Nob1p interaction with precursor subunits

2.3.1 Nob1p co-purified pre-SSU rRNA independent of the in vivo assembly of some platform or head domain r-proteins

As previously mentioned, yeast Nob1p presumably harbors the endonuclease activity converting 20S pre-rRNA into mature 18S rRNA. To directly address the association of Nob1p after shutdown of various r-proteins of the small subunit, pre-rRNAs co-purifying with TAP-tagged Nob1p in the corresponding pGAL-RPSX strains were analyzed (Figure 48 A-E).

The C-terminal fusion of Nob1p with the TAP-epitope obviously resulted in a phenotype itself.

Either by indirect or direct influence of the TAP-tag, final 18S rRNA maturation was slowed down and 20S pre-RNA was heavily accumulating (Figure 48 F and G, compare always lane 1 and 5). This observation might be the reason that TAP-tagged Nob1p in the pGAL-RPS5 strain led to roughly 20% lowered doubling times (Supplemental Figure 1 A). Upon in vivo depletion of rpS5 or rpS14 for at least 2 hours, only strongly reduced levels of 20S pre-rRNA were detectable (Figure 48 F and G, compare 20S in lanes 1 to 4). TAP-fusion of Nob1p in these strain delayed occurrence of the depletion phenotype by more than twice the normal time (Figure 48 F and G, compare always 20S in lanes 2 and 6, 3 and 7, etc.).

In summary, TAP-tagged Nob1p still co-purified very efficiently 20S pre-rRNA after shutdown of rpS2, rpS5, rpS14 and rpS20 (Figure 48 A-D). Additionally, the immunoprecipitation efficiencies suggest that Nob1p exists only sub-stoichiometrically amounts in yeast. A high level of 20S pre-rRNA in the input fractions did not correlate with an elevated rate of co-precipitation. The IP ratio rather looks like Nob1p-TAP could be saturated with 20S pre-rRNA and could only co-precipitate a certain amount of 20S pre-rRNA (Figure 48 A-D, compare 20S levels in IP lanes).

To further characterize the interaction of Nob1p with precursor subunits after shutdown of rpS5, Nob1p-TAP associated pre-rRNAs were purified in a yeast strain expressing HA-tagged alleles of rpS5, rpS5-ΔC, rpS5-Δloop or rpS5-short-loop (Figure 48 E). As shown before, Nob1p was able to interact with pre-SSU rRNA independent of in vivo assembly of rpS5 (Figure 48 B). Co-expression of the HA-tagged variants didn't alter this co-precipitation pattern (Figure 48 E).

These results support the data obtained by mass spectrometry (see 2.2.4.7 and 2.2.6.4), namely that the virtually blocked conversion of 20S pre-rRNA to mature 18S rRNA in the rpS5-ΔC (and rpS5-short-loop) as well as SAS20 containing SSU precursors is not due to the absence of the putative endonuclease Nob1p.

Results

Figure 48. Analysis of Nob1p-TAP interactions with SSU precursors after depletion of various r-proteins of the small subunit

(A)-(E) Northern blot analysis of RNA co-purified with TAP-tagged Nob1p. RNA was extracted from Input (In) and immuno-purified (IP) fractions. Probes used for detection of (pre-) rRNA species are depicted right-hand. 200 mM salt (KCl, see 5.2.7.1) was used for cell breakage, binding and washing of the immunoprecipitations. Cells were grown overnight in YP-galactose, diluted in YP-galactose and subsequently expression of pGAL-RPSX was shut down in YP-glucose medium for the time indicated. (A) Yeast strain ToY2065, in which RPS2 is under control of the GAL1 promoter and Nob1p is TAP-tagged. (B) Yeast strain ToY1765, in which RPS5 is under control of the GAL1 promoter and Nob1p is TAP-tagged. (C) Yeast strain ToY2067, in which RPS14 is under control of the GAL1 promoter and Nob1p is TAP-tagged. (D) Yeast strain ToY2066, in which RPS20 is under control of the GAL1 promoter and Nob1p is TAP-tagged. (E) Yeast strain ToY1765, in which RPS5 is under control of the GAL1 promoter and Nob1p is TAP-tagged was transformed either with an empty vector (YEplac181) or vectors coding for HA-tagged full length rpS5 (ToP1162), rpS5-ΔC (ToP1156), rpS5-Δloop (ToP1157) or rpS5-short-loop (ToP1158) under the control of a constitutive promoter. Cells were grown overnight in selective media containing galactose, diluted in YP-galactose and subsequently expression of pGAL-RPS5 was shut down for 3 hours in YP-glucose medium. Nob1p-TAP associated SSU precursors were purified from these strains as described before.

