Eukaryotic 5S rRNA biogenesis (original) (raw)

. Author manuscript; available in PMC: 2012 Feb 14.

Published in final edited form as: Wiley Interdiscip Rev RNA. 2011 Feb 25;2(4):523–533. doi: 10.1002/wrna.74

Abstract

The ribosome is a large complex containing both protein and RNA which must be assembled in a precise manner to allow proper functioning in the critical role of protein synthesis. 5S rRNA is the smallest of the RNA components of the ribosome, and although it has been studied for decades, we still do not have a clear understanding of its function within the complex ribosome machine. It is the only RNA species that binds ribosomal proteins prior to its assembly into the ribosome. Its transport into the nucleolus requires this interaction. Here we present an overview of some of the key findings concerning the structure and function of 5S rRNA and how its association with specific proteins impacts its localization and function.

INTRODUCTION

5S rRNA is a small RNA (120 nt) with a molecular mass of 40 kDa. The secondary and tertiary structures are generally conserved across phylogeny. The secondary structure is composed of five helices,1 four loops (two hairpin and two internal), and one hinge (Figure 1) which usually fold into a Y structure. 5S rRNA is unique among the rRNAs in that it forms a preribosomal particle with the ribosomal protein L5 in eukaryotes.2 Prior to joining the ribosome, 5S rRNA has a complicated biogenesis pathway that may involve several other proteins (Figure 2). Unlike the other eukaryotic rRNAs, it is not generally transcribed in the nucleolus. Therefore, its interaction with L5 in its function as a targeting protein may be required to bring it to the site of ribosomal assembly within the nucleolus (Figure 3). The presence of 5S rRNA is required for normal translation in most ribosomes although its presence in some mitochondrial ribosomes has not been demonstrated. 5S rRNA sits in the junction between the large ribosomal subunit (LSU or 60 S in eukaryotes) and small ribosomal subunit (SSU or 40 S in eukaryotes) and forms part of the central protuberance (CP).3 Although its exact function within the ribosome is unclear, it is thought to play a critical role in both protein–RNA and RNA–RNA interactions within the ribosome.

FIGURE 1.

FIGURE 1

Structure of 5S rRNA. Loops are labeled A–E and helices I–V. Translucent blue ovals represent contacts with TFIIIA and L5 (data used by permission of author).4

FIGURE 2.

FIGURE 2

Preribosomal interactions of 5S rRNA with its predominant binding partners. This figure indicates both the transient and more stable interaction of 5S rRNA (La, TFIIIA, and L5, respectively). The nuclear export of 5S rRNA can occur together with either TFIIIA or L5 but return to the nucleus occurs only in conjunction with L5.

FIGURE 3.

FIGURE 3

Ribosomal biogenesis pathway. This diagram depicts the biogenesis of yeast 60S ribosomal subunits, including proteins that have known association at specific stages in this process. We have also included two of the known 60S subunit export factors: exportin 1 and Nmd3. Dashed arrows indicate known biogenesis and transport processes. (Reprinted with permission from Ref 5. Copyright 1999 American Society for Microbiology; and from Ref 6. Copyright 2009 American Society for Microbiology.)

5S rRNA Function

Early structural analysis of prokaryotic 5S rRNA led to speculation that a direct interaction of the–CGAAC–motif of 5S rRNA with the common arm of tRNA (GTΨCG) could lead to a transduction of rotational motion (coiling–uncoiling) in the 5S rRNA into linear motion (necessary for ‘walking’ along the mRNA).7 Prokaryotic 5S rRNA is indeed critical for tRNA binding to the aminoacyl–tRNA binding site of the ribosome.8 In Escherichia coli, it has been possible to reconstitute partially functional ribosomes without 5S rDNA, 5S rRNA, or 5S rRNA-binding proteins.9,10 However, cells with these defects exhibit growth defects and their ribosomal particles have limited ability to synthesize natural protein which is dramatically improved upon complementation with 5S rRNA.8 In Saccharomyces cerevisiae, mutant versions of 5S rRNA expressed in the absence of wild-type 5S rRNA led to inviable phenotypes.11 It has been proposed for yeast that, since 5S rRNA provides a physical connection between the different functional regions of the ribosome, it serves as a signal transducer to facilitate communication between these regions thus helping to direct their coordination of translation events.12 Furthermore, in E. coli the site of peptidyltransferase activity and the EF-G binding site may communicate through 5S rRNA by its interaction with LSU rRNA (varies in size from 23 to 28S depending on the organism).13 Recruitment of 5S rRNA is needed for the processing of the LSU rRNA and may assure that stoichiometric amounts of the other three rRNAs are present.14

