Ribosome biogenesis factors and snoRNPs

The main difference between bacterial and eukaryotic (archaeal) ribosome biogenesis is the requirement of a vast number of non-ribosomal proteins and small nucleolar RNAs (snoRNA) in the production of ribosomes.

Prokaryotic ribosomal RNA species are nevertheless modified. They are subjected to isomerization of uridine to pseudouridine and chemically modified by addition of methyl groups, just like the archaeal and eukaryotic ones (see 1.4.3.2, reviewed in (Kaczanowska and Rydén-Aulin, 2007)). Though the mechanism of pseudouridine formation is independent of snoRNAs in Bacteria (Charette and Gray, 2000). And in addition, most of the chemical

Introduction

modifications, with exception of some 23S modification in the proximity of the peptidyl-transferase center, are dispensable (Krzyzosiak et al., 1987; Green and Noller, 1996). Complete in vitro reconstitution of prokaryotic ribosomes from E. coli is independent of any non-ribosomal factors. But, to create functional subunits, heating steps are required that most likely prevent the rRNAs from blundering in thermodynamically folding traps (Mizushima and Nomura, 1970; Herold and Nierhaus, 1987). In vivo, ribosome maturation factors probably assume responsibility for correct folding. There are about 50 known biogenesis factors in E. coli, however most of them are non-essential (reviewed in (Kaczanowska et al., 2007)).

A common feature of Archaea and Eukarya is that selectivity of base modification is achieved using small non-coding RNAs, the snoRNAs. The increasing number of sequenced archaeal genomes since the turn of the millennium allowed the identification of first C/D-box snoRNAs (called sRNA in Archaea) and later also H/ACA type snoRNAs (among others (Gaspin et al., 2000; Omer et al., 2000a; Tang, Bachellerie, et al., 2002)). The C/D-box snoRNAs are complementary in sequence to the respective modification site and “guide” the RNP, which methylates nucleotides in rRNA and tRNA species. The mechanism of target site selection of H/ACA type snoRNAs is the same as of C/D-box snoRNAs, but the consequence of RNP binding is the isomerization of uridine to pseudouridine (reviewed in (Dennis et al., 2001)).

The first steps in eukaryotic rRNA processing (see 1.4.3.1) largely depend on the U3 snoRNA (Hughes and Ares, 1991a). This snoRNA together with about 30 proteins form the 90S pre-ribosome, also named SSU processome in yeast, because it consists of mostly SSU biogenesis factors (Dragon et al., 2002a). Further modification of the ribosomal RNAs involves about 70 additional snoRNPs (reviewed in (Henras et al., 2008)). Like in prokaryotes, most of the single modifications are not essential, though absence of a subset alters e.g. ribosome fidelity and/or function (King et al., 2003; Liang et al., 2007) (see also 1.4.3.2).

The U3 snoRNP is thought to assemble co-transcriptionally at the nascent 35S pre-rRNA transcript, forming a 90S particle, which is visible in Miller chromatin spreads of actively transcribed rRNA genes as “terminal balls” (Dragon et al., 2002a). Mainly biogenesis factors that are required for maturation of the SSU are associated within this 90S pre-ribosomes.

Endonucleolytic processing in the internal transcribed spacer 1 (ITS1) region (see 1.4.3.1) splits the following maturation pathways of pre-40S and pre-60S particles. In general, biogenesis factors can be divided into many different classes: endoribonucleases (e.g.

RNase MRP, maybe Nob1p), exoribonucleases (e.g. Rat1p, Xrn1p), helicases (e.g. Dbp2p, Prp43p), kinases (e.g. Hrr25p), ATPases (e.g. Rea1p), GTPases (e.g. Nog1p, Nog2p), methyl-transferases (e.g. Nop2p, Dim1p), peptidyl-proline-isomerases (e.g. Fpr3p), r-protein assembly factors (e.g. Rrp7p), intra-nucleolar/nuclear transport factors (e.g. Noc1p, Noc2p, Noc3p) and nuclear-export factors (e.g. Crm1p, Rrp12p, Mex67p/Mtr2p, Ltv1p).

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In total about 150 non-ribosomal trans-acting factors are engaged in eukaryotic ribosome biogenesis (reviewed in (Tschochner and Hurt, 2003; Nazar, 2004; Henras et al., 2008)).

Recent analysis in yeast showed that the high efficiency in ribosome production is maybe boosted by association of biogenesis factors with pre-ribosomes not as single proteins, but as multiprotein “building blocks” (Merl et al., 2010). These protein complexes seem to assemble independent of pre-rRNA and offer kinetic advantages in contrast to a sequential binding of each component. One putative multiprotein complex of particular interest in this work was purified via Rio2p. Rio2p itself is a serine kinase that efficiently co-purifies 20S pre-rRNA and is required for final cytoplasmic pre-18S rRNA processing (Vanrobays et al., 2003; Geerlings et al., 2003). TAP-tag purification and subsequent protein analysis by mass spectrometry identified 6 proteins (Krr1p, Ltv1p, Enp1p, Tsr1p, Dim1p, Hrr25p) that associate independent on pre-rRNA with Rio2p. Several components of this complex are required for late pre-40S biogenesis (Gelperin et al., 2001; Seiser et al., 2006a). Nob1p and Pno1p/Dim2p were constantly co-purified but their stable association is dependent on concurrent pre-rRNA incorporation into the Rio2p-RNP (Merl et al., 2010). Pno1p (partner of Nob1p), alias Dim2p, is a nucleolar/nuclear-cytoplasmic shuttling protein with homology to Krr1p and involved in pre-18S rRNA processing and modification events (Vanrobays et al., 2004). Up to now, there is no absolute evidence of the endoribonuclease that mediates final pre-18S rRNA processing (see 1.4.3.1). One of the two proposed ribonucleases is Fap7p, because it was found to transiently interact with rpS14, a component of the head-platform interface in proximity to the 18S 3'-end and to exhibit NTPase activity (Granneman et al., 2005). The other, more likely candidate is Nob1p, which contains a PIN domain and co-purifies with pre-SSU particles. Additionally Nob1p interacts in vitro with rpS14 and rpS5, both localized in the head-platform interface (Fatica et al., 2003, 2004). Recently it was shown that Nob1p is able to bind solely with certain specifity to artificial RNA constructs that mimic its potential substrate, though its endonucleolytic activity is rather low under these conditions (Lamanna and Karbstein, 2009; Pertschy et al., 2009a).

In a genetic suppressor screen of the cold-sensitive phenotype caused by depletion of Ltv1p, Nob1p, Prp43 and Pfa1p were found (Pertschy et al., 2009b). The helicase Prp43p and its co-factor Pfa1p participate in spliceosome dissasembly (Arenas and Abelson, 1997; Martin et al., 2002), are needed to break up snoRNA-rRNA hybrids (Bohnsack et al., 2009) and maybe are involved in a conformational change of pre-40S subunits, preceding final 18S maturation (Pertschy et al., 2009a).

Introduction

1.4.3 Maturation of ribosomal RNAs