Ribosome biogenesis

Ribosomes are essential ribonucleoprotein complexes that are responsible for protein synthesis in all three domains of life. The eukaryotic ribosome sediments at 80S and is composed of four rRNAs and approximately 80 ribosomal proteins (RPs) that are assembled into two asymmetric subunits. In human cells, the 40S small subunit (SSU) contains the 18S rRNA and 33 RPs, while the 60S large subunit (LSU) is composed of the 5S, 5.8S and 28S rRNAs together with 47 RPs. During translation, decoding of the mRNA sequence takes place in the SSU and peptide bond formation is accomplished in a catalytic center consisting of rRNA that is located in the LSU. The functional core of the ribosome is highly conserved in all organisms, but eukaryotic ribosomes have an increased size and complexity compared to their bacterial counterparts due to the presence of rRNA expansion segments, additional RPs and RP extensions (Melnikov et al., 2012; Wilson and Doudna Cate, 2012; Yusupova and Yusupov, 2014).

The production of eukaryotic ribosomes starts with the transcription of ribosomal RNA precursors (pre-rRNAs), which undergo processing, folding and modification and are concurrently assembled with RPs to generate the mature ribosomal subunits. This requires the assistance of hundreds of assembly factors, which bind transiently and in a defined order and generally perform irreversible reactions that drive the process forward (Strunk and Karbstein, 2009; Kressler et al., 2010). Thus, ribosome biogenesis is a highly regulated and energy-consuming process. The pathway of ribosome assembly is best studied in Saccharomyces cerevisiae (S. cerevisiae) and, while most features are conserved across eukaryotes, in human cells there are specific differences in pre-rRNA processing as well as

a larger number of assembly factors (Tafforeau et al., 2013; Henras et al., 2015; Tomecki et al., 2017).

Figure 1.2. Overview of ribosome biogenesis in human cells. (A) Schematic representation of the 47S pre-rRNA transcript, which contains the sequences of the mature 18S, 5.8S and 28S rRNAs flanked by external transcribed spacer (5′ETS and 3′ETS) and separated by internal transcribed spacer (ITS1 and ITS2) regions.

This precursor is processed by endonucleolytic cleavage at sites that are marked above. The position of the first and last nucleotide of the mature rRNAs within the precursor are indicated below. This panel is based on Mullineux and Lafontaine, 2012 and Henras et al., 2015. (B) During ribosome assembly, a multitude of factors associate with the nascent transcript to generate the 90S pre-ribosome, which undergoes a pre-rRNA cleavage event that separates the precursors of the two ribosomal subunits. These pre-ribosomal complexes are further processed in the nucleus and cytoplasm to produce the mature 40S and 60S subunits. This panel was adapted from Martin, 2014.

In human cells, the 18S, 5.8S and 28S rRNAs are co-transcribed in the nucleolus by RNA polymerase I to generate the 47S pre-rRNA transcript, in which the sequences of the mature rRNAs are interspersed with external transcribed spacer (5′ETS and 3′ETS) and internal transcribed spacer (ITS1 and ITS2) regions (Figure 1.2A). Multiple assembly factors and several RPs are recruited co-transcriptionally to the nascent pre-rRNA, leading to the formation of the earliest biogenesis precursor, the 90S pre-ribosome, which contains the full-length transcript and predominantly proteins required for SSU maturation (Grandi et al., 2002; Phipps et al., 2011; Figure 1.2B). Structures of 90S pre-ribosomes from S. cerevisiae and Chaetomium thermophilum have revealed that this macromolecular complex assembles as a scaffold around the pre-rRNA and ensures that its maturation takes place

in a coordinated and sequential manner (Kornprobst et al., 2016; Kressler et al., 2017; Sun et al., 2017a). The processing pathways of the SSU and LSU diverge after an endonucleolytic cleavage in ITS1. The precursors of the two subunits undergo additional maturation steps that involve the dynamic association and release of assembly factors, the incorporation of RPs and further pre-rRNA processing and structural rearrangement events.

These pre-ribosomal complexes transition through the nucleolus and nucleoplasm and are then exported to the cytoplasm, where final maturation occurs and the two subunits associate for translation (Kressler et al., 2017; Pena et al., 2017; Chaker-Margot, 2018).

The 5S rRNA precursor is transcribed separately by RNA polymerase III at sites adjacent to the nucleolus and joins the assembly pathway at an early stage (Ciganda and Williams, 2011).

The processing of the 47S pre-rRNA transcript into the mature 18S, 5.8S and 28S rRNAs involves sequential endonucleolytic cleavages that take place at defined sites in the spacer regions and are followed by exonucleolytic trimming (Henras et al., 2015; Aubert et al., 2018; Figure 1.2A). The initial precursor is processed first at sites A′ in the 5′ETS and 02 in the 3′ETS to produce the 45S pre-rRNA. The cleavage at site A′ was shown to not be required for the downstream steps and, while its role is not known, it is interesting to note that this site is only present in metazoans (Sloan et al., 2014). Two parallel pathways exist for processing of the 45S precursor that differ in the relative order of the 5′ETS removal and ITS1 cleavage events and give rise to different pre-rRNA species. Cleavage of the 45S pre-rRNA at site 2 in ITS1 prior to 5′ETS excision generates the 30S and 32.5S precursors.

