UV-Induced Cross-linking in RNA-Protein Complexes

2.2.5.1 UV-Cross-linking of Brat-NHL protein with hb RNA

The Brat-NHL-hb RNA complex was assembled by incubating 1 nmol of in vitro transcribed hb RNA with 1 nmol of recombinant Brat-NHL protein (provided by Inga Loedige from Dr. Gunter Meister’s lab of RNA Biology, Biochemistry Center Regensburg, University of Regensburg, Germany) making volume upto 100 µl with the buffer (20 mM Tris-Cl pH 8, 150 mM NaCl) for 1 h on ice. For UV-cross-linking, the assembled complex was taken in a microtiter plate placed on aluminum block on ice at a distance of 1 cm from the lamps and UV-irradiated at 254 nm for 10 min. The sample was then ethanol precipitated for overnight. The pellet was dissolved in 50 µl of 4 M Urea in 50 mM Tris-Cl pH 7.9 and diluted to 1M Urea by adding 150 µl of 50 mM Tris-Cl pH 7.9. The RNA was hydrolyzed using 1 µl of RNase A (1 µg/µl) and T1 (1 U/µl) at 52 °C for 1 h followed by 1 µl of Benzonase (25 U/µl) in the presence of 1 mM MgCl2 at 37 °C for 1 h with continuous shaking at 500 rpm. The protein/peptide was digested using trypsin in enzyme to protein ratio of 1:20 (w/w) at 37 °C for 16 h with shaking at 500 rpm.

The sample was then desalted by C18 reversed phase chromatography and enriched by TiO2 solid phase extraction as described by Kramer et al., 2011 (details in materials and methods heading 2.2.5.3). The control (non-UV-irradiated) sample was also processed in parallel to the UV-irradiated sample.

51 For mass spectrometric analysis, the samples were dried in the SpeedVac and reconstituted in 12 µl of 5% (v/v) acetonitrile, 0.1% (v/v) formic acid. The samples were analyzed by LTQ Orbitrap Velos mass spectrometer (Thermo Scientific).

2.2.5.2 UV-Cross-linking of CWC2 protein with U4 and U6 snRNAs

The UV-cross-linking analysis of CWC2 protein with U4 and U6 snRNAs was done according to the protocol published by Schmitzová et al., 2012. The CWC2-U4 snRNA and CWC2-U6 snRNA complexes were reconstituted by incubating 100 µg of CWC2 for 30 min on ice with 3 µg of U4 and U6 snRNAs separately making volume upto 100 µl with the buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM DTT). The sample was UV-irradiated at 254 nm for 10 min in a microtiter plate kept on an aluminum block on ice at a distance of 1 cm from the light source. The sample was ethanol precipitated for overnight. The pellet was dissolved in 50 µl of 4 M Urea in 50 mM Tris-Cl pH 7.9 and adjusted to final concentration of 1 M Urea by adding 150 µl of 50 mM Tris-Cl pH 7.9. The hydrolysis of RNA was carried out by 1 µl of RNase A (1 µg/µl) and T1 (1 U/µl) at 52 °C for 1 h followed by 1 µl of Benzonase (25 U/µl) in the presence of 1 mM MgCl2 at 37 °C for 1 h with shaking at 500 rpm. The protein was digested using trypsin in enzyme to protein ratio of 1:50 (w/w) at 37 °C for 16 h with continuous shaking at 500 rpm. The desalting and enrichment of the sample were carried out by C18 reversed phase chromatography and TiO2 solid phase extraction respectively according to the protocol described by Kramer et al., 2011 (detail in materials and methods heading 2.2.5.3). The control (non-UV-irradiated) samples were also processed in parallel to the UV-irradiated samples. For mass spectrometric analysis, the samples were dried in the SpeedVac and reconstituted in 12 µl of 5% (v/v) acetonitrile, 0.1% (v/v) formic acid. The samples were analyzed by Q-Exactive mass spectrometer (Thermo Scientific).

