, MgCl2) supplemented with protease inhibitors (Aprotinin 2µg.mL-1 , Leupeptin 1µg.mL-1 , Pepstatin 1µg.mL-1 , PMSF 50µg.mL-1 ) then lysed using sonication for 4 minutes on ice, and centrifuged at 18000 rpm for 25 minutes at 4?C

, Clontech) preequilibrated in lysis buffer, and incubated for 2 hours in 50ml falcons on a rotator at 4 ?C. Beads were collected and washed with lysis buffer then transferred on Bio-Spin columns (Biorad) pre-washed in lysis buffer, Columns were further washed with lysis buffer followed by a wash buffer (1.5x PBS, 50mM Imidazole, 250mM NaCl, 0.1% Igepal, 10% Glycerol, 1mM MgCl2)

, Nmd4 was further dialyzed against calmodulin binding buffer (1x PBS, 100mM NaCl, 0.1% Igepal, 10% Glycerol, 1mM MgCl2, 4mM CaCl2, 1mM DTT) overnight at 4?C in Spectrapor-4, pp.12-14

, then mixed with 500?l Calmodulin Affinity Resin (= 1ml of 50% slurry, Agilent) preequilibrated in binding buffer, MWCO)

. Igepal,

, Proteins were finally dialyzed against 1.5x PBS, 150mM NaCl, 10% (w/v) glycerol, 1mM DTT and 1mM MgCl2 in Spectrapor-4 (12-14 MWCO) then stored at-80?C

, In vitro pull-down assays

, Pull-down was performed using preblocked calmodulin affinity beads (Agilent)

, Preblocking beads. Briefly, in order to preblock beads, 1 ml calmodulin sepharose beads (50% Slurry) were spun, and resuspended in 20mM Hepes, 500mM NaCl, 0.1% Igepal, 0.08mg.ml-1 glycogen carrier, 0.08mg.ml-1 tRNA and 0.8mg.ml-1 BSA. After 2 hours at 4°C, beads were washed 3 times (20mM Hepes, 150mM NaCl, 0.1% Igepal) then resuspended in 500µl 1x binding buffer 250/10 (20mM Hepes

, Each mix contained 2µg of each protein in 30µl reaction mixes. NaCl and glycerol concentrations were adjusted to 150mM and 15% respectively. Five microliters (1/6) aliquots were REFERENCES Boratyn GM, vitro pull-down assay. Recombinant CBP-Nmd4, yeast Upf1 and human Upf1 proteins were thawed on ice, vol.7, p.12, 2012.

J. M. Cherry, E. L. Hong, C. Amundsen, R. Balakrishnan, G. Binkley et al., Saccharomyces Genome Database: the genomics resource of budding yeast, Nucleic Acids Res, vol.40, pp.700-705, 2012.

J. Cox and M. Mann, MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification, Nat Biotech, vol.26, pp.1367-1372, 2008.

J. Cox, N. Neuhauser, A. Michalski, R. A. Scheltema, J. V. Olsen et al., Andromeda: A Peptide Search Engine Integrated into the MaxQuant Environment, J. Proteome Res, vol.10, pp.1794-1805, 2011.

A. Dobin, C. A. Davis, F. Schlesinger, J. Drenkow, C. Zaleski et al., STAR: ultrafast universal RNA-seq aligner, Bioinformatics, vol.29, pp.15-21, 2013.

C. Fu, W. P. Donovan, O. Shikapwashya-hasser, X. Ye, and R. H. Cole, Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase, PLoS One, vol.9, 2014.

E. Garí, L. Piedrafita, M. Aldea, and E. Herrero, A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae, Yeast, vol.13, pp.837-848, 1997.

S. Ghaemmaghami, W. Huh, K. Bower, R. W. Howson, A. Belle et al., Global analysis of protein expression in yeast, Nature, vol.425, pp.737-741, 2003.

G. Giaever, A. M. Chu, L. Ni, C. Connelly, L. Riles et al., Functional profiling of the Saccharomyces cerevisiae genome, Nature, vol.418, pp.387-391, 2002.

V. V. Kushnirov, Rapid and reliable protein extraction from yeast, Yeast, vol.16, pp.857-860, 2000.

M. I. Love, W. Huber, and S. Anders, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, Genome Biology, vol.15, p.550, 2014.

M. Oeffinger, K. E. Wei, R. Rogers, J. A. Degrasse, and B. T. Chait, Comprehensive analysis of diverse ribonucleoprotein complexes, Aitchison JD & Rout MP, vol.4, pp.951-956, 2007.

L. P. Pryszcz, J. Huerta-cepas, and T. Gabaldón, MetaPhOrs: orthology and paralogy predictions from multiple phylogenetic evidence using a consistency-based confidence score, Nucleic Acids Res, vol.39, p.32, 2011.

C. Saveanu, J. Rousselle, P. Lenormand, A. Namane, A. Jacquier et al., The p21-activated protein kinase inhibitor Skb15 and its budding yeast homologue are 60S ribosome assembly factors, Mol. Cell. Biol, vol.27, pp.2897-2909, 2007.
URL : https://hal.archives-ouvertes.fr/pasteur-01404696

J. C. Silva, M. V. Gorenstein, G. Li, J. Vissers, and S. J. Geromanos, Absolute Quantification of Proteins by LCMSE A Virtue of Parallel ms Acquisition, Mol Cell Proteomics, vol.5, pp.144-156, 2006.

J. A. Vizcaíno, A. Csordas, N. Del-toro, J. A. Dianes, J. Griss et al., 2016 update of the PRIDE database and its related tools, Nucleic Acids Res, vol.44, pp.447-456, 2016.

D. Wessel and U. I. Flügge, A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids, Anal. Biochem, vol.138, pp.141-143, 1984.