Impact of two neighbouring ribosomal protein clusters on biogenesis factor binding and assembly of yeast late small ribosomal subunit precursors

Abstract

Many of the small ribosomal subunit proteins are required for the stabilisation of late small ribosomal subunit (SSU) precursors and for final SSU rRNA processing in S. cerevisiae. Among them are ribosomal proteins (r-proteins) which form a protein cluster around rpS0 (uS2) at the "neck" of the SSU (S0-cluster) and others forming a nearby protein cluster around rpS3 (uS3) at the SSU "beak". Here we applied semi-quantitative proteomics together with complementary biochemical approaches to study how incomplete assembly of these two r-protein clusters affects binding and release of SSU maturation factors and assembly of other r-proteins in late SSU precursors in S. cerevisiae. For each of the two clusters specific impairment of the local r-protein assembly state was observed in Rio2 associated SSU precursors. Besides, cluster-specific effects on the association of biogenesis factors were detected. These suggested a role of S0-cluster formation for the efficient release of the two nuclear export factors Rrp12 and Slx9 from SSU precursors and for the correct incorporation of the late acting biogenesis factor Rio2. Based on our and on previous results we propose the existence of at least two different r-protein assembly checkpoints during late SSU maturation in S. cerevisiae. We discuss in the light of recent SSU precursor structure models how r-protein assembly states might be sensed by biogenesis factors at the S0-cluster checkpoint.

Release of the ribosome biogenesis factor Bud23 from small subunit precursors in yeast

“…This analysis revealed that the levels of most pre-40S factors remain relatively unchanged (log2 fold change < 0.5) while Rrp12, Slx9, Bud23, and its binding partner Trm112 increased in response to Rps2depletion (log2 fold change > 0.5) (Figure 3D). The accumulation of Rrp12 and Slx9 is consistent with a recent report that showed these proteins accumulate on pre-40S particles in response to Rps2-depletion (Linnemann et al 2019). Together, these results suggest that the binding of the Rps0-cluster proteins is needed for the release of the Bud23 (and Trm112) from the pre-40S, consistent with predictions based on the structures of human pre-40S intermediates (Figure 1) (Ameismeier et al 2018).…”

“…In State C, the atypical kinase/ATPase Rio2 associates with the pre-40S and binds to the Psite where Bud23 was previously located. This scenario is consistent with a recent report from yeast that found that Rio2 is recruited after the Rps0-cluster proteins (Linnemann et al 2019). The transition between States B and C also coincides with the release of Rrp12 from the pre-40S as well as the incorporation of several RPs and some additional rRNA remodeling (Figures 1 & S1C).…”

“…Rps2 appears to enter the pre-40S concurrently with Rps0 and Rps21 (Figure 1) (Ameismeier et al 2018) as part of a heterotrimeric cluster of ribosomal proteins known as the Rps0-cluster proteins (Linnemann et al 2019). We wondered if Rps0 and Rps21, like Rps2, would be reduced in pre-40S particles from Bud23-depleted cells.…”

Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME

“…It can be detected by western blotting in 90S particles purified via Krr1-TAP (34), suggesting it associates at the stage of 90S particles. Its binding to pre-ribosomes is important for the efficient release of AFs Rrp12 and Slx9 from 40S precursors, as well as for efficient binding of the AF Rio2 (51). In the Rps2-TAP purification, one of the most enriched proteins was Tsr4 (Figure 2B).…”

Conformational proofreading of distant 40S ribosomal subunit maturation events by a long-range communication mechanism

“…Interestingly, additional examples lead to the proposal that communication across nascent ribosomal subunits may play a role in coordinating ribosome biogenesis (see, for example, refs. 27,42,43 ); however, the molecular mechanisms underlying these processes remain subject to future investigations. Moreover, it is likely that similar long-distance communication mechanisms, as described in our study, are also used to ensure proper assembly and functional regulation of other large ribonucleoprotein complexes.…”