5S rRNA Structure

5S rDNA genes are present in eukaryotic genomes as clusters of tandem repeats.15 The number of these genes is highly variable and their sequences have been used extensively as phylogenetic markers.16 The primary structure of the transcribed product, 5S rRNA, consists of approximately 120 nucleotides and has a molecular mass of 40 kDa. Several methodologies, including enzymatic and chemical probing,17,18 accessible base-specific chemical modifications,19 thermal melting analysis,18–21 and biochemical exchange of divergent or modified RNAs22 as well as theoretical base-pairing considerations18 have shed light on the secondary structure of 5S rRNA from various eukaryotic sources. It comprises five stems or helices (I–IV) and four loops (A–E; Figure 1). Two of these are hairpin loops (C and D), two are internal loops (B and E), and loop A acts as a hinge1 between three stems (I, II, and V). Helix III contains two conserved bulged adenosine residues.23,24 Helix II in Xenopus contains an extrahelical cytosine that points away from the duplex in two different configurations and which can establish major or minor groove contacts in base-triple formations, bridging RNA helices.25 Phylogenetic analysis of 5S rRNA has shown that helices I and III (Figure 1) are ancestral structural motifs.26

The secondary structure motifs fold into a characteristic tertiary structure. Loop A/B/C together with stems I/II/III form a ‘head’ or top domain, stems IV/V and loop E constitute a middle domain, and stems IV/loop D form a bottom or ‘toe’ domain.27 It has been hypothesized28 that helix V can also adopt a hairpin structure in the 5S rRNA gene, aiding in the formation of the transcription complex through interaction with TFIIIA. Structural elements have been described as more important than sequence elements in the binding to proteins (Figure 1).29

Assembly of 5S rRNA into the Ribosome

Whether 5S rRNA is immediately brought to the site of ribosomal assembly or arrives from a storage particle in the cytoplasm, it is brought to the nuclear pre-60S complex in a complex with the L5 ribosomal protein (Figures 2 and 3, discussed below). Reports differ on the stage at which 5S rRNA is integrated into the ribosome. Most recently, Woolford and colleagues30 have shown that they are associated in the very early 90S step in ribosomal assembly, although earlier work suggested that association occurred much later.31 In Trypanosoma brucei, it has been shown that 5S rRNA and L5 are both present in the 90S precursor particle.6 In S. cerevisiae, accessory factors Rpf2 and Rrs1 recruit 5S rRNA together with the ribosomal proteins L5 and L11 into the preribosomal 90S particle.30

5S rRNA Within the Ribosome

Several structural domains of 5S rRNA have been crystallized32 and X-ray scattering studies in solution have determined that free 5S rRNA is elongated in the absence of bound proteins but becomes more compact when it is in the ribosome33,34 (Figure 4). NMR studies performed on Xenopus laevis 5S rRNA have also shown that while loop E is fully folded in the absence of protein (approximating an A-formed helix), the junction of helix V and loop A is conformationally heterogenous in the free molecule.1 The major groove of the A-formed loop E is further kept open by an extrahelical guanosine that interacts with a reversed Hoogsteen A:U pair, presumably defining an interacting surface similar to the sarcin/ricin loop motif of 23S (LSU) rRNA.35–37 Most interactions with ribosomal proteins occur through the 2-amino group of a guanine in the minor groove. The major groove in A-form is more inaccessible and needs considerable distortion to provide intermolecular contacts.38

FIGURE 4.

