Characterization of the UTP-A complex

To gain knowledge about the interaction partners of the tUTP/UTP-A complex, we affinity purified Pol5 and several tUtps using a similar workflow as described by Krogan et al. (2004) (see 4.2.2.13 and 4.2.2.16) (Figure 27).

Figure 27: Workflow for tUtp purifications adapted from Krogan et al. (2004).

According to the workflow presented in Figure 27, the non-tagged control strain and strains expressing the TAP-tagged proteins Utp4, Pol5, and Utp17 were cultivated in rich medium containing glucose (YPD). The obtained cell pellets were processed for affinity purification with IgG-coupled beads. Since the TAP tag consists of two elements for affinity purification, two protein A domains and the calmodulin binding peptide (CBP), separated by a cleavage site for the TEV protease, the elution of affinity purified protein complexes was performed by TEV cleavage and the eluted samples were analyzed by SDS- PAGE followed by either western blotting (see 4.2.5.4) (Figure 28A) or Coomassie staining (see 4.2.5.3) (Figure 28B).

Western blot analysis and detection with anti-CBP antibody showed the enrichment of the bait proteins Utp4, Pol5, and Utp17 in the eluates of the heavy fractions after ultracentrifugation. In lanes corresponding to the eluates of the light fractions, we could not detect any signal, probably due to the presence of bait proteins below the detection limit for the antibody against CBP. In agreement, Coomassie staining revealed a similar enrichment of proteins in the eluted samples from the heavy fractions, whereas only few prominent bands were visible in the eluted samples from the light fractions. This result would suggest the existence of low amounts of ribosome biogenesis factors not associated with pre-ribosomal particles under optimal growth conditions.

Gel lanes containing the tagged proteins were excised, digested with trypsin in gel, and analyzed by HPLC-coupled electron spray ionization mass spectrometry (HPLC-ESI-MS),

as indicated in section 4.2.2.16, by Dr. Astrid Bruckmann and Eduard Hochmuth (department of Biochemistry I). The obtained ESI-MS datasets for purifications obtained from light and heavy fractions were filtered by the peptide coverage, arbitrarily sorted from the highest to the lowest peptide coverage in Utp4, and only proteins related to the synthesis of ribosomes were plotted (Figure 29A and 29B). For light fractions, a peptide coverage higher than 30% was chosen, whereas the limit for heavy fractions was set to 50% peptide coverage both corresponding to the top 50 proteins identified by ESI-MS. The graph of the light fraction exclusively filtered for the peptide coverage is illustrated in supplemental Figure 57.

Figure 28: Analysis by western blotting and Coomassie staining of eluates from control and TAP- tagged strains Utp4, Pol5, and Utp17 obtained using the protocol adapted from Krogan et al. (2004).

Affinity purification using cell extracts from non-tagged control (no tag) and the TAP-tagged strains Utp4 (U4), Pol5 (P5), and Utp17 (U17) was performed as described in Figure 27. Elution of purified proteins was performed by digestion with the TEV protease. (A) 10% of the eluates from light fractions (LF) and heavy fractions (HF) were resolved in 8% SDS-PAGE followed by western blotting. Bait proteins were detected with an antibody against CBP still present in the TAP tag. (B) 30% of the eluates from light (LF) and heavy fractions (HF) were resolved in NuPAGE 4-12% Bis-Tris gradient gel system and stained with commercially available Coomassie staining solution (SimplyBlue SafeStain from Invitrogen). Lanes corresponding to purifications of Utp4, Pol5, and Utp17 (U4, P5, and U17, respectively) were excised, in-gel digested with trypsin, and analyzed by HPLC-coupled electron spray ionization mass spectrometry (HPLC-ESI-MS). Respective identified bait proteins in supernatant lanes were labeled with *.

In the light fractions, proteins engaged in ribosome synthesis were only purified with Utp4 and Utp17 but not with Pol5. Interestingly, Pol5 was not found in the Utp4 and Utp17 affinity purifications. As expected, Utp4 and Utp17 showed association with a very similar set of proteins, which mainly belonged to the tUTP complex and other components of the SSU processome. This result suggests that most of the proteins purified from the light fractions might correspond to disassembly complexes, to the unspecific degradation of pre-rRNA during the affinity purification, or small precursors of the SSU processome. The proteins detected in all three samples were predominantly comprised of heat shock proteins, cell cycle proteins, ATPases, and kinases, unless we filtered by ribosome AFs (see Figure 57). These very abundant proteins are frequently co-purified from yeast cells as background contaminants and their enrichment in all purifications was most likely unrelated to a direct interaction with the bait proteins.

Figure 29: HPLC-ESI-MS datasets of light and heavy fractions obtained from TAP-tagged and affinity purified Utp4, Utp17, and Pol5.

Light and heavy fractions obtained after ultracentrifugation were used for affinity purification with the indicated TAP-tagged proteins. After TEV elution, samples were resolved in Bis-Tris gradient gel system (see Figure 28B), digested with trypsin in gel, and analyzed by HPLC-ESI-MS. (A) MS datasets were obtained from the eluates of the affinity purified light fractions of TAP-tagged Utp4 (black), Utp17 (gray), and Pol5 (white). All proteins found with a peptide coverage higher than 30% and related to the synthesis of ribosomes were plotted. (B) MS datasets were obtained from the eluates of the affinity purified heavy fractions of TAP-tagged Utp4 (black), Utp17 (gray), and Pol5 (white). All proteins found with a peptide coverage higher than 50% and related to the synthesis of ribosomes were plotted. (C) Cartoon depiction of the in vitro-architecture of the tUTP complex as published by Pöll et al. in 2014. Nan1 is the alternative name of Utp17.

