Stage-specific assembly events of the 6-MDa small-subunit processome initiate eukaryotic ribosome biogenesis

Chaker-Margot, Malik; Hunziker, Mirjam; Barandun, Jonas; Dill, Brian D.; Klinge, Sebastian

doi:10.1038/nsmb.3111

Cited by 105 publications

(170 citation statements)

References 35 publications

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“…Thus, the UTP-A and ÀB modules might predominantly contribute to the structural nucleation of the evolving 90S preribosome ( Figures 2B,C and 3). To obtain more insight into the timing of 90S assembly, two groups expressed various 3 0 truncations of the pre-18S rRNA and analyzed the associated proteome, thereby revealing the gradual binding of distinct clusters of biogenesis factors [61,62]. Notably, relatively few biogenesis factors dissociate or show reduced association as formation of the 90S preribosome reaches completion [61].…”

Section: Assembly Of the Ssumentioning

confidence: 99%

A Puzzle of Life: Crafting Ribosomal Subunits

Kressler

Hurt

Baßler

2017

Trends in Biochemical Sciences

164

127

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The biogenesis of eukaryotic ribosomes is a complicated process during which the transcription, modification, folding, and processing of the rRNA is coupled with the ordered assembly of $80 ribosomal proteins (r-proteins). Ribosome synthesis is catalyzed and coordinated by more than 200 biogenesis factors as the preribosomal subunits acquire maturity on their path from the nucleolus to the cytoplasm. Several biogenesis factors also interconnect the progression of ribosome assembly with quality control of important domains, ensuring that only functional subunits engage in translation. With the recent visualization of several assembly intermediates by cryoelectron microscopy (cryo-EM), a structural view of ribosome assembly begins to emerge. In this review we integrate these first structural insights into an updated overview of the consecutive ribosome assembly steps. Synopsis of Eukaryotic Ribosome AssemblyRibosomes are the molecular machines that translate the genetic information from the intermediary mRNA templates into proteins [1]. Eukaryotic 80S ribosomes comprise two unequal subunits that contain four different rRNAs and around 80 r-proteins ( Figure 1). The small 40S subunit (SSU) comprises the 18S rRNA and 33 r-proteins (referred to as RPS or S). The large 60S subunit (LSU) comprises the 25S/28S, 5.8S, and 5S rRNA and, in most eukaryotic species, 47 r-proteins (RPL or L); a notable exception is budding yeasts, which lack eL28The act of building a ribosome begins in the nucleolus, where the ribosomal DNA (rDNA) is transcribed into a long pre-rRNA precursor (35S pre-rRNA in yeast) and involves the ordered assembly of the r-proteins with the pre-rRNA, which is concomitantly processed into the mature rRNA species [5][6][7][8] (Figure 1 and Box 1). These assembly and processing events are tightly coupled and occur within preribosomal particles (see Glossary) that travel, as maturation progresses, from the nucleus across nuclear pore complexes (NPCs) to the cytoplasm, where they are ultimately converted into translation-competent ribosomal subunits [5,[9][10][11][12][13] (Figure 2, Key Figure). Given the gargantuan complexity of this process, it is unsurprising that the assembly of eukaryotic ribosomes strictly requires the assistance of a plethora (>200) of mostly essential ribosome biogenesis factors, which are also called trans-acting or assembly factors [5,14,15].Our current knowledge has been mainly obtained by studying ribosome biogenesis in the yeast Saccharomyces cerevisiae. Research conducted in the 1970s defined the r-protein composition of ribosomes, revealed the major pre-rRNA processing intermediates, and uncovered the existence of the 90S, 43S, and 66S preribosomal particles. The following 25 years witnessed the identification of numerous biogenesis factors and attributed functional roles TrendsCryoelectron microscopy analyses of the 90S preribosome show that the biogenesis factors create a casting mold that encloses the nascent pre-40S subunit.Structures of nuclear pre-60S particles reveal that a...

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Section: Assembly Of the Ssumentioning

confidence: 99%

A Puzzle of Life: Crafting Ribosomal Subunits

Kressler

Hurt

Baßler

2017

Trends in Biochemical Sciences

164

127

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“…Applied to cases where the resolution is not fully sufficient for de novo tracing of protein chains and building of complete atomic models, the combination of cryo-EM and CX-MS (Walzthoeni et al 2013) holds great promise for the detailed analysis of even highly dynamic molecular assemblies, including biogenesis intermediates that are located far upstream in the pathway. In addition to state-of-the-art structural biology methods, innovative techniques may need to be developed and applied to trap and isolate these early intermediates (Chaker-Margot et al 2015).…”

Section: Future Perspectivesmentioning

confidence: 99%

“…Ribosome biogenesis starts in the nucleolus, where RNA polymerase I transcribes the 35S pre-rRNA, which contains the sequences for the 18S, 25S, and 5.8S rRNAs as well as internal and external transcribed spacers (Henras et al 2008(Henras et al , 2014. The nascent 35S transcript assembles with the U3 small nucleolar ribonucleoprotein (U3 snoRNP), additional box C/D and H/ACA snoRNPs, 40S subunit assembly factors, and early-binding ribosomal proteins to produce the first pre-ribosomal particle, which is termed the SSU processome, and sediments at 90S (Dragon et al 2002;Grandi et al 2002;Phipps et al 2011;Woolford and Baserga 2013;Chaker-Margot et al 2015;Kornprobst et al 2016;Zhang et al 2016). The U3 snoRNP in the SSU processome is important for pre-rRNA cleavage at site A 2 in the internal transcribed spacer 1 (ITS1) (Hughes and Ares 1991), which has been suggested to be carried out by the PIN domain nuclease Utp24 (Wells et al 2016) and can occur co-or post-transcriptionally in yeast (Udem and Warner 1972;Osheim et al 2004;Kos and Tollervey 2010).…”