The 5′ETS region of the 30S pre-rRNA is subsequently removed by coordinated cleavages at sites A0 and 1, giving rise to the 21S intermediate, which is then processed at its 3′

terminus through the combined action of endo- and exonucleases to produce the 18SE pre-rRNA. This precursor is exported to the cytoplasm where a final cleavage at site 3 in ITS1 generates the mature 18S rRNA (Henras et al., 2015; Aubert et al., 2018).

Alternatively, excision of the 5′ETS region in the 45S pre-rRNA leads to the formation of the 41S intermediate, which can be further processed via two pathways. In the major pathway, cleavage takes place at site 2 in ITS1 and creates the 21S and 32.5S precursors, while in the minor pathway processing occurs instead at site E in ITS1 and produces the 18SE and 36S pre-rRNAs (Preti et al., 2013; Sloan et al., 2013). The 21S and 18SE precursors of the small ribosomal subunit are matured as described above. The 36S pre-rRNA is trimmed at its 5′ end by the 5′-3′ exonuclease XRN2 to produce the 32.5S intermediate, which is the common LSU biogenesis precursor for all the alternative pathways. The 5′ end of the 32.5S pre-rRNA is further digested by XRN2 to generate the abundant 32S intermediate, which contains the sequences of the 5.8S and 28S rRNAs. Cleavage at site 4 in ITS2 followed by

exonucleolytic digestion releases the mature forms of these rRNAs (Henras et al., 2015;

Aubert et al., 2018). Interestingly, a second ITS2 cleavage has been reported at site 4a, which leads to the excision of a fragment corresponding to the 4a-4 region that is degraded by XRN2 (Schillewaert et al., 2012). Other pre-rRNA spacer regions are released during processing and XRN2 has also been linked to the turnover of the 5′-A′, A0-1 and E-2 fragments (Wang and Pestov, 2011; Sloan et al., 2013; Sloan et al., 2014).

In addition to nucleases that participate directly in pre-rRNA processing, the ribosome assembly pathway requires the action of a multitude of other factors, such as RNA helicases, GTPases, kinases, structural proteins and snoRNAs that associate with proteins into snoRNPs. These assembly factors are essential for a wide range of processes, which include, among others, folding and modification of pre-rRNAs, remodeling and export of pre-ribosomal complexes, acting as structural scaffolds within pre-ribosomes or chaperoning and assisting the integration of RPs (Strunk and Karbstein, 2009; Kressler et al., 2010; Watkins and Bohnsack, 2012; Konikkat and Woolford, 2017; Pillet et al., 2017).

The role of RNA helicases in ribosome biogenesis has been mainly characterized in yeast, where 21 helicases participate in this process. These enzymes were suggested to remodel RNA-RNA and protein-RNA interactions within pre-ribosomes and were recently shown to also mediate the export of pre-ribosomal complexes and the acetylation of pre-rRNA (Martin et al., 2013; Rodriguez-Galan et al., 2013; Neumann et al., 2016; Sharma et al., 2017). The RNA/RNP remodeling function of RNA helicases is exerted in diverse ways during ribosome biogenesis. For example, multiple RNA helicases were suggested to mediate the release of snoRNPs from pre-ribosomes by unwinding snoRNA-rRNA interactions. This includes Dbp4, Rok1, Has1, Dhr1 and Prp43, whose depletion or inactivation led to the accumulation of specific snoRNPs in pre-ribosomal particles (Kos and Tollervey, 2005; Liang and Fournier, 2006; Bohnsack et al., 2008; Bohnsack et al., 2009; Sardana et al., 2015). RNA helicases can also unwind secondary structures in pre-rRNAs that facilitate the binding of snoRNPs to their target sites as has been suggested for Prp43. Another role proposed for Prp43 is the remodeling of late pre-ribosomal complexes to enable access of the endonuclease Nob1 to its cleavage site (Lebaron et al., 2009; Pertschy et al., 2009).

Therefore, in addition to snoRNPs, RNA helicases might also regulate the binding or dissociation of ribosome assembly proteins either in a direct or indirect manner. A remodeling function was also described for the RNA helicase Mtr4, which is required to unfold structured pre-rRNA substrates and facilitate their processing or degradation by the nuclear exosome (Thoms et al., 2015; Schuller et al., 2018; Weick et al., 2018).

RNA helicases in higher eukaryotes are expected to perform similar functions in ribosome biogenesis as their yeast counterparts (Martin et al., 2013; Rodriguez-Galan et al., 2013).

Consistent with this, some mammalian helicases have already been implicated in snoRNA release/association within pre-ribosomes (Srivastava et al., 2010; Sloan et al., 2015).

However, the function of most RNA helicases in human ribosome biogenesis is poorly characterized and, given the increased complexity of this pathway compared to yeast, additional roles might be revealed.