FIGURE 4

Interactions of 5S rRNA within the ribosome. (a) Position of 5S rRNA (blue) in the CP of the ribosome. (b) Close-up view of the interactions between 5S rRNA (blue) and motifs in the LSU 26S rRNA; red: helix 38 (A-site finger), green: helix 39, black: helix 42, violet: helix 89, cyan: helix 91. PDB accession numbers: 3JYV, 3JYW, 3JYX.

Much of the structural information on 5S rRNA within the ribosome comes from bacteria, where 5S rRNA has been shown to form the CP of the 70S ribosomal subunit close to domains V and II of the 23S rRNA.39 Loop D contacts a cleft in the middle of helices 39 and 40 of 23S rRNA (domain II), whereas loop E and stem V contact helix 38 of 23S rRNA, the longest stem in the largest domain of this rRNA.38 However, most interactions between 5S rRNA and 23S rRNA occur indirectly, through ribosomal proteins. The bacterial 5S rRNA is linked to domains V of 23S rRNA through L5, L21e, and L10e and to domain II through L5, L21e, and L30.40

Through cryo-electron microscopy studies, it has observed that in S. cerevisiae 5S rRNA also sits in the CP of the 60S subunit and that it contacts helix 42 and helix 89 of the 28S rRNA (Figure 4), the former linking 5S rRNA to the GTPase associated center3,41 (involving contacts with protein factors and GTP hydrolysis stimulation). Helix 89 in turn contacts helix 91 which is linked to the elongation factor binding site through the sarcin/ricin loop of 28S rRNA. Therefore 5S rRNA, through direct or indirect contacts, reaches far into relevant functional sites in ribosomal subunit 60S (Figure 4).

5S rRNA and Ribosomal Biogenesis

Ribosomal biogenesis (Figure 3) is a process that requires the concerted processing and assembly of four ribosomal RNAs with over 80 ribosomal proteins.42,43 Recent large-scale proteomic approaches to the study of ribosome biogenesis have revealed an unexpectedly high number of enzymatic activities and _trans_-acting factors, including GTPases, ATPases, RNA exonucleases, RNA helicases, enzymes which modify RNA by methylation and pseudouridylation as well as many small RNAs that guide these processes.44,45 Biogenesis of both 60S and 40S subunits begins in the nucleolus46 with transcription of the 35S rRNA precursor47 by RNA polymerase I. In contrast, 5S rRNA is transcribed by RNA polymerase III from independent genes usually located in a different chromosomal locus. Yeast constitutes an exception, where 5S rRNA genes are interspersed between other nucleolar ribosomal genes, a situation that raises the question of whether and how their expression is affected by their positions within this locus. The individual subunit precursor particles, 66S (containing precursors to 25, 5.8, and 5S rRNAs) and 43S (containing precursors to 18S rRNA), are released from the 90S precursor upon cleavage of the 35S pre-rRNA.5,48 The two subunit precursors follow separate maturation pathways, although both are exported to the cytoplasm using the exportin 1 nuclear export pathway.49–51 The final steps of maturation occur in the cytoplasm, where joining of the 60S and 40S subunits occurs to form active 80S ribosomes.52,53

5S rRNA Processing

Processing of the 3′ ends of 5S rRNA involves participation of several yeast exonucleases (Rex1p, Rex2p, and Rex3p) with overlapping roles in the processing of other nuclear RNAs.54 Surveillance of readthrough transcripts and defective 5S rRNA species may occur by interaction with the Ro protein, a factor that binds a heterogeneous population of 69–112 nt RNA polymerase III transcripts.55 5S rRNA can be polyadenylated and subsequently degraded by the exosome.56,57 Nucleotides in eukaryotic 5S rRNA are rarely modified but there are reports of pseudouridinylation in some ascomycetes.58

PRERIBOSOMAL 5S rRNA BINDING PARTNERS

The La Protein

In eukaryotes, 5S rRNA metabolism begins with association of the primary transcript with the La protein (Figure 2).59–62 This association is transient, and only about 1–2% of free 5S rRNA containing particles contains the La protein.63,64 The La protein was first described in humans as an autoantigen but has since been found in all eukaryotes.62 La is an abundant protein of approximately 47 kDa that is primarily localized to the nucleus.65,66 It associates with a diverse group of RNAs which are transcribed by RNA pol III, including precursors to tRNAs and 5S rRNA. La interacts with these RNAs through recognition of their 3′ uridylates67 and functions as a chaperone in the maturation and stability of its RNA-binding partners62,68 and is thought to be involved in folding.

