Puf6 and Loc1 Are the Dedicated Chaperones of Ribosomal Protein Rpl43 in Saccharomyces cerevisiae

Abstract

Ribosomal proteins are highly expressed, and the quality of ribosomal proteins must be rigorously controlled to build up a functional ribosome. Rpl43, ribosomal protein large subunit 43, is located nearby the E-site of ribosomes. In our previous study, we found that Puf6, Loc1, and Rpl43 form a trimeric complex in Saccharomyces cerevisiae. Rpl43 protein levels are under-accumulated in the absence of PUF6 or LOC1. However, why the loss of Puf6 or Loc1 decreased the protein levels of Rpl43 remained unclear. In the present study, we further dissected the connections among these three proteins and found that the processing defects of pre-ribosomal RNA in puf6Δ and loc1Δ are similar to those of the mutant with depletion of Rpl43. The stability of newly synthesized Rpl43 protein decreased slightly in puf6Δ and significantly in loc1Δ. We also found that Puf6 and Loc1 could interact with nascent Rpl43 co-translationally via the N-terminus of Rpl43. While the association and dissociation of Rpl43 with karyopherins did not depend on Puf6 and Loc1, Puf6 and Loc1 interacted with nascent Rpl43 in collaboration. While the N-terminus of Puf6 contained nuclear localization signals for transport, the PUF (Pumilio) domain was essential to interaction with Loc1, Rpl43, and 60S subunits. The C-terminus of Loc1 is more important for interaction with Puf6 and Rpl43. In this study, we found that Puf6 and Loc1 are the dedicated chaperones of ribosomal protein Rpl43 and also analyzed the potential interaction domains among the three proteins. Correct formation of the Puf6, Loc1, and Rpl43 ternary complex is required to properly proceed to the next step in 60S biogenesis.

Mechanism of 5S RNP recruitment and helicase‐surveilled rRNA maturation during pre‐60S biogenesis

“…Similar to Spb1, the RNA‐binding protein Puf6 (Yang et al , 2016; Liang et al , 2019; Gerhardy et al , 2021) is another pre‐60S factor that conformationally traps an extended 25S rRNA subdomain in a premature position (state Puf6 ). Specifically, Puf6 connects H66 to H69, the L1 stalk module, and the B factors, reminiscent of how Dbp10 acts in upstream pre‐60S particles.…”

“…Notably, domain IV consists of several rRNA motifs essential for later ribosome function, such as (i) the long expansion segment (ES27) located near the peptide tunnel exit, with an important role during early protein synthesis (Knorr et al , 2019), (ii) H64–H70, in which the mature 60S subunit form the PTC, and (iii) H69, which, together with h44 form the 40S subunit, contributes to the formation of an important inter‐subunit bridge. To date, several pre‐60S factors have been implicated in participating in the maturation of domain IV, including (i) the essential DEAD‐box helicases Dbp10 and Spb4 (de la Cruz et al , 1998; Burger et al , 2000; Manikas et al , 2016; Bruning et al , 2018; Choudhury et al , 2021; Sailer et al , 2022); (ii) along with Loc1, the Pumilio‐like RNA‐binding protein Puf6 (Pumilio homology domain family member 6) can be cross‐linked to domain IV, and both are required for the stable binding of ribosomal uL14 and eL43 to H66 (Urbinati et al , 2006; Yang et al , 2016; Liang et al , 2019; Gerhardy et al , 2021; Sailer et al , 2022); and the methyltransferase Spb1, which methylates a universally conserved guanine nucleotide in the A‐loop of the 25S rRNA (Kressler et al , 1999; Lapeyre & Purushothaman, 2004), a modification critical to the transition of the pre‐60S particle from the nucleolus to the nucleoplasm (Sekulski et al , 2023; Yelland et al , 2023).…”

Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation

“…In order to understand the specific contribution of the peptide motif, we recorded 1D S4C,D). In presence of the first motif Loc1p (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) the melting curves of the imino signals of U3, U5, and G24 were shifted to higher temperatures, indicating a stabilization of the stem-loop. In summary, melting points of imino signals at the 5´and 3´region, and in the loop region were increased (Figure 4D).…”

“…Expression and purification of Loc1p (1-20)10 was identical to the procedures of wild-type Loc1p (6). Loc1p (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) was expressed with an N-terminal GST tag in E. coli strain BL21 (DE3) Gold pRARE using M9 minimal media supplemented with 15 N ammonium chloride and 13 C glucose. After induction with 0.25 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) in the mid-logarithmic growth phase the cells were cultured for 16 h at 18 °C and subsequently harvested.…”

Eukaryotic ribosome quality control system: a potential therapeutic target for human diseases

“…Meanwhile, dedicated chaperones specifically mediate the transportation of RPs to ensure the successful implementation of this process. Recently, study by Liang et al 38 found that both Puf6 and Loc1 were the dedicated chaperones of ribosomal protein Rpl43, which could form a ternary complex required in biogenesis of 60S LSU. Similarly, Black et al 39 reported that Tsr4 was a cytoplasmic chaperone dedicated to Rps2; Rossler et al 40 carried out a tandem-affinity purification-based screen and identified Nap1 and Tsr4 as direct binding partners of Rps6 and Rps2, respectively.…”