Abstract
During eukaryotic 60S biogenesis, the 5S RNP requires a large rotational movement to achieve its mature position. Cryo-EM of the Rix1-Rea1 pre-60S particle has revealed the post-rotation stage, in which a gently undulating α-helix corresponding to Cgr1 becomes wedged between Rsa4 and the relocated 5S RNP, but the purpose of this insertion was unknown. Here, we show that cgr1 deletion in yeast causes a slow-growth phenotype and reversion of the pre-60S particle to the pre-rotation stage. However, spontaneous extragenic suppressors could be isolated, which restore growth and pre-60S biogenesis in the absence of Cgr1. Whole-genome sequencing reveals that the suppressor mutations map in the Rpf2–Rrs1 module and Rpl5, which together stabilize the unrotated stage of the 5S RNP. Thus, mutations in factors stabilizing the pre-rotation stage facilitate 5S RNP relocation upon deletion of Cgr1, but Cgr1 itself could stabilize the post-rotation stage.
Highlights
During eukaryotic 60S biogenesis, the 5S ribonucleoprotein particle (5S RNP) requires a large rotational movement to achieve its mature position
Among the many other structural peculiarities, the Rix1–Rea[1] particle exhibited a 114 Å long, slightly undulating, αhelix inserted between the β-propeller domain of Rsa[4] and the rotated 5S RNP, thereby clamping H38 of the 25S rRNA (A-site finger) at a new position (Fig. 1a)[24]. We suspected that this αhelix corresponds to the small, 120-amino-acid-long protein Cgr[1] (Fig. 1b), which has been suggested to perform a role in pre-60S biogenesis[42,43,44]
Consistent with a predominantly nucleolar/nuclear localization of GFP–Cgr[1] (Fig. 1c), the two different Cgr[1] purifications were co-enriched for ribosome assembly factors that are typically present on intermediate pre-60S particles (i.e. Nog[2], Rix[1] and Arx1), and, Cgr[1] was not found on early nuclear (Ssf[1] and Nsa1) or later cytoplasmic (Lsg1) particles (Fig. 1d, e, Supplementary Fig. 1a, b)
Summary
During eukaryotic 60S biogenesis, the 5S RNP requires a large rotational movement to achieve its mature position. Subsequent cytoplasmic pre-60S maturation steps include the Rei1–Jjj1–Ssa1-dependent dissociation of the export factor Arx[129,30], assembly of the P-stalk and incorporation of Rpp[0] ( known as uL10)[31,32], removal of Nmd[3] by the GTPase Lsg[1] coupled to the incorporation of Rpl[10] ( known as uL16)[33,34,35], and release of the anti-association factor Tif[6] promoted by Efl[1] and Sdo[130,36], which activates the 60S subunit to enter the pool of functionally translating ribosomes. Owing to the nature of these suppressor mutations, which bypass Cgr1’s function in this process, we were able to gain insight into the mechanism of 5S RNP rotation, revealing how untying of the twisted 5S RNP from its surrounding assembly factor network can drive 5S RNP rotation
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