Characterisation of an mRNA encoding a human ribosomal protein homologous to the yeast L44 ribosomal protein

We describe the isolation and characterisation of a full-length cDNA sequence (pZH-21) of a human ribosomal protein (rp) mRNA isolated from a cDNA library constructed from the human ZR-75-1 mammary tumour cell-line. The predicted protein is highly basic and shows 72% homology at the amino acid (aa) level with yeast rp L44. Comparative RNA blotting of ZR-75-1 poly(A)+ RNA isolated from cells cultured in the presence of the anti-oestrogen tamoxifen demonstrates the presence of a number of mRNA species whose concentration is elevated co-ordinately 5-6-fold in the presence of 17beta-oestradiol. Insulin in the presence of tamoxifen, also enhanced rp mRNA levels suggesting increased levels are a reflection of cell proliferation as opposed to specific hormonal regulation. Genomic analysis demonstrates the presence of a family of related human sequences, and homology with rat and guinea pig rp genes, but not yeast DNA. The conservation of rp aa sequence, in the absence of detectable homology at the nucleotide (nt) level, points to an important common functional role of the L44 protein in ribosome structure and function in man and yeast.     

Structure/function analysis of yeast ribosomal protein L2

Ribosomal protein L2 is a core element of the large subunit that is highly conserved among all three kingdoms. L2 contacts almost every domain of the large subunit rRNA and participates in an intersubunit bridge with the small subunit rRNA. It contains a solvent-accessible globular domain that interfaces with the solvent accessible side of the large subunit that is linked through a bridge to an extension domain that approaches the peptidyltransferase center. Here, screening of randomly generated library of yeast RPL2A alleles identified three translationally defective mutants, which could be grouped into two classes. The V48D and L125Q mutants map to the globular domain. They strongly affect ribosomal A-site associated functions, peptidyltransferase activity and subunit joining. H215Y, located at the tip of the extended domain interacts with Helix 93. This mutant specifically affects peptidyl-tRNA binding and peptidyltransferase activity. Both classes affect rRNA structure far away from the protein in the A-site of the peptidyltransferase center. These findings suggest that defective interactions with Helix 55 and with the Helix 65-66 structure may indicate a certain degree of flexibility in L2 in the neck region between the two other domains, and that this might help to coordinate tRNA-ribosome interactions.     

Identification of the yeast nuclear gene for the mitochondrial homologue of bacterial ribosomal protein L16.

An open reading frame encoding a member of the L16 family of ribosomal proteins is adjacent to the URA7 gene on the left arm of chromosome II in Saccharomyces cerevisiae. The predicted L16-like polypeptide is basic (pl 11.12), contains 232 amino acids (26.52 kDa) and has 36% amino acid sequence identity to E. coli L16. Immunoblot analysis with polyclonal antibodies to the L16-like polypeptide showed specific cross-reaction with a 22,000 Mr mitochondrial polypeptide that co-sediments with the large subunit of the mitochondrial ribosome in sucrose density gradients. The levels of the L16 mRNA and protein varied in response to carbon source. In [rho degree] cells lacking mitochondrial rRNA, the L16 mRNA accumulated at normal levels, but the protein was barely detectable, indicating RNA-dependent accumulation of the L16 protein. Gene disruption experiments demonstrated that the yeast mitochondrial L16 is an essential ribosomal protein in vivo.     

Identification of a protein mediating respiratory supercomplex stability

The complexes of the electron transport chain associate into large macromolecular assemblies, which are believed to facilitate efficient electron flow. We have identified a conserved mitochondrial protein, named respiratory supercomplex factor 1 (Rcf1-Yml030w), that is required for the normal assembly of respiratory supercomplexes. We demonstrate that Rcf1 stably and independently associates with both Complex III and Complex IV of the electron transport chain. Deletion of the RCF1 gene caused impaired respiration, probably as a result of destabilization of respiratory supercomplexes. Consistent with the hypothetical function of these respiratory assemblies, loss of RCF1 caused elevated mitochondrial oxidative stress and damage. Finally, we show that knockdown of HIG2A, a mammalian homolog of RCF1, causes impaired supercomplex formation. We suggest that Rcf1 is a member of an evolutionarily conserved protein family that acts to promote respiratory supercomplex assembly and activity.     

Local folding coupled to RNA binding in the yeast ribosomal protein L30.

The ribosomal protein L30 from yeast Saccharomyces cerevisiae auto-regulates its own synthesis by binding to a structural element in both its pre-mRNA and its mRNA. The three-dimensional structures of L30 in the free (f L30) and the pre-mRNA bound (b L30) forms have been solved by nuclear magnetic resonance spectroscopy. Both protein structures contain four alternating alpha-helices and four beta-