Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly

Abstract

Numerous ribosomal proteins have a striking bipartite architecture: a globular body positioned on the ribosomal exterior and an internal loop buried deep into the rRNA core. In eukaryotes, a significant number of conserved r-proteins have evolved extra amino-or carboxy-terminal tail sequences, which thread across the solvent-exposed surface. The biological importance of these extended domains remains to be established. In this study, we have investigated the universally conserved internal loop and the eukaryote-specific extensions of yeast L4. We show that in contrast to findings with bacterial L4, deleting the internal loop of yeast L4 causes severely impaired growth and reduced levels of large ribosomal subunits. We further report that while depleting the entire L4 protein blocks early assembly steps in yeast, deletion of only its extended internal loop affects later steps in assembly, revealing a second role for L4 during ribosome biogenesis. Surprisingly, deletion of the entire eukaryotespecific carboxy-terminal tail of L4 has no effect on viability, production of 60S subunits, or translation. These unexpected observations provide impetus to further investigate the functions of ribosomal protein extensions, especially eukaryote-specific examples, in ribosome assembly and function.

The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site

“…In agreement with an involvement in 60S biogenesis, polysome profile analysis revealed that Δacl4 cells contained reduced levels of 60S subunits, as evidenced by a shortage of free 60S subunits and the accumulation of half-mer polysomes, resulting in a substantial decrease in overall polysome content ( Fig 4E ). Compared to cells that were genetically depleted for Rpl4, which showed as previously reported a striking decrease in 27SB and 7S pre-rRNAs ( S8 Fig ) [ 43 , 48 ], the reduction of these pre-rRNA species was clearly observable but less pronounced in Δacl4 cells ( S8 Fig ). In line with the in vivo purification, sucrose gradient fractionation revealed that Acl4-TAP was exclusively present in the soluble fractions ( Fig 4F ); thus, providing further evidence that Acl4 is not stably associated with pre-60S or mature 60S subunits.…”

Insights into the origin of the nuclear localization signals in conserved ribosomal proteins

“…These contacts suggest that having evolved NLSs at the interface with conserved rRNA allows to use the NLSs to not only promote protein trafficking, but also to facilitate rRNA folding during ribosome biogenesis, thereby coordinating delivery of ribosomal proteins to the nascent rRNA with the rRNA folding. This notion is indirectly supported by recent studies of the ribosome biogenesis in bacteria and yeast 18 19 20 . For instance in E. coli , deletion of the N-terminal extension of protein uS12 causes defects of the 16S rRNA architecture and provokes lethality 18 .…”

Role of the yeast ribosomal protein L16 in ribosome biogenesis

“…In contrast, impairment of 27SA 3 and 27SB L pre-rRNA processing, accompanied by the turnover of the downstream 27SB S and 7S S/L pre-rRNA species, seems to be more specifically related to the depletion of L16. These pre-rRNA processing defects closely resemble those described upon loss-of-function mutations in or depletion of different sets of 60S r-subunit biogenesis factors: (a) the Pwp1 subcomplex (Pwp1, Nop12, Ebp2 and Brx1) [63][64][65], (b) the A 3 factors (Erb1-Nop7-Ytm1 subcomplex, Nop15, Nsa3, Rlp7, Rrp1) ( [66] and references therein) and (c) Has1 [67], and upon depletion of distinct 60S r-proteins including: (a) L8 and L36, which, together with L15, are close neighbours in a region of domain I of 25S/5.8S rRNA, where the 5 0 end of 25S rRNA basepairs with the 3 0 end of 5.8S rRNA [60,65] and (b) L4, L6, L7, L18, L20, L32 and L33, which, together with L16 and L14 are clustered around domain II of 25S/5.8S rRNA [20,22,37,54,68]. Decreased levels of 27SB S pre-rRNA relative to 27SB L pre-rRNA and absence of both 7S pre-rRNA species have also been described upon depletion of L3 [22,29].…”