In the work presented here we created 26 yeast strains each of which conditionally expresses one of the 46 ribosomal proteins of the large ribosomal subunit. None of the mutant strains exhibited significant growth under restrictive conditions, indicating that production of the majority of LSU r-proteins is essential for yeast growth. After long term in vivo
depletion of most LSU proteins, we observed not only a decrease in cellular content of LSUs and a resulting ribosomal subunit imbalance but also a clear, albeit less pronounced decrease in the amount of SSUs per cell. The latter phenotype was seen before in conditional mutants of RPL25 and of several LSU biogenesis factors (see 
and discussion therein). On the other hand, soon after shifting to restrictive conditions, we could see specific effects on production of new LSUs in most conditional LSU r-protein gene mutant strains whereas biogenesis of SSUs remained largely unaffected. Therefore we conclude that a primary effect of shortage of LSU r-protein expression is on pre-LSU maturation.
In most cases, shutdown of individual LSU r-protein gene expression led to rather strong defects in different, specific steps of LSU maturation, namely 5′ maturation of 5.8S pre-rRNA, endo-and exonucleolytic processing of the ITS2 region of pre-LSU rRNA and cytoplasmic accumulation of LSU precursors. Despite the fact that current 3-D models of the LSU clearly indicate a complex network of interactions between its structural components, individual groups of LSU r-proteins seem to have specific impact on different aspects of rRNA maturation.
In principle, various molecular functions in rRNA maturation and transport can be envisioned for r-proteins as for ribosome biogenesis factors. They could have intrinsic exo- or endonucleolytic activity required for rRNA maturation or facilitate by themselves passage through nuclear pores. They could directly mediate the interaction of pre-LSUs with rRNA maturation/transport factors, as was suggested for rpL10 
or they could be involved in building up local or global structures that allow the interaction of rRNA maturation/transport factors. In addition they could trigger the release of maturation/transport factors from pre-LSUs, as might be the case for the nonessential rpL24 
and for rpL10 
, or they could be involved in protecting pre-LSUs from degradation by endo- and exonucleases.
Structural models may help to predict molecular functions of r-proteins: Primary rRNA maturation/transport phenotypes observed in strains carrying conditional r-protein-gene mutants can be compared with the exact positioning of the corresponding proteins in current atomic resolution structure models of eukaryotic ribosomes.
Down-regulation of expression of r-proteins rpL3, rpL4, rpL7, rpL8, rpL16, rpL18, rpL20, rpL32 and rpL33 resulted in inefficient production of pre-rRNAs with a matured 5′ end of 5.8S pre-rRNA. More specifically, the maturation pathway leading in yeast to the major 5′ end of 5.8S pre-rRNA was strongly affected in these mutant strains. This pathway is initiated by an endonucleolytic cut about 80 nucleotides upstream of the 5′ end of 5.8S rRNA at site A3 and involves then an exonucleolytic trimming reaction mediated by the general 5′–3′ exonucleases Rat1p/Xrn1p. These enzymes stop exonucleolytic digestion at site B1S which is the 5′ end of about 80% of mature 5.8S rRNA in wildtype conditions. Detailed analyses of current 3D-folding models of eukaryotic LSU rRNAs 
suggests that the 5′end of 5.8S rRNA forms an extended secondary structure network involving a part of domain II of 25S rRNA and that formation of these interactions requires correct folding of domain I and domain II sequences positioned inbetween these two elements (). Interestingly, rpL3, rpL4, rpL7, rpL8, rpL16, rpL18, rpL20, rpL32 and rpL33 are LSU r-proteins that contact this area of LSU rRNA domains I and II or are closely positioned near the 5′ end of 5.8S rRNA (). Thereby it seems plausible that 1) these r-proteins help establish the 3-dimensional organisation of LSU rRNA domains I and II leading to the extensive rRNA-rRNA interaction network at the 5′ end of 5.8S rRNA, and 2) the establishment of rRNA-rRNA interactions at the 5′ end of 5.8S rRNA is important to restrict the exonucleolytic action of Rat1p/Xrn1p to correctly trim LSU pre-rRNAs rather than allowing Rat1p/Xrn1p to degrade LSU pre-rRNAs.
rRNA – r-protein interactions as indicated by atomic resolution structure models of the eukaryotic mature cytoplasmic LSU.
Down-regulation of expression of another group of LSU r-proteins resulted in a pronounced delay of endonucleolytic cleavage in the ITS2 region of pre-rRNA at site C2. According to current 3D-models of the eukaryotic LSU rpL19, rpL25 and rpL35 are positioned at the bottom of the LSU, and rpL23 and rpL9 on an axis spanning from there towards the base of the (L7/L12-)phospho-protein stalk (see ). According to secondary structure models 
the ITS2 region of pre-rRNA is predicted to fold in several helical segments with site C2 about 135 nucleotides away from the 5.8S rRNA 3′ end and 100 nucleotides away from the 25S rRNA 5′ end. How the ITS2 region of pre-rRNA folds in space and how it orients toward other parts of the LSU precursor particle is currently not known.
