Coordinated Ribosomal ITS2 RNA Processing by the Las1 Complex Integrating Endonuclease, Polynucleotide Kinase, and Exonuclease Activities

Gasse, Lisa; Flemming, Dirk; Hurt, Ed

doi:10.1016/j.molcel.2015.10.021

Cited by 98 publications

(134 citation statements)

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“…During the subsequent maturation steps, the 27SB pre-rRNA is processed within the ITS2 region: consecutive endo-and exonucleolytic cleavages catalyzed by the Las1 complex result in the formation of the 7S and 25S 0 pre-rRNAs [95] (Figure 2L). Interestingly, cryo-EM structures of pre-60S ribosomes (e.g., the Nog2 particle) show in great detail how several biogenesis factors are associated with the 7S pre-rRNA to form the foot structure [25,[96][97][98] (Figures 2M and 4).…”

Section: Assembly Of the Lsumentioning

confidence: 99%

A Puzzle of Life: Crafting Ribosomal Subunits

Kressler

Hurt

Baßler

2017

Trends in Biochemical Sciences

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165

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 Lsumentioning

confidence: 99%

A Puzzle of Life: Crafting Ribosomal Subunits

Kressler

Hurt

Baßler

2017

Trends in Biochemical Sciences

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165

127

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“…4G; Leidig et al 2014;Wu et al 2016). During ITS2 processing, the components of the four-subunit Las1 complex perform the Las1-mediated initial incision at site C 2 as well as the Rat1-dependent trimming of the 5 ′ -end of the resulting 25.5S rRNA (Geerlings et al 2000;Gasse et al 2015). The A 3 -factors in the foot may regulate ITS2 processing by controlling the access of these nucleases to the packaged ITS2 RNA , in agreement with the finding that the Rat1 exonuclease, one of the subunits of the Las1 complex (Gasse et al 2015), degrades rather than properly processes pre-rRNAs in A 3 -factor mutants (Sahasranaman et al 2011).…”

Section: Molecular Mechanisms Of 60s Subunit Biogenesismentioning

confidence: 99%

“…During ITS2 processing, the components of the four-subunit Las1 complex perform the Las1-mediated initial incision at site C 2 as well as the Rat1-dependent trimming of the 5 ′ -end of the resulting 25.5S rRNA (Geerlings et al 2000;Gasse et al 2015). The A 3 -factors in the foot may regulate ITS2 processing by controlling the access of these nucleases to the packaged ITS2 RNA , in agreement with the finding that the Rat1 exonuclease, one of the subunits of the Las1 complex (Gasse et al 2015), degrades rather than properly processes pre-rRNAs in A 3 -factor mutants (Sahasranaman et al 2011). The biogenesis factor Nop53 found in the pre-60S foot binds the exosome-associated helicase Mtr4 , thereby mediating the pre-ribosomal recruitment of the nuclear exosome, which participates in the conversion of the 7S pre-rRNA produced by C 2 cleavage to the mature 5.8S rRNA (Mitchell et al 1996(Mitchell et al , 1997Henras et al 2014;Fernandez-Pevida et al 2015).…”

Section: Molecular Mechanisms Of 60s Subunit Biogenesismentioning

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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“…For example, the discovery of the ability to program CRISPR-associated nucleases at specific sites by guide RNAs has led to an explosion in gene editing (2,3). Another recently identified nuclease is the endoribonuclease Las1 (Las1L in mammals), which plays a vital role in eukaryotic ribosome assembly (4). Mutations in the LAS1L gene have been linked to a congenital motor neuron disease (5) and X-linked intellectual disability disorders (6), highlighting the need to further understand the activity of this essential enzyme.…”

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confidence: 99%

Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage

Pillon

Sobhany

Borgnia

et al. 2017

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

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Las1 is a recently discovered endoribonuclease that collaborates with Grc3-Rat1-Rai1 to process precursor ribosomal RNA (rRNA), yet its mechanism of action remains unknown. Disruption of the mammalian Las1 gene has been linked to congenital lethal motor neuron disease and X-linked intellectual disability disorders, thus highlighting the necessity to understand Las1 regulation and function. Here, we report that the essential Las1 endoribonuclease requires its binding partner, the polynucleotide kinase Grc3, for specific C2 cleavage. Our results establish that Grc3 drives Las1 endoribonuclease cleavage to its targeted C2 site both in vitro and in Saccharomyces cerevisiae. Moreover, we observed Las1-dependent activation of the Grc3 kinase activity exclusively toward single-stranded RNA. Together, Las1 and Grc3 assemble into a tetrameric complex that is required for competent rRNA processing. The tetrameric Grc3/Las1 cross talk draws unexpected parallels to endoribonucleases RNaseL and Ire1, and establishes Grc3/ Las1 as a unique member of the RNaseL/Ire1 RNA splicing family. Together, our work provides mechanistic insight for the regulation of the Las1 endoribonuclease and identifies the tetrameric Grc3/ Las1 complex as a unique example of a protein-guided programmable endoribonuclease.pre-rRNA processing | endoribonuclease | polynucleotide kinase | HEPN domain | Las1 N ucleases are found throughout all walks of life and are involved in numerous biological processes, including DNA replication and repair, RNA processing and maturation, and cell defense and death (1). Despite their long history, new nucleases continue to be discovered. For example, the discovery of the ability to program CRISPR-associated nucleases at specific sites by guide RNAs has led to an explosion in gene editing (2, 3). Another recently identified nuclease is the endoribonuclease Las1 (Las1L in mammals), which plays a vital role in eukaryotic ribosome assembly (4). Mutations in the LAS1L gene have been linked to a congenital motor neuron disease (5) and X-linked intellectual disability disorders (6), highlighting the need to further understand the activity of this essential enzyme.Ribosome assembly begins within the nucleolus of the cell with the transcription of the pre-ribosomal RNA (rRNA) by RNA Pol I, which is transcribed as a long polycistronic precursor, known as the 35S pre-rRNA in Saccharomyces cerevisiae (Sc) (7,8). The 35S pre-rRNA includes the 18S, 5.8S, and 25S rRNA as well as two external sequences [5′-and 3′-external transcribed spacers (ETSs)] and two internal sequences [internal transcribed spacers (ITS1 and ITS2)]. Removal of these four spacer sequences is a complex process requiring numerous exonucleases and endonucleases (9). Cleavage within ITS1 separates the 40S and 60S maturation pathways, whereas the subsequent removal of the ITS2 is an important step in the 60S maturation pathway. The first step in the removal of ITS2 is the separation of the precursors of the 5.8S and 25S rRNA by cleavage at the C2 site, which lies alo...

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Coordinated Ribosomal ITS2 RNA Processing by the Las1 Complex Integrating Endonuclease, Polynucleotide Kinase, and Exonuclease Activities

Cited by 98 publications

References 33 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

Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage

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