Section: Introductionmentioning

confidence: 99%

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

Greber

2016

RNA

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Eukaryotic ribosomes, the protein-producing factories of the cell, are composed of four ribosomal RNA molecules and roughly 80 proteins. Their biogenesis is a complex process that involves more than 200 biogenesis factors that facilitate the production, modification, and assembly of ribosomal components and the structural transitions along the maturation pathways of the preribosomal particles. Here, I review recent structural and mechanistic insights into the biogenesis of the large ribosomal subunit that were furthered by cryo-electron microscopy of natively purified pre-60S particles and in vitro reconstituted ribosome assembly factor complexes. Combined with biochemical, genetic, and previous structural data, these structures have provided detailed insights into the assembly and maturation of the central protuberance of the 60S subunit, the network of biogenesis factors near the ribosomal tunnel exit, and the functional activation of the large ribosomal subunit during cytoplasmic maturation.

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“…We propose that the extent of guide-substrate pairing provides a mechanism to control the association time of C/D RNP with rRNA. In eukaryotes, the precursor rRNA is cotranscriptionally modified by C/D RNPs and H/ACA RNPs and assembled into preribosomes (40)(41)(42)(43). The association of guide RNAs could suppress misfolding of the target site sequence when other sequences required for assembling native structures have not been transcribed.…”

Section: Discussionmentioning

confidence: 99%

Box C/D guide RNAs recognize a maximum of 10 nt of substrates

Yang

Lin

2016

Proc. Natl. Acad. Sci. U.S.A.

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Box C/D RNAs guide site-specific 2′-O-methylation of RNAs in archaea and eukaryotes. The spacer regions between boxes C to D′ and boxes C′ to D contain the guide sequence that can form a stretch of base pairs with substrate RNAs. The lengths of spacer regions and guide-substrate duplexes are variable among C/D RNAs. In a previously determined structure of C/D ribonucleoprotein (RNP), a 12-nt-long spacer forms 10 bp with the substrate. How spacers and guide-substrate duplexes of other lengths are accommodated remains unknown. Here we analyze how the lengths of spacers and guide-substrate duplexes affect the modification activity and determine three structures of C/D RNPs assembled with different spacers and substrates. We show that the guide can only form a duplex of a maximum of 10 bp with the substrate during modification. Slightly shorter duplexes are tolerated, but longer duplexes must be unwound to fit into a capped protein channel for modification. Spacers with <12 nucleotides are defective, mainly because they cannot load the substrate in the active conformation. For spacers with >12 nucleotides, the excessive unpaired sequences near the box C/C′ side are looped out. Our results provide insight into the substrate recognition mechanism of C/D RNA and refute the RNAswapped model for dimeric C/D RNP.B ox C/D RNAs are a large family of noncoding RNAs conserved in archaea and eukaryotes (1-3). The majority of these RNAs function as guides in site-specific 2′-O-methylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (4-7). A few special members, including U3 and U14, small nucleolar RNAs are required for pre-rRNA processing and ribosome assembly. One of the most abundant modifications in rRNAs, 2′-O-methylated nucleotides cluster in functionally important regions and are believed to fine-tune ribosome structure and function (8)(9)(10)(11).Box C/D RNAs have a bipartite structure and contain the C (RUGAUGA; R is a purine) and D (CUGA) box motifs at two ends and the related C′ and D′ box motifs in the internal regions. Each pair of boxes C and D combine into a characteristic kink turn (K-turn) or K-loop structure (12-15). The spacer regions located between boxes D and C′ (D spacer) and between boxes D′ and C (D′ spacer) harbor the guide sequence. The guide sequence can potentially form 10-21 bp with complementary sequences in substrate RNAs and select the fifth nucleotide apart from box D′/D for modification (5).To form a functional ribonucleoprotein (RNP) enzyme, each C/D RNA associates with four core proteins [Snu13, fibrillarin (Nop1 in yeast), Nop56, and Nop58] in eukaryotes (16-23) and with three proteins (L7Ae, fibrillarin, and Nop5) in archaea (24). The homologous eukaryotic Nop56 and Nop58 proteins are replaced by a single protein, Nop5, in archaea. Fibrillarin is the catalyst that transfers a methyl group from S-adenosylmethionine to the target ribose (21, 25). The RNA-guided methylation activity has been reconstituted for archaeal C/D RNPs (24), but not yet for eukaryotic complexes (26).Structural st...

show abstract

Stage-specific assembly events of the 6-MDa small-subunit processome initiate eukaryotic ribosome biogenesis

Cited by 105 publications

References 35 publications

A Puzzle of Life: Crafting Ribosomal Subunits

A Puzzle of Life: Crafting Ribosomal Subunits

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

Box C/D guide RNAs recognize a maximum of 10 nt of substrates

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