RNA-protein interactions of stored 5S RNA with TFIIIA and ribosomal protein L5 during Xenopus oogenesis

We studied the pathway of 5S RNA during oogenesis in Xenopus laevis from its storage in the cytoplasm to accumulation in the nucleus, the sequence requirements for the 5S RNA to follow that pathway, and the 5S RNA-protein interactions that occur during the mobilization of stored 5S RNA for assembly into ribosomes. In situ hybridization to sections of oocytes indicates that 5S RNA first becomes associated with the amplified nucleoli during vitellogenesis when the nucleoli are activity synthesizing ribosomal RNA and assembling ribosomes. When labeled 5S RNA is microinjected into the cytoplasm of stage V oocytes, it migrates into the nucleus, whether microinjected naked or complexed with the protein TFIIIA as a 7S RNP storage particle. During vitellogenesis, a nonribosome bound pool of 5S RNA complexed with ribosomal protein L5 (5S RNPs) is formed, which is present throughout the remainder of oogenesis. Immunoprecipitation assays on homogenates of microinjected oocytes showed that labeled 5S RNA can become complexed either with L5 or with TFIIIA. Nucleotides 11 through 108 of the 5S RNA molecule provide the necessary sequence and conformational information required for the formation of immunologically detectable complexes with TFIIIA or L5 and for nuclear accumulation. Furthermore, labeled 5S RNA from microinjected 7S RNPs can subsequently become associated with L5. Such labeled 5S RNA is found in both 5S RNPs and 7S RNPs in the cytoplasm, but only in 5S RNPs in the nucleus of microinjected oocytes. These data suggest that during oogenesis a major pathway for incorporation of 5S RNA into nascent ribosomes involves the migration of 5S RNA from the nucleus to the cytoplasm for storage in an RNP complex with TFIIIA, exchange of that protein association for binding with ribosomal protein L5, and a return to the nucleus for incorporation into ribosomes as they are being assembled in the amplified nucleoli.     

Two zinc finger proteins from Xenopus laevis bind the same region of 5S RNA but with different nuclease protection patterns.

Immature oocytes from Xenopus laevis contain a 42S ribonucleoprotein particle (RNP) containing 5S RNA, tRNA, a 43 kDa protein, and a 48 kDa protein. A particle containing 5S RNA and the 43 kDa protein (p43-5S) liberated from the 42S particle upon brief treatment with urea can be purified by anion exchange chromatography. The purified p43-5S RNA migrates as a distinct species during electrophoresis on native polyacrylamide gels. Radiolabeled 5S RNA can be incorporated into the p43-5S complex by an RNA exchange reaction. The resulting complexes containing labeled 5S RNA have a mobility on polyacrylamide gels identical to that of purified p43-5S RNPs. RNP complexes containing 5S RNA labeled at either the 5' or 3' end were probed with a variety of nucleases in order to identify residues protected by p43. Nuclease protection assays performed with alpha-sarcin indicate that p43 binds primarily helices I, II, IV, and V of 5S RNA. This is the same general binding site observed for TFIIIA on 5S RNA. Direct comparison of the binding sites of p43 and TFIIIA with T1 and cobra venom nucleases reveals striking differences in the protection patterns of these two proteins.     

A Role for Ribosomal Protein L5 in the Nuclear Import of 5S rRNA inXenopusOocytes

Prior to ribosome assembly, 5S ribosomal RNA (5S rRNA) binds to ribosomal protein L5 to form a stable ribonucleoprotein particle (5S RNP). We have analyzed the role of L5 binding in the nuclear targeting of 5S rRNA inXenopusoocytes, and have compared the nuclear import pathway of 5S RNPs with other karyophilic molecules. Nuclear import ofin vitro-generated 5S RNPs was found to be sensitive to three general inhibitors of nuclear pore complex-mediated translocation: ATP depletion, chilling, and wheat germ agglutinin. The initial rate and extent of net nuclear import was threefold greater with preassembled 5S RNPs than with 5S rRNA microinjected alone, suggesting that L5 binding is a prerequisite for nuclear accumulation. Nuclear import of 5S rRNA/5S RNPs is a facilitated process dependent on limiting factors, since nuclear import exhibited saturation kinetics. Not only was nuclear import of labeled 5S rRNA reduced in the presence of excess unlabeled 5S rRNA, but also in the presence of the synthetic karyophilic protein P(lys)-BSA. In contrast, import was not inhibited by U1 small nuclear RNA (snRNA) or U3 small nucleolar RNA (snoRNA). 5S rRNA/5S RNP nuclear import therefore appears to follow a pathway of molecular interactions similar to many karyophilic proteins, but not the methylguanosine cap-dependent U1 snRNA pathway or the cap-independent U3 snoRNA pathway.     

