RNA interactome capture in yeast

RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365 nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL) and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3 days to complete. Next to a comprehensive explanation of the method, we focus on our findings about the choice of crosslinking in yeast and discuss the rationale of individual steps in the protocol.


Considerations before you begin
In vivo crosslinking in yeast is challenging compared to eukaryotic cell culture samples, but has been successfully applied [Granneman et al., 2009[Granneman et al., , 2010. PAR-CL (the usage of 4-thio-modified nucleotide analogs in combination with UV light at 365 nm wavelength) has been performed in human cell culture [Hafner et al., 2010] but also in yeast [Creamer et al., 2011].
For in vivo labeling of RNA in yeast, we use 4-thiouracil which is dissolvable in DMSO. Unlike in tissue culture, samples containing 4-thiouracil do not have to be kept in darkness during growth or lysis as daylight is not leading to background crosslinking in yeast according to our observations. Please note that we use the following abbreviations: • 4SU: 4-thiouridine (dissolvable in water, for cultured mammalian cell lines, e.g. HeLa) • 4tU: 4-thiouracil (dissolvable in DMSO, for yeast) Both compounds are suited for incorporation and will result in the same RNA-modification; however we had better success using 4tU in yeast. Another detailed discussion of the protocol (for HeLa cells) is described by Castello et al. [Castello et al., 2013] 2 Cell culture and in vivo labeling • Start a 5 ml YPAD preculture by inoculating with a colony from a plate (use freshly streaked yeast) and grow overnight • Start a 3 times 1 liter SC Ura120µM culture from the preculture (starting OD 600 should be 0.01-0.05) and grow to OD 600 0.4-0.5 at 30°C with 160-180 rpm • Add 500 µM 4-thiouracil (4tU) (10 ml of a 50 mM stock) • Let the cells grow and harvest after 3 hrs • Pellet the cells by centrifugation (4,000 rpm, 15 min, 4°C ) • Resuspend the cells in 40 ml cold water 3 In vivo crosslinking and cell lysis During these steps, keep the cells on ice whenever possible.
• Distribute the sample to 2 petri-dishes (on ice) • Crosslink cells in a Stratalinker device emmiting UV light at 365 nm with a distance of ⇠ 5 cm from the UV bulb • Energy settings: -Set time: 3 min up to 30 min (3 times 10 min with 20 sec pauses in between) or -Set energy: Start from 0.7 J/cm 2 up to 7 J/cm 2 • Harvest the cells from the petri dish with a pipette • Spin down the cells by centrifugation (4,000 rpm, 5 min, 4°C ) • Resuspend the pellet in 2 ml of lysis solution • Distribute the mixture in 2-3 2 ml screw-capped tubes with ⇠ 300 µl acid washed glass beads • Use a FastPrep machine for cell disruption (6 m/s, 60 sec): 5 repetitions with 30 sec pause in between to allow cooling of the cells • Clear the lysate by centrifugation (12,000 rpm, 2 min at 4°C ) and transfer the supernatant to 50 ml tubes • Remove a 10-50 µl aliquot as input control and freeze at -80°C • Snap-freeze the rest in N 2 and store at -80°C

Bead preparation
• Take 1 ml oligo d(T) 25 magnetic beads (per litre of starting culture) and place in a magnetic separator to remove the supernatant • Add 1 ml lysis bu↵er and let stand for 1 min at room temperature • Remove the supernatant again and repeat this wash step 2 times