2.2.5.3 UV-Cross-linking of RNA-Protein Complex from HeLa Nuclear Extract Assembled on PM5/MINX pre-mRNAs

The purified RNA-protein complex (H/E complex) from HeLa nuclear extract was UV-cross-linked according to the protocol described by Qamar et al., 2015. The

52 sample was taken in a volume of 1 ml (protein concentration 0.3 mg/ml) in pre-cooled custom-made glass dishes, with a planar surface and an inner diameter of 3.5 cm, so the depth of the sample solution was approximately 1 mm. The dishes were kept on an aluminum block on ice at a distance of 1 cm from the light source. The sample was UV-irradiated at 254 nm for 10 min. The sample was pooled in the Corex glass tube and ethanol precipitated for overnight. The pellet was dissolved in 100 µl of 1% (w/v) SDS in size exclusion (SE) running buffer by shaking and diluted to final concentration of 0.1% (w/v) SDS with size exclusion (SE) running buffer. The protein was digested with trypsin in 1:50 (w/w) enzyme to protein ratio at 37 °C for 16 h with continuous shaking at 500 rpm. The sample was then again ethanol precipitated. The pellet was re-dissolved in 5 µl of 1%

(w/v) SDS in size exclusion (SE) running buffer and diluted upto 0.1% (w/v) SDS with SE running buffer. The sample was injected into the SMART system equipped with Superdex 75 PC 3.2/30 column running in SE running buffer with a flow rate of 40 μl/min at room temperature. The fractions containing RNA were pooled together and ethanol precipitated overnight. The pellet was dissolved in 4M Urea, in 50 mM Tris-Cl pH 7.9 and adjusted to final concentration of 1 M Urea by adding 150 µl of 50 mM Tris-Cl pH 7.9. Digestion of RNA was carried out by 1µl of RNase A (1 µg/µl) and T1 (1 U/µl) at 52 °C for 1 h followed by 1 µl of Benzonase (25 U/µl) in the presence of 1 mM MgCl2 at 37 °C for 1 h with shaking at 500 rpm. The protein was digested by using trypsin in 1:20 (w/w) enzyme to protein ratio at 37 °C for 16 h with continuous shaking at 500 rpm. The sample was desalted and enriched by C18 reversed phase chromatography and TiO2

solid phase extraction respectively according to the protocol described by Kramer et al., 2011. For the C18 reversed phase chromatography the columns were prepared in-house by fitting 2 mm² piece of coffee filter paper at the end of 10 µl pipette tip as frit material. The columns were packed with the C18 material suspended in 100% (v/v) methanol with the help of 1 ml combitip to give a bed height of 3 mm. The columns were fitted in 2 ml microcentrifuge tubes by making holes in the lid and washed by applying 60 µl of 95% (v/v) acetonotrile, 0.1% (v/v) formic acid, then 80% (v/v) acetonitrile, 0.1% (v/v) formic acid followed by 50%

(v/v) acetonitrile, 0.1% (v/v) formic acid and finally by 0.1% (v/v) formic acid with

53 centrifugation at 5000 rpm for 5 min after each step. Meanwhile, 10 µl of 100%

(v/v) acetonitrile and 2 µl of 10% (v/v) formic acid were added into the sample to make an end concentration to 5% (v/v) acetonitrile and 0.1% (v/v) formic acid respectively. The sample was vortexed and centrifuged for 2 min at 13,000 rpm at room temperature to remove precipitates. The sample was then applied onto the column in portions of 60 µl by centrifugation at 5000 rpm for 5 min after each step. The column was then washed twice with 60 µl of 0.1% (v/v) formic acid and the sample was eluted, once with 60 μl of 20% (v/v) acetonitrile, 0.1% (v/v) formic acid, twice with 50% (v/v) acetonitrile, 0.1% (v/v) formic and once with 80% (v/v) acetonitrile, 0.1% (v/v) formic acid by centrifugation at 5000 rpm for 5 min. The eluate was then dried in the SpeedVac for 45 min. In order to remove the non-cross-linked peptides TiO2 solid phase extraction was performed. The TiO2

columns were prepared with the TiO2 suspension in the same manner as for C18 reversed phase chromatography. The columns were washed twice with 60 μl of buffer B by centrifugation at 3000 rpm for 5 min. Meanwhile, the sample was dissolved in 100 μl of buffer A by vortexing and ultrasonification for 1 min and loaded onto the column in the portion of 60 μl by centrifugation at 3000 rpm for 5min. The column was then washed three times with buffer A and four times with buffer B, each time with centrifugation at 3000 rpm for 5 min. The sample was eluted thrice with 40 μl of buffer C by centrifugation at 3000 rpm for 5 min. The control (non-irradiated) sample was also processed in parallel to the UV-irradiated sample. For mass spectrometric analysis, the samples were dried in the SpeedVac and reconstituted in 12 µl of 5% (v/v) acetonitrile, 0.1% (v/v) formic acid. The samples were analyzed by LTQ Orbitrap Velos mass spectrometer (Thermo Scientific).

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