The Evolutionarily Conserved Protein LAS1 Is Required for Pre-rRNA Processing at Both Ends of ITS2

Schillewaert, Stéphanie; Wacheul, Ludivine; Lhommé, Frédéric; Lafontaine, Denis L. J.

doi:10.1128/mcb.06019-11

Cited by 76 publications

(107 citation statements)

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“…5F). The accumulation of 27S pre-rRNA underscores the importance of the Grc3 C terminus for C2 cleavage, whereas the abundance of the 7S confirmed previous findings suggesting a processing role for Las1 at the 3′-end of the 7S pre-rRNA (25). We also measured synthesis of ribosome particles by polysome profile analyses.…”

Section: Resultssupporting

confidence: 83%

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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Section: Resultssupporting

confidence: 83%

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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“…ITS2 removal is initiated by cleavage at the C 2 site in ITS2 by the endonuclease Las1 [130–132] and requires the association of many AFs with pre-ribosomes (reviewed in ref. [13]).…”

Section: The Middle Stage: Remodeling Events Required To Trigger Remomentioning

confidence: 99%

Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast

Konikkat

Woolford

2017

Biochemical Journal

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Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ~76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae. We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly.

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“…Steady-state and kinetic analyses of pre-rRNA processing Steady-state analyses of mature rRNAs and their precursors by primer extension and Northern hybridization and metabolic pulsechase analysis were carried out as previously described (Schillewaert et al 2012;Gamalinda et al 2013;Ohmayer et al 2013). …”

Section: Construction Of Yeast Strains and Depletion Of Rpls In Vivomentioning

confidence: 99%

A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains

et al. 2014

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Despite having high-resolution structures for eukaryotic large ribosomal subunits, it remained unclear how these ribonucleoprotein complexes are constructed in living cells. Nevertheless, knowing where ribosomal proteins interact with ribosomal RNA (rRNA) provides a strategic platform to investigate the connection between spatial and temporal aspects of 60S subunit biogenesis. We previously found that the function of individual yeast large subunit ribosomal proteins (RPLs) in precursor rRNA (pre-rRNA) processing correlates with their location in the structure of mature 60S subunits. This observation suggested that there is an order by which 60S subunits are formed. To test this model, we used proteomic approaches to assay changes in the levels of ribosomal proteins and assembly factors in preribosomes when RPLs functioning in early, middle, and late steps of pre-60S assembly are depleted. Our results demonstrate that structural domains of eukaryotic 60S ribosomal subunits are formed in a hierarchical fashion. Assembly begins at the convex solvent side, followed by the polypeptide exit tunnel, the intersubunit side, and finally the central protuberance. This model provides an initial paradigm for the sequential assembly of eukaryotic 60S subunits. Our results reveal striking differences and similarities between assembly of bacterial and eukaryotic large ribosomal subunits, providing insights into how these RNA-protein particles evolved.

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The Evolutionarily Conserved Protein LAS1 Is Required for Pre-rRNA Processing at Both Ends of ITS2

Cited by 76 publications

References 50 publications

Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage

Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage

Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast

A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains

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