/AS: BS-AS/AS linearized with EcoRI, and AS/AS : BS-AS/AS linearized with EcoRI. After synthesis (1 h) the template was removed by DNase I treatment (1 U in 20 m l reaction mixture for 15 min at 37 C) and the RNA was separated from unincorporated nucleotides by Sephadex G25 spin dialysis. Subsequently, the samples were phenol/chloroform extracted, ethanol precipitated, washed in 70% ethanol and renatured in renaturation buffer (20 mM TrisHCl pH 7.5, 100 mM KCl, 2 mM MgCl2), 5 min at 90 C and 30 min on ice. The integrity of the RNAs was checked by gel electrophoresis. Analysis of RNAprotein interactions For electrophoretic mobility shift assays (EMSAs), radiolabeled RNA probes (105 c.p.m.) were incubated in cellular extract with a protein content of 10 m g in a 10 m l reaction mixture containing 20 mM HEPESKOH pH 7.5, 100 mM KCl, 1 mM MgCl2, 1 mM DTT and 0.01% NP-40 for 45 min on ice. In competition experiments, the competitor RNAs were preincubated with the extract for 5 min before addition of the probe. After the binding reaction the samples were incubated at room temperature for 20 min with a mixture of 12.5 m g/ml RNase A and (...truncated)