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Supplementary Information
Methods
Purification of the U3 snoRNP
Unless otherwise indicated, yeast strains were derived from YPH499 (Mata, ura3-52, lys 280, ade2-101, trp1-∆63, his3-∆200, leu2-∆1) or YPH500 (Mat, ura3-52, lys 2-80, ade2-101,
trp1-∆63, his3-∆200, leu2-∆1). Using YPH500, a FLAG-6XHIS tag was inserted at the Cterminus of Mpp10 by chromosomal integration of a PCR fragment bearing the HIS3 marker.
Similarly, the TAP tag 1 was inserted at the C-terminus of Nop5/58 in the Mpp10-FLAG tagged
strain using the TRP marker. This strain thus bears genes encoding Mpp10-FLAG-6XHis and
Nop5/58-TAP expressed from their chromosomal loci. The doubly tagged strain was grown to
mid-log phase (A600 = 0.5) in YPD and harvested. The cell pellet was washed twice with cold
water and resuspended in one volume of TMK150 (25 mM Tris-HCl, pH 7.6, 10 mM MgCl2,
150 mM KCl, 1 mM DTT, 0.1% NP-40) containing Complete™ protease inhibitors (Roche
Molecular Biochemicals). Typically, a 5-liter culture gave 15 g of yeast, which were
resuspended in 15 ml of TMK150. Droplets of resuspended cells were flash frozen in liquid
nitrogen and stored at -80˚C. Frozen cells were ground in a stainless steel Waring blender in the
presence of liquid nitrogen. After addition of another volume of TMK150, the mixture was
thawed at 4˚C. The cell debris were removed by centrifugation at 30,000 X g for 30 min. The
extract was incubated in batch with 400 µl IgG beads (Amersham Pharmacia Biotech) for 2
hours at 4˚C. The beads were poured in a Poly-Prep® column (Bio-Rad) and the flow through
was collected. The beads were first washed with 90 ml (225 column volume) TMK150 buffer
with protease inhibitors, and then washed with 30 ml (75 column volume) TEV protease buffer
(TMK150 buffer containing 0.5 mM EDTA). 300 U of TEV protease (Gibco BRL) were diluted
in 3 ml TEV protease buffer and added to the beads. The column was placed on a nutator and
incubated for 2 hours at 16˚C. The eluate was then incubated in batch with 200 µl anti-FLAG
beads (Sigma) on a nutator for 2 hours at 4˚C. The flow through was collected, the beads were
washed with 40 ml (200 column volume) TMK150 lacking NP-40, and the complex was eluted
with 0.5-1 ml of this buffer containing FLAG peptide (1 mg/ml; Sigma). Aliquots of the
different purification steps were flash frozen in liquid nitrogen. The presence of Mpp10 in these
fractions was determined by western blotting with anti-Mpp10 polyclonal antibodies 2. Analysis
of RNAs present in the extract and in the purified fractions was performed by [5’-32P] pCp
labeling 3.
Identification of proteins by mass spectrometry
The sample was digested with trypsin (1 mg; 48 hrs; Promega), acidified with glacial acetic
acid (Sigma) and the resulting mixture of tryptic peptides was then analyzed by on-line,
nanoflow HPLC/electrospray ionization mass spectrometry on an LCQ ion trap instrument
(ThermoFinnigan, San Jose, CA) 4,5. A linear HPLC gradient ( 0-100 % B in 1.5 hr where A =
0.1% acetic acid and B = acetonitrile with 0.1% acetic acid) was used for the mass spectrometry
analysis. MS/MS spectra were extracted, like scans grouped, charge states assigned and poor
quality spectra eliminated using a suite of in-house software termed Proteofarm. MS/MS spectra
were searched against the yeast nrpep FASTA database using SEQUEST (ThermoFinnigan).
All sequence assignments were verified by manual inspection of the MS/MS spectra.
Bioinformatics analysis
The novel protein sequences were analyzed using YPD 6, by BLAST searches 7 and for
motifs using the PROSITE database 8 and the Coils program 9.
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