Eukaryotic rpL10 drives ribosomal rotation

Abstract

Ribosomes transit between two conformational states, non-rotated and rotated, through the elongation cycle. Here, we present evidence that an internal loop in the essential yeast ribosomal protein rpL10 is a central controller of this process. Mutations in this loop promote opposing effects on the natural equilibrium between these two extreme conformational states. rRNA chemical modification analyses reveals allosteric interactions involved in coordinating intersubunit rotation originating from rpL10 in the core of the large subunit (LSU) through both subunits, linking all the functional centers of the ribosome. Mutations promoting rotational disequilibria showed catalytic, biochemical and translational fidelity defects. An rpL3 mutation promoting opposing structural and biochemical effects, suppressed an rpL10 mutant, re-establishing rotational equilibrium. The rpL10 loop is also involved in Sdo1p recruitment, suggesting that rotational status is important for ensuring late-stage maturation of the LSU, supporting a model in which pre-60S subunits undergo a ‘test drive’ before final maturation.

Mechanistic insight into eukaryotic 60S ribosomal subunit biogenesis by cryo-electron microscopy

“…6). Both of these release reactions depend on the previous incorporation of ribosomal protein uL16 West et al 2005;Bussiere et al 2012;De Keersmaecker et al 2013;Sulima et al 2014a;Weis et al 2015), which is cotranslationally captured by its dedicated chaperone Sqt1 and then assembled into the maturing pre-60S particle Pausch et al 2015). Because Sqt1 can be trapped on pre-60S particles by certain GTPase-deficient mutants of Kre35, Sqt1 itself may also be transiently incorporated into the pre-60S particle during uL16 insertion and may be released together with Nmd3 in a Kre35-dependent manner Pausch et al 2015).…”

“…Because uL16 has been found to be required for the recruitment of SBDS and the release of eIF6 (Bussiere et al 2012;Sulima et al 2014a;Weis et al 2015), and because eIF6-bound D. discoideum late pre-60S subunits contain uL16 and lack Nmd3 (Weis et al 2015), the release of eIF6 has recently been proposed to occur after removal of Nmd3 (Weis et al 2015). Furthermore, available structural data (Pausch et al 2015;Weis et al 2015) and the mapping of the binding sites of Nmd3 on the 60S subunit (Sengupta et al 2010;Matsuo et al 2014) raise the possibility that the presence of Sqt1 and Nmd3 might interfere with the binding of the eIF6-releasing factors due to steric hindrance or overlap of binding sites.…”

Bypass of the pre-60S ribosomal quality control as a pathway to oncogenesis

“…In addition to its role in catalysis (13,14), rpL10/ uL16 plays an important role in the late stages of 60S subunit biogenesis. After initial production of the separate ribosomal subunits in the nucleus, immature and functionally inactive pre-60S subunits are exported to the cytoplasm, where they undergo additional maturation events (15), including incorporation of rpL10/ uL16, before they can associate with mature 40S subunits and engage in protein synthesis (16).…”

“…Tif6 release requires the tRNA structural mimic Sdo1p (19) and the GTPase Efl1, a paralog of eukaryotic elongation factor 2 (eEF2) (20). We have suggested that structural rearrangements of the internal loop of rpL10/uL16 coordinate this final maturation process, resulting in a test drive of the pre-60S subunit to ensure that only properly functioning subunits are allowed to enter the pool of translationally active ribosomes (13,21). Defective ribosomes carrying mutations in rpL10/uL16 specifically fail in this test drive, leading to their degradation through a molecular pathway that is yet to be characterized.…”

The SBP2 protein central to selenoprotein synthesis contacts the human ribosome at expansion segment 7L of the 28S rRNA

“…SBP2 binding site on the human ribosome www.rnajournal.org 1053 conformational rearrangements through the ribosome (Petrov et al 2008;Sulima et al 2013). Gonzalez-Flores et al (2012) propose that the conformational changes in H89 provide the ability of H89 to interact with Domain IV of the elongation factor eEFSec in order to dissociate the ternary complex eEFSec•Sec-tRNA Sec •GTP and to deliver the Sec-tRNA Sec to the A site.…”