In the case of the heavy fractions, Utp4 and Utp17 shared a high number of common interaction partners, as expected from proteins belonging to the same protein complex (Figure 29C). In contrast, Pol5 co-purified with only few SSU-processome related proteins like Utp22, Nop56, and Bfr2 with a similar peptide coverage as in Utp4 or Utp17 purifications (Krogan et al., 2004; Lafontaine and Tollervey, 2000; Pérez-Fernández et al., 2007; Soltanieh et al., 2014). Moreover, neither Pol5 was identified in the affinity purifications of tUtps nor the tUtps were identified in Pol5 purified samples. Interestingly, several proteins corresponding to RNA Pol I subunits (Rpa190 and Rpa135) and AFs involved in the synthesis of both ribosomal subunits (Rrp5, Has1, and Prp43) were found in all purified samples (Bohnsack et al., 2009; Dembowski et al., 2013a; Liang and Fournier, 2006; Venema and Tollervey, 1996). In contrast, some LSU AFs, like Nug1, Nsa1, or Drs1, were only enriched with Pol5 (Baßler et al., 2001; Harnpicharnchai et al., 2001; Ripmaster et al., 1992).

Since we could not exclude that the obtained data differed from published results (Krogan et al., 2004) due to the use of different bait proteins in both affinity purifications (Utp4 and Utp17 instead of Utp8, Utp9, and Utp10), we repeated the experiment using TAP- tagged Utp10 and Pol5 as bait proteins.

Figure 30: Analysis by western blotting and Coomassie staining of eluates from control and TAP- tagged strains Utp10 and Pol5 obtained using the protocol adapted from Krogan et al. (2004).

Affinity purification using cell extracts from non-tagged control (nt) and the TAP-tagged strains Utp10 (U10) and Pol5 (P5) was performed as described in Figure 27. Elution of purified proteins was performed by digestion with the TEV protease. (A) We resolved in 8% SDS-PAGE, 0.05% of the whole cell lysate (WCL), 0.2% of the light fraction obtained after ultracentrifugation (LF), 0.2% of the fraction not bound to the beads (flow-through, FT), 4.5% of the last washing step (W), 5.5% of the eluate (E), and 7% of the beads obtained after elution (BaE). After western blotting bait proteins were detected using an antibody against CBP still present in the TAP tag. (B) 17% of eluates (E) and 14% of beads obtained after elution (BaE) were resolved in NuPAGE 4-12% Bis-Tris gradient gel system and stained with commercially available Coomassie staining solution (SimplyBlue SafeStain from Invitrogen). Lanes corresponding to the beads after elution fractions (BaE) obtained from non-tagged control, Utp10, and Pol5 purifications were excised, in-gel digested with trypsin, and analyzed by HPLC-coupled ESI-MS. Respective identified bait proteins in supernatant lanes were labeled with *.

Cell growth and affinity purification were again performed with the workflow described in Figure 27 and the TEV eluates were analyzed by western blotting (Figure 30A) and Coomassie straining (Figure 30B).

In western blot analysis, we analyzed all steps of the affinity purification performed from the light fractions. Bands for Utp10 and Pol5 were detected in whole cell lysates before and after ultracentrifugation as well as in the beads obtained after the elution. A significant part of the bait proteins present in the light fraction was effectively and strongly bound to the beads (5 to 10% of the total). Since the TEV protease could not release the purified proteins from the beads, elution was achieved by heat denaturation of proteins. In agreement, Coomassie staining only showed a few bands in the TEV-eluted fractions, and many bands were detected in the heat-eluted fraction. As observed for the tUTP components Utp4 and Utp17, the protein patterns from the Pol5-TAP and Utp10- TAP lanes differed completely. Despite the high stringency of the second elution step, we did not observe any clear enrichment of proteins in the sample corresponding to the control strain. Therefore, we decided to analyze the lanes corresponding to the heat- eluted fractions by in gel digestion with trypsin and HPLC-ESI-MS analysis.

Figure 31: HPLC-ESI-MS datasets of light fractions obtained from TAP-tagged and affinity purified Utp10 and Pol5.

Light fractions obtained after ultracentrifugation were used for affinity purification with the indicated TAP- tagged proteins. After TEV elution, samples were resolved in Bis-Tris gradient gel system (see Figure 30B), digested with trypsin in gel, and analyzed by HPLC-ESI-MS. MS datasets were obtained from the bead fractions of the affinity purified light fractions of TAP-tagged Utp10 (gray) and Pol5 (black). All proteins found with a peptide coverage higher than 30% and related to the synthesis of ribosomes were plotted.

The obtained MS datasets of supernatants from Utp10 and Pol5 purifications were filtered, sorted, and plotted as described previously (30% of peptide coverage for proteins involved in ribosome biogenesis; Figure 31). Many of the proteins co-purified with Utp10, most notably Utps, were also identified in the previous affinity purifications of Utp4 and Utp17 (see Figure 29A). In contrast, Pol5 was neither identified in the Utp10 affinity purification nor vice versa. Neither tUTP- nor SSU-processome components were found in the lane corresponding to the Pol5 purification. In addition, several proteins found with Utp10 and Pol5 were also detected to be co-purified in the non-tagged strain. These proteins are very abundant in the yeast cells and are therefore often co-purified as background contaminants (see Figure 58).