In vitro assembly of yeast 5S rRNA and a fusion protein containing ribosomal protein L5 and maltose binding protein

Binding of yeast ribosomal protein L5 with 5S rRNA has long been considered a promising model for studying molecular mechanisms of protein-RNA interactions. However, in vitro assembly of a ribonucleoprotein (RNP) complex from purified yeast ribosomal protein L5 (also known as L1, L1a, or YL3) and 5S rRNA proved to be difficult, thus limiting the utility of this model. In the present report, we present data on the successful in vitro assembly of a RNP complex using a fusion (MBP-L5) protein consisting of the yeast ribosomal protein L5 fused to the carboxyl terminus of the E. coli maltose-binding protein (MBP). We demonstrated that: 1) the MBP-L5 protein binds yeast 5S rRNA but not 5.8S rRNA in vitro; 2) the MBP protein itself does not bind yeast 5S rRNA; 3) formation of the RNP complex is proportional to the concentration of MBP-L5 protein and 5S rRNA; and 4) the MBP moiety of the fusion protein in the RNP complex can be removed with factor Xa. The electrophoretic mobility of the resultant RNP complex is indistinguishable from that of L5-5S rRNA complex isolated from the ribosome. Using this new experimental approach, we further showed that the RNA binding capability of a mutant L5 protein is decreased by 60% compared to the wild-type protein. Additionally, the mutant RNP complex migrates slower than the wild-type RNP complex suggesting that the mutant RNP complex has a less compact conformation. The finding provides a probable explanation for an earlier observation that the 60S ribosomal subunit containing the mutant protein is unstable.     

The participation of 5S rRNA in the co-translational formation of a eukaryotic 5S ribonucleoprotein complex

The eukaryotic ribosomal 5S RNA–protein complex (5S rRNP) is formed by a co-translational event that requires 5S rRNA binding to the nascent peptide chain of eukaryotic ribosomal protein L5. Binding between 5S rRNA and the nascent chain is specific: neither the 5S rRNA nor the nascent chain of L5 protein can be substituted by other RNAs or other ribosomal proteins. The region responsible for binding 5S rRNA is located at positions 35–50 with amino acid sequence RLVIQDIKNKYNTPKYRM. Eukaryotic 5S rRNA binds a nascent chain having this sequence, but such binding is not substantive enough to form a 5S-associated RNP complex, suggesting that 5S rRNA binding to the nascent chain is amino acid sequence dependent and that formation of the 5S rRNP complex is L5 protein specific. Microinjection of 5S rRNP complex into the cytoplasm of Xenopus oocytes results in both an increase in the initial rate and also in the extent of net nuclear import of L5. This suggests that the 5S rRNP complex enhances nuclear transport of L5. We propose that 5S rRNA plays a chaperone-like role in folding of the nascent chain of L5 and directs L5 into a 5S rRNP complex for nuclear entry.     

Yeast 5S rRNA binding to ribosomal protein L1a alters the fluorescence of tryptophan residues lying outside the binding site.

The yeast ribosomal protein L1a contains two tryptophan residues located at positions 95 and 183. Spectrofluorometric analysis showed that the average tryptophan environment is moderately polar. Quenching studies of the yeast 5S rRNA-L1a protein complex (RNP) with acrylamide and iodide revealed tryptophan heterogeneity. The two tryptophan residues are located in the non-RNA-binding region of the L1a molecule. However, dissociation of the yeast 5S rRNA-L1a protein RNP complex to its components resulted in a decline of tryptophan fluorescence. The observation implied that the environment of the tryptophan-containing L1a regions which were not known to be involved in RNA binding was influenced by association with the 5S rRNA molecule.     