Binding and purification
• Increase the volume of the frozen eluate to 40 ml (for 3 liters of starting yeast culture or at least 25 ml if less starting material was used) using lysis bu↵er • Use ⇠ 1 ml cell lysate to resupend the beads, add the beads to the lysate and rotate for 1 h at 4°C • Collect the beads by placing the tubes in a magnetic separator at 4°C , wait until all beads are e ciently concentrated at the tube wall (takes up to 30 min) • Save the supernatant and store on ice • Resuspend the beads in 40 ml lysis bu↵er and rotate for 5 min at 4°C • Discard the supernatant and wash 2 times with each bu↵er (40 ml / 25 ml) at room temperature for 5 min: -Wash bu↵er 1 -Wash bu↵er 2 -Low salt bu↵er • Elute the mRNP-complexes by adding 1 ml elution bu↵er and shake at 55°C for 10 min • Remove a 50 µl aliquot as elution control and freeze at -80°C • Re-activate the beads for a second round of binding and purification by washing three times with lysis bu↵er before adding to the supernatant from the first round and repeat the complete procedure We usually perform 2-3 rounds of purification with the same beads. Elutions maybe pooled afterwards or analysed seperately. RNA and proteins are still covalently bound. For analysis of purified proteins, RNase treatment is necessary and for analysis of purified (m)RNA, proteinase K treatment is advisable.
6 RNase treatment and concentration • Add 5 x RNase bu↵er to the elution fraction and adjust volume with water • Add 0.5 µl RNase A (10 mg/ml) and 0.5 µl RNase T1 (100 U/µl) and incubate for 1 h at 37°C • Load sample in 500 µl fractions onto an Amicon filter unit with a cut-o↵ of 3 kD and centrifuge for 30 min at full speed in a table-top centrifuge; repeat until all sample is concentrated • Load 400 µl 50 mM NaCl and centrifuge to remove residual bu↵er components • Recover concentrated samples (⇠ 25 µl) At this point, the purified proteins can be analysed by SDS-PAGE or proteomics techniques.

B.1 Crosslinking e ciency
Determination of crosslinking e ciency is complex and varies from protein to protein (own observation).
RNA analysis Analyse total RNA using a NanoDrop or other UV-spectrophotometer at 260 nm wavelength. Measure RNA concentration of all elution fractions. Normally, the crosslinked fraction contains less RNA than non-crosslinked controls (factor in yeast ⇠ 1.6) RNA-labeling e ciency We used a dot-blot-based assay to evaluate 4tU-incorporation in RNA of yeast using the method described by Dölken and colleagues [Dölken et al., 2008]. Also, HPLC-based methods exist, e.g. as described by Spitzer et al. [Spitzer et al., 2014].

B.2 Quality control
During the purification, some indicators show if the crosslinking was successful. You can use these for in-process quality control.

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QC2: Bead appearance After washing with wash bu↵er 2 (5), a brownish "halo" appears around the magnetic beads when proteins were successfully crosslinked.
QC2: Bead appearance After washing with wash bu↵er 2 (5), a brownish "halo" appears around the magnetic beads when proteins were successfully crosslinked.

B.2 Problems during purification
White clouds/precipitates during binding and washes These white precipitates are most likely chromatin or DNA complexes. Increase the volume of bu↵er during the lysis and washes until they dissapear.

B.3 Problems during purification
White clouds/precipitates during binding and washes These white precipitates are most likely chromatin or DNA complexes. Increase the volume of bu↵er during the lysis and washes until they dissapear.
Brownish film on tube wall after low salt bu↵er washes or elution Beads sticking to the tube wall may impact e ciency of protein purification. One solution is to add NP40 to a final concentration of 0.05% to wash bu↵ers 1 and 2. However, adding NP40 in too high concentrations or adding to the low salt bu↵er may render the elusions incompatible with any downstream mass spectrometry applications!

B.4 Timing
The protocol described here to purify mRBPs from yeast cells requires a lot of "hands-on" time and is usually done in three days: • Day 1: cell growth, labeling, crosslinking and cell lysis • Day 2: purification (2-3 rounds) • Day 3: RNase digestion, concentration, SDS-PAGE, silverstaining or western blot Two or three rounds of purification from 1-3 litre cultures usually takes a complete day which is mainly due to waiting time for complete magnetic bead separation. Moreover, during the many short-timed wash-magnet cycles during the washing steps, it becomes di cult to do other experiments in between.