La is essential in flies and mammals but is not essential in yeast66 (except against a background of mutated Sm and Sm-like proteins involved in snRNP metabolism). It has been postulated that in S. cerevisiae, where La is missing, the cells are still viable because of the existence of unique alternative pathways.65 However, this is not simply a case of differences between unicellular eukaryotes and metazoans. RNA interference directed against the La homolog in T. brucei, an early branching unicellular eukaryote, causes a lethal phenotype in contrast to the results in yeast.69 It is unclear which of the many possible functions of La may be critical to the parasites’ survival but not required for the yeast cells to survive.

The La protein possesses both a La motif which is a winged-helix structure and an RRM1 which forms a typical RRM structure (Figure 5). Structural analysis of the La motif from human and trypanosomes indicates that it is conserved.70,71 Both the La and RRM motifs are required for La to bind UUUOH with high affinity,70,71 although La does not bind the substrate in the fashion typical for these motifs.72 La traffics to the nucleus via a nuclear localization signal (NLS) and also has a nuclear retention signal (NRE) as well as a nucleolar localization signal.

FIGURE 5.

FIGURE 5

Modular structure of eukaryotic characterized 5S rRNA binding proteins. Interpro signatures are as follows: L18p: IPR005484 Ribosomal_L18/L5; C2H2: IPR015880 Zn finger C2H2-like, LaHTH: IPR006630; RRM: IPR000504 (RNP1); RRM3: IPR014886; Vertical blue flags indicate nuclear export signals (NES) as defined by NetNES 1.1. The six rectangles beneath rL5 define the ribosomal L5 signature.

The Ribosomal L5 Protein

Following association with La and subsequent maturation, eukaryotic 5S rRNA associates with the ribosomal protein L5 (Figure 2). L5 functions to stabilize 5S rRNA73 and the levels of L5 also regulate the levels of 5S rRNA.74 5S rRNA is unique among the ribosomal RNAs because it can be found separately from the ribosome as part of a ribonucleoprotein particle (RNP). In bacteria there are three proteins found in this preribosomal complex: L5 (not the homolog of the eukaryotic L5), L18 (the eukaryotic L5 homolog), and, in some cases, L25. In contrast, in eukaryotes there is a single protein L5 (YL3 or Rpl5p in yeast, ortholog to bacterial L18). A complex of 5S rRNA and L5 can also be dissociated from the ribosome as was shown in early experiments.75 While other proteins can bind to 5S rRNA to translocate it to the cytoplasm or within the cytoplasm, only L5 can form a complex with 5S rRNA that can be transported to the nucleus for ribosomal assembly. Mutagenesis of the L5 protein or 5S rRNA can cause the RNP complex to be unstable and also render the assembled ribosomal subunits unstable64 supporting a critical role for the L5–5S rRNA complex. Phosphorylation of L5 by human CKII, a ubiquitous conserved Ser/Thr kinase, decreases binding to 5S rRNA76 and may play a regulatory function.

The 34 kDa L5 protein is localized to both the nucleus and the cytoplasm of eukaryotic cells and its dual localization plays an important part in the nucleocytoplasmic shuttling of L5–5S rRNA. Loss of L5 prevents the nuclear localization of 5S rRNA and affects subsequent ribosomal assembly. The NLS is complex, involving two distinct regions of the L5 sequence (aa 21–37 and aa 255–265), whereas the NES has been mapped to aa 101–11177

In S. cerevisiae L5, it has been postulated that a basic region toward the C-terminus is important in the binding to 5S rRNA2 and in effective ribosome biogenesis.78 These basic amino acids (R282, R285, and K289) lie on the same side of a putative _α_-helix. Single mutants of these residues interfere with 5S rRNA binding and double mutants are lethal. Eukaryotic 5S rRNA shows a high frequency of uncompensated G-U wobble pairs (pyrimidine at 5′ of G) that have been proposed to play a role in binding to L5, as well as to TFIIIA (see below).79