Characterization of a novel association between two trypanosome-specific proteins and 5S rRNA

P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are essential and are involved in ribosome biogenesis. Here, we show that these proteins interact in vitro with the 5S rRNA with nearly identical binding characteristics in the absence of other cellular factors. The T. brucei 5S rRNA has a complex secondary structure and presents four accessible loops (A to D) for interactions with RNA-binding proteins. In other eukaryotes, loop C is bound by the L5 ribosomal protein and loop A mainly by TFIIIA. The binding of P34 and P37 to T. brucei 5S rRNA involves the LoopA region of the RNA, but these proteins also protect the L5 binding site located on LoopC.     

A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells

A novel 5S RNA-protein (RNP) complex in human and mouse cells has been analyzed using patient autoantibodies. The RNP is small (approximately 7S) and contains most of the nonribosome-associated 5S RNA molecules in HeLa cells. The 5S RNA in the particle is matured at its 3' end, consistent with the results of in vivo pulse-chase experiments which indicate that this RNP represents a later step in 5S biogenesis than a previously described 5S*/La protein complex. The protein moiety of the 5S RNP has been identified as ribosomal protein L5, which is known to be released from ribosomes in a complex with 5S after various treatments of the 60S subunit. Indirect immunofluorescence indicates that the L5/5S complex is concentrated in the nucleolus. L5 may therefore play a role in delivering 5S rRNA to the nucleolus for assembly into ribosomes.     

Local tertiary structure probing of ribonucleoprotein particles by nuclease fusion proteins

Analyses of the conformational dynamics of the numerous cellular ribonucleoprotein particles (RNP) significantly contribute to the understanding of their modes of action. Here, we tested whether ribonuclease fusion proteins incorporated into RNPs can be used as molecular probes to characterize the local RNA environment of these proteins. Fusion proteins of micrococcal nuclease (MNase) with ribosomal proteins were expressed in S. cerevisae to produce in vivo recombinant ribosomes which have a ribonuclease tethered to specific sites. Activation of the MNase activity by addition of calcium led to specific rRNA cleavage events in proximity to the ribosomal binding sites of the fusion proteins. The dimensions of the RNP environment which could be probed by this approach varied with the size of the linker sequence between MNase and the fused protein. Advantages and disadvantages of the use of MNase fusion proteins for local tertiary structure probing of RNPs as well as alternative applications for this type of approach in RNP research are discussed.     

Interactions between the Translation Machinery and Microtubules

Microtubules are components of eukaryotic cytoskeleton that are involved in the transport of various components from the nucleus to the cell periphery and back. They also act as a platform for assembly of complex molecular ensembles. Ribonucleoprotein (RNP) complexes, such as ribosomes and mRNPs, are transported over significant distances (e.g. to neuronal processes) along microtubules. The association of RNPs with microtubules and their transport along these structures are essential for compartmentalization of protein biosynthesis in cells. Microtubules greatly facilitate assembly of stress RNP granules formed by accumulation of translation machinery components during cell stress response. Microtubules are necessary for the cytoplasm-to-nucleus transport of proteins, including ribosomal proteins. At the same time, ribosomal proteins and RNA-binding proteins can influence cell mobility and cytoplasm organization by regulating microtubule dynamics. The molecular mechanisms underlying the association between the translation machinery components and microtubules have not been studied systematically; the results of such studies are mostly fragmentary. In this review, we attempt to fill this gap by summarizing and discussing the data on protein and RNA components of the translation machinery that directly interact with microtubules or microtubule motor proteins.     

RNA binding strategies of ribosomal proteins.

Structures of a number of ribosomal proteins have now been determined by crystallography and NMR, though the complete structure of a ribosomal protein-rRNA complex has yet to be solved. However, some ribosomal protein structures show strong similarity to well-known families of DNA or RNA binding proteins for which structures in complex with cognate nucleic acids are available. Comparison of the known nucleic acid binding mechanisms of these non-ribosomal proteins with the most highly conserved surfaces of similar ribosomal proteins suggests ways in which the ribosomal proteins may be binding RNA. Three binding motifs, found in four ribosomal proteins so far, are considered here: homeodomain-like alpha-helical proteins (L11), OB fold proteins (S1 and S17) and RNP consensus proteins (S6). These comparisons suggest that ribosomal proteins combine a small number of fundamental strategies to develop highly specific RNA recognition sites.