(F) and (G) Steady state (pre-) rRNA processing analyses of pGAL-RPS5 or pGAL-RPS14 with or without TAP-tagged NOB1. Cells were grown overnight in YP-galactose, diluted in YP-galactose and subsequently expression of pGAL-RPSX was shut down in YP-glucose medium for the time indicated. At each time point 1 OD(600) of cells was harvested, RNA was extracted and analyzed by Northern blotting. Probes used for detection of (pre-) rRNA species are depicted right-hand. (F) Yeast strains pGAL-RPS5 (ToY1659) and pGAL-RPS5 + NOB1-TAP (ToY1765). (G) pGAL-RPS14 (ToY1658) and pGAL-RPS14 + NOB1-TAP (ToY2067).

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2.3.2 20S pre-rRNA might be stabilized through TAP-tag fusion of Nob1p

In strains depleted for rpS5 or rpS14, a potential 20S pre-rRNA processing delay or protection of this pre-rRNA species from degradation was already visible on steady state rRNA analysis (Figure 48 F and G, see also 2.3.1). Several generation times after depletion of GAL1-promoter driven rpS14 expression this effect is plainest visible, since a small amount of 20S pre-rRNA could still be detected (Figure 48 G, lane 8).

A possible explanation for the higher ratio of newly synthesized 20S to 18S rRNA when full length rpS5 was expressed (compare 20S to 18S ratio in Figure 49 A and B, lanes 5 to 8) might be reduced endonucleolytic activity of TAP-tagged Nob1p. If one compares the levels of newly made 20S pre-rRNA over time, in the strains expressing rpS5-ΔC with or without TAP-tagged Nob1p, while in both no final pre-18S rRNA maturation occurred, 20S pre-rRNA accumulated in the Nob1p-TAP strain (compare 20S levels in Figure 49 A and B, lanes 9 to 12).

It is tempting to speculate that Nob1p mediated D-site processing still occurs in all these cases, but maybe due to misfolded rRNAs or unstable assembly of r-proteins, the immature subunits become degraded. TAP-tag fusion of Nob1p may lead to reduced nuclease activity and thereby to an apparent stabilization of precursor subunits (see also discussion in 3.3).

Figure 49. Pulse-chase analysis of newly synthesized rRNA in pGAL-RPS5 with or without NOB1-TAP

(A) and (B) The subsequently described strains were transformed with an empty vector (YEplac181) or vectors coding for HA-tagged full length rpS5 (ToP1162) or rps5-ΔC (ToP1156) under the control of a constitutive promoter. Cells were grown overnight in selective media, diluted in YP-galactose and expression of pGAL-RPS5 was shut down for 2 hours in YP-glucose medium. The cells were pulsed with 5’,6’-[³H] uracil for 5 minutes at 30°C and chased with non-marked uracil for the time indicated. Total RNA was extracted and separated by gel electrophoresis, radio-labeled RNA was visualized by fluorography (see also 5.2.5.4). (A) Yeast strain ToY1659 (pGAL-RPS5) was transformed with the above described HA-tagged alleles and analyzed by pulse-chase labeling.

(B) Yeast strain ToY1765 (pGAL-RPS5 + NOB1-TAP) was transformed with the above described HA-tagged alleles and analyzed by pulse-chase labeling.