P34 and P37

In T. brucei, the causative agent of African trypanosomiasis, two novel, abundant- and closely related RNA-binding proteins, termed P34 and P37, have been identified.80,81 P34 and P37 (predicted 28.8 and 30.3 kDa, respectively), each contain three distinct domains as determined from their primary amino acid sequence (Figure 5). The N-terminal region consists of an alanine-, proline-, and lysine-rich domain which may serve a role in mediating protein–protein interactions. The internal regions of P34 and P37 contain two RBDs of the RRM family type, which consist of several basic amino acid residues. The last nine amino acids of the second RBD comprise a leucine-rich NES. The C-terminal portion is comprised of a lysine–lysine–aspartic acid-‘X’ motif, which includes both NLS and NES.

It has been shown that P34 and P37 are essential to the survival of both bloodstream forms (the stage present within the mammalian host) and procyclic forms (the replicative stage in the invertebrate) of T. brucei and the loss of these proteins leads to a 25-fold decrease in 5S rRNA.82 This change was specific to 5S rRNA and was not seen for other rRNAs. These results are similar to those observed in yeast cells where L5 plays a role in stabilizing 5S rRNA.73,74 Recent work has shown that P34 and P37 undergo nucleocytoplasmic shuttling6 as suggested by the presence of the NES and NLS (above). P34 and P37 have been shown to interact specifically with 5S rRNA in nuclear extracts6,83 and with 5S rRNA directly in in vitro filter binding assays (Ciganda and Williams, unpublished). Immunoprecipitation studies have shown that P34 and P37 also interact with T. brucei ribosomal protein L5 in nuclear extracts.6 These associations are indicative of potential role(s) of P34 and P37 in the stabilization and transport of 5S rRNA, and in the biogenesis of ribosomes, a process that remains poorly characterized. Moreover, in trypanosomes cells lacking P34 and P37 also show phenotypic alterations with respect to the formation of 80S ribosomes and overall cellular translation levels.84 Interestingly, these two 5S rRNA binding proteins are differentially expressed. P34 is dominantly expressed in the procyclic stage, while P37 is dominantly expressed in the bloodstream stage. It is not yet known if this reflects differences in function of these two proteins and their interaction with 5S rRNA between these stages. The discovery of additional, organism-specific 5S rRNA binding factors (see below, p43) expands our view of the interactions involved in 5S rRNA entry into the ribosomal biogenesis pathway originally described.

TFIIIA

TFIIIA has been carefully studied predominantly in its role as a transcription factor. It is a zinc finger protein (38.5 kDa) with nine C2H2 zinc finger repeats within the two thirds of the protein’s N-terminal (Figure 5). This region binds the internal control region of the 5S rDNA as well as mature 5S rRNA. Transcription of 5S rRNA requires TFIIIA, TFIIIB, and TFIIIC. TFIIIA has been identified in all eukaryotes to date, although it has not yet been identified in the genome of the kinetoplastids. In yeast, 5S rRNA transcription (but not stabilization) is the only essential function of TFIIIA. In the ascomycete Yarrowia lipolytica, TFIIIA-independent transcription of 5S rRNA can occur via dicistronic units of tRNA-5S rRNA.85 Xenopus TFIIIA interacts with approximately 50% of the 5S rRNA to form the 7S particle to function in transport of 5S rRNA from the nucleus to the cytoplasm for storage (see below). Fingers 4–7 bind RNA with high affinity (1 nM Kd) although the tertiary structure and some regions of secondary structure appear to be critical. Specific residues including a lysine in the _α_-helix of finger 4 and the threonine–tryptophan–threonine (TWT) in the _α_-helix of finger 6 are thought to be important to 5S rRNA binding through a central core comprised of loop A, helix V, region E, and helix IV, while finger 7 appears to bind helix II.86 This broad region of interaction suggests a role of TFIIIA in protecting 5S rRNA from turnover. Microinjection of mutant RNAs into Xenopus oocytes and localization studies have been used to correlate structural characteristics of 5S rRNA to domains necessary for nuclear import, present mainly in helix II, loop C, and loop E87 although no protein ligands have been identified for this specific role.