We also found in this study that the expression of another group of LSU r-proteins was specifically required for efficient final 3′ maturation of 5.8S rRNA precursors (rpL5, rpL21, rpL2, rpL43) and/or productive nuclear export of LSU precursor particles (rpL10, rpL13, rpL17, rpL21, rpL28). In current 3D models these proteins are distributed all over the LSU () with one cluster around the LSUs central protrusion (rpL5, rpL21, rpL10), with rpL28 near the L1 stalk, rpL17 near the exit tunnel and rpL2 and rpL43 at the subunit interface close to the 3′ end of 5.8S rRNA. Several of these proteins are prototypic examples of r-proteins that fold in a globular domain with a protruding extension (rpL2, rpL28, rpL17, rpL5). These extensions are characterised by a high content of basic aminoacids that reach inside the RNA core of the LSU and are responsible for a disproportionally high amount of RNA-protein interactions found in the LSU 
. Our data suggest, that rpL2, rpL28, rpL17 and rpL5, which are as mentioned above examples of r-proteins carrying these extension domains, are not strictly required for all (rpL28, rpL17, see lanes 47–48 and lanes 43–44) or most (rpL2, rpL5, see lanes 35–38) of the LSU pre-rRNA processing steps. In support of this, recent work showed that in prokaryotes the extension domains of some r-proteins, including the rpL17 homologue L22, are not required for in vivo
assembly of ribosomal subunits 
A remarkable characteristic of the r-proteins identified here to be involved in final nuclear steps of pre-rRNA maturation and/or cytoplasmic accumulation of LSUs is that most of them (rpL2, rpL5, rpL10, rpL17, rpL21 and rpL28) interact both with domains II and V of LSU rRNA 
. rpL43, the only exception, interacts with LSU rRNA domain II, not with domain V but, on the other hand, is in close contact with rpL2. LSU rRNA domains II and V, together with rpL10, rpL21 and rpL7 build an interaction platform for the 5S rRNA - rpL5 - rpL11 RNP, the major constituent of the LSUs central protruberance. Therefore it seems, that correct positioning of the 5S rRNA – rpL5 – rpL11 RNP in the LSU is specifically required for efficient final nuclear 3′ processing of 5.8S pre-rRNA and / or cytoplasmic accumulation of nascent LSUs. Future analyses will have to show whether in this group of conditional r-protein-gene mutants the 5S RNP is physically excluded from newly synthesised LSUs as was observed before in cells depleted in vivo
for rpL5, rpL11 or one of the ribosome biogenesis factors Rpf2p and Rrs1p 
. In any case, inspection of LSU 3D structure suggests that the 5S RNP and the extended protein folds found in rpL2, rpL17 and rpL28 promote the correct positioning of LSU rRNA domains towards each other. Apparently, late nuclear pre-rRNA maturation events and productive nucleo-cytoplasmic translocation correlate with the establishment of a highly ordered structural organisation of the nascent LSUs that is not strictly required for other nuclear steps of LSU pre-rRNA maturation.
The observation that nuclear export of nascent LSUs was not blocked, but detectably delayed when expression of rpL1 was downregulated, furthermore suggests that also local changes in nascent LSU structure can detectably affect its nucleo-cytoplasmic translocation efficiency: rpL1 is the only protein constituent of one of the LSUs two lateral protruberances (). Lowered expression of rpL1 does neither lead to significant pre-rRNA processing phenotypes (see above, 
) nor does the omission of its prokaryotic homologue L1 affect the assembly of the residual LSU in in vitro
reconstitution experiments with purified prokaryotic LSU components 
Altogether the LSU pre-rRNA maturation phenotypes observed here in strains conditionally expressing LSU r-proteins do not match exactly most of the ones observed in yeast strains in which components of the endo- and exonucleases involved in LSU pre-rRNA processing, namely RNAse MRP 
, Rat1p, Xrn1p 
, Rnt1 
, Ngl2p 
, Rex1p, Rex2p, Rex3p 
or the exosome 
, were inactivated or in vivo
depleted. Strikingly, individual and/or combinational deletions of several of the genes coding for RNAses involved in pre-rRNA processing are not lethal (Xrn1p, Rnt1p, Ngl2p, Rex1p, Rex2p, Rrp6) but lead to rather strong accumulation of LSUs containing immature rRNA precursors 
. In contrast, in vivo
depletion of most of the yeast LSU r-proteins was lethal and resulted in nuclear restricted newly synthesized LSUs which contained partial or fully processed rRNA and were ultimately substrates for degradation. Similar phenotypes were observed in a large number of conditional alleles of LSU biogenesis factors whose primary structure does not contain obvious indications for their direct role as pre-rRNA processing enzymes ( see 
for a review). Whether and how some of these factors promote the assembly of r-proteins 
remains in most cases to be answered. We assume that this work can help in future studies to understand better the interplay of ribosome assembly factors, individual transient or non-transient assembly events on nascent subunits and LSU rRNA precursor maturation in eukaryotic cells.