Johann Stojko1,2, Adrien Dupin3,4, Stéphane Chaignepain5, Lionel Beaurepaire3,4, Amélie Vallet-Courbin4, Alain Van Dorsselaer1,2, Jean-Marie Schmitter5, Lionel Minvielle-Sébastia4, Sébastien Fribourg3,4, and Sarah Cianférani1,2
- BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France
- IPHC, CNRS, UMR7178, 67087 Strasbourg, France
- IECB, Université de Bordeaux, IECB, 33607 Pessac, France
- INSERM U1212, UMR CNRS 5320, 33076 Bordeaux, France
- CBMN, UMR 5248, CNRS, Université de Bordeaux, INP Bordeaux, 33607 Pessac, France
The cleavage/polyadenylation factor IA (CF IA) is a yeast multiprotein complex that consists of Rna14, Rna15, Pcf11 and Clp1 proteins, and is involved in the 3′-end maturation of mRNAs. Structural data have been reported for the individual protein partners and binary complexes; however, little is known about the molecular architecture of the entire CF IA assembly. Here, we report a thorough characterization of complete recombinant CF IA assembly and its subcomplexes using a combination of mass spectrometry (MS) approaches. We first focused on the Rna14p:Rna15p and Pcf11p:Clp1p subcomplexes in order to obtain a detailed picture of their interactions. Native MS and crosslinking MS showed that the intact CF IA assembly exists in solution as pentameric and hexameric species, composed of two copies of Rna14p, one each of Pcf11p and Clp1p, and one or two of Rna15p, respectively. Partial denaturation experiments followed by native MS along with crosslinking analysis revealed two building blocks: Rna14p:Rna15p multimer subcomplexes assemble with Pcf11p:Clp1p heterodimers to form the CF IA complex. We then used ion mobility-MS (IM-MS) to investigate the conformational changes induced upon CF IA assembly. The new information on the CF IA assembly process provided by this combination of MS approaches (native MS, crosslinking MS and IM-MS) allowed us to discuss a topological model of the CF IA assembly.
CovalX Technology Used (Click each option to learn more)
PCR was performed on yeast genomic DNA in order to obtain the full-length protein-coding sequence before the sequences were cloned into a modified version of pET-15b, pET28b, pCDF, and pLysS plasmids. BL21(DE3) cells were transformed for co-expression and plated on Petri dishes with 50% of the antibiotic concentration used in mini-cultures (10 ml of LB medium, 37 °C, antibiotics) before the induction of protein expression with the addition of 1 mM IPTG. The induced samples were incubated overnight at 15 °C before cells were harvested by centrifugation and lysed by sonication with buffer (1.5x PBS, 1 mM MgAc2, O.1% NP-40, 20 mM imidazole, 10% (v/v) glycerol) so that the supernatant could be removed and incubated. The supernatant was incubated with His-affinity resin for 30 minutes at 4 °C and then the resin was washed for SDS-PAGE analysis of the bound proteins with loading buffer.
Samples were purified using Terrific Broth that had been inoculated with pre-culture and incubated. The harvested cell pellet was lysed by using a homogenizer 3 times and then the extraction was centrifuged for 1 hour at 50,000g 4 °C. The supernatant was incubated with His-affinity resin and then contaminants were removed by washing with a buffer (2 mM Tris-HCl, pH 7.5, 150 mM NaCl) in a column. The proteins were extracted from the column with an imidazole gradient up to 150 mM and then loaded onto a 5 ml column for separation using a NaCl gradient (up to 1 M in 25 mM Tris-Hcl, pH 7.5). Using another column and an NaCl gradient (up to 500 mM in 25 mM Tris-Hcl, pH 7.5), the His-Pcf1 1pΔ Q20:Clp1p, His-Pcf11p[454-563]:Clp1p and Rna14p complexes were purified.
Disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), disuccinimidyl glutarate (DSG), CyanurBiotinDimercaptoPropionylSuccinimide (CBDPS H8/D8), and glutaraldehyde were used as cross-linkers. BS3 and glutaraldehyde were solubilized in water while the others were solubilized in pure dimethylformamide. Using a range of 1.25 to 10 μM in 10 μl, the entire CF IA ΔQ20 complex was cross-linked at a final concentration of 0.2 mg/ml (glutaraldehyde was at 0.1% (w/v)). The samples with added cross-linker were incubated for 1,3,6 or 15 hours at 25 °C. After their allotted incubation time, the samples were mixed with matrix (sinapic acid (10 mg/ml), 50% acetonitrile diluted in deionized water, 0.1% TFA) in a 1:1 (v/v) ratio. 1 μl of these mixtures was sampled and dropped on a MALDI target. 1 μL of each sample was also analyzed without the addition of the cross-linker. The target was analyzed using a mass spectrometer that had been modified with a CovalX HM2 detection system. The data were background subtracted and smoothed using the CovalX Complex Tracker software.