P43

Although TFIIIA in Xenopus oocytes binds to 50% of 5S rRNA as the 7S particle, a zinc finger protein p43 binds to the other 50% of the 5S rRNA in the form of a 42S RNP, together with a 48 kDa protein with EF-1_α_-like activity and tRNAs.88,89 TFIIIA and p43 do not share significant sequence homology, although p43, like TFIIIA, is a zinc finger protein (Figure 5) and both possess a TWT motif. Unlike TFIIIA, p43 does not bind directly to 5S rDNA.89 Zinc fingers 1–4 are essential for specific binding to 5S rRNA.90 Uncharacterized homologs of Xenopus P43 can be found in the genomes of Danio rerio (zebrafish) and Oryzias latipes (ricefish). Further studies on these factors are needed to clarify their roles and address the question of why these organisms need additional 5S rRNA binding proteins.

Mitochondrial 5S rRNA

Examination of mitochondrial ribosomes from many eukaryotes suggests that they lack 5S rRNA, although 5S rRNA has been reported in the mitochondria of a number of organisms (reviewed in Ref 91). No clear role for this mitochondrial 5S rRNA is yet defined. Although many mitochondrial genes including that for 5S rRNA have been transferred to the nucleus in the course of eukaryotic evolution, it is still present in the mitochondria of land plants and subsets of algae (reviewed in Ref 91). The existence of nuclearly encoded 5S rRNA in the mitochondria of eukaryotes has been a point of contention. Although some organisms have the mitochondrial gene for 5S rRNA and do not import nuclear 5S rRNA, mammalian mitochondria do import 5S rRNA.92,93 Helices I and IV (in particular G-U wobble pairs present in them) are necessary for high-efficiency import of human 5S rRNA into the mitochondrion.94

Cytoplasmic Storage of 5S rRNA

One of the most extensively characterized 5S rRNA pathways has been described for Xenopus oogenesis. Xenopus generates a huge excess of 5S rRNA in the early stages of oogenesis, the majority of which is found in the cytoplasm stored as a non-ribosomal RNP.95 Two types of 5S rRNA are present in these cells; a somatic type which is constitutively expressed and the oocyte specific type which is transcribed only in oogenesis.96 These two classes differ in sequence and appear to have differing affinity for TFIIIA and L5 with the oocyte specific type having a higher affinity for TFIIIA and the somatic having a higher affinity for L5. It is however the difference in abundance of the two proteins at different stages of oogenesis that has the greatest impact.

In the early stages, TFIIIA is present in excess of the 5S genes and is predominantly present in the cytoplasm as part of the 7S RNP. Later in oogenesis, TFIIIA levels drop and L5 levels rise in the middle stages of oogenesis concomitant with expression of other ribosomal components. 5S rRNA associates with L5 as part of the 5S RNP. Nuclear export of 5S rRNA appears to occur in association with either TFIIIA or L5 while the return to the nucleus in later stages of oogenesis requires association with L5 alone. This is because of the masking of the NLS of TFIIIA upon binding 5S rRNA.97–99

CONCLUSIONS

Although 5S rRNA has been studied for decades, both in its role in the ribosome and as a phylogenetic marker, we still have many unanswered questions concerning this small RNA. Even though the ribosome has garnered much attention since the awarding of the Nobel Prize in 2009, 5S rRNA remains the most elusive of all ribosomal RNAs regarding its precise function and how this correlates with structure. It is not immediately evident why 5S rRNA is necessary for eukaryotic translation, while its presence enhances but is not strictly required for prokaryotes. Whether or not it is present in eukaryotic mitochondria and what role it plays are still unresolved questions. We do not know the significance of the unusual chromosomal location of 5S rRNA genes in yeast and its implication for regulation. The lack of a requirement of the La protein in yeast raises the question of what alternative maturation pathways might be present in yeast but are not present in other unicellular eukaryotes requiring the La protein. We are also learning about previously unsuspected functions involving 5S rRNA, including its role in the p53 pathway. Finally, as the number of identified binding partners for this RNA increases (i.e., P43 and P34/P37), we begin to see that the 5S rRNA biogenesis pathway may not be as limited as once thought.

References