The chaperone Tsr2 regulates Rps26 release and reincorporation from mature ribosomes to enable a reversible, ribosome-mediated response to stress

Abstract

Although ribosome assembly is quality controlled to maintain protein homeostasis, different ribosome populations have been described. How these form, especially under stress conditions that impact energy levels and stop the energy-intensive production of ribosomes, remains unknown. Here we demonstrate how a physiologically relevant ribosome population arises during high Na+ and pH stress via dissociation of Rps26 from fully assembled ribosomes to enable a translational response to these stresses. The chaperone Tsr2 releases Rps26 in the presence of high Na or pH in vitro and is required for Rps26 release in vivo. Moreover, Tsr2 stores free Rps26 and promotes re-incorporation of the protein, thereby repairing the subunit after the stress subsides. Our data implicate a residue in Rps26 involved in Diamond Blackfan Anemia in mediating the effects of Na+. These data demonstrate how different ribosome populations can arise rapidly, without major energy input, and without bypass of quality control mechanisms.

Chaperone-Directed Ribosome Repair after Oxidative Damage

“…2A ). Release was specific for Rps26 and not observed for Rps10, and required the ability of Tsr2 to bind both Rps26 (Tsr2_DWI, ( 20, 22 )) and the 40S subunit (Tsr2_K/E, ( 20 )), demonstrating active release, rather than ‘catching’ of released Rps26 ( Fig. 2B) .…”

“…Rps26 can be selectively released from ribosomes by the chaperone Tsr2 when its binding is weakened by loss of a Mg ion at the RNA-Rps26 interface, thereby producing Rps26-deficient ribosomes ( 20 ), which regulate metal ion homeostasis ( 21 ). Because the Zn-finger forms part of the 40S interface ( Fig.…”

“…1D ), we hypothesized that Tsr2 could similarly release oxidized Rps26 lacking Zn. To examine this idea, we incubated recombinant, purified Tsr2 with untreated 40S, or 40S exposed to 1 or 10 mM H 2 O 2 , and then assayed for Rps26 release ( 20 ). After oxidation, Rps26 was released from 40S subunits, and the extent of release increased with H 2 O 2 concentration ( Fig.…”

“…S3E ). Pre-existing ribosomes were isolated using Rps3-TAP affinity purification ( 20, 21 ) and incorporation of newly-made Rps26-HA was measured by Western blot with or without treatment with 1 mM H 2 O 2 . T: total lysate; E: elution.…”

“…To investigate this possibility we carried out a different pulse-chase experiment ( Fig. 3F & S3E ), where exposure to H 2 O 2 was coupled to a switch from expression of untagged Rps26 to HA-tagged Rps26, which is fully functional ( 20 ) and can be readily distinguished by Western blotting. Moreover, ribosomes made prior to H 2 O 2 addition were traced with Rps3-TAP, which allows for affinity purification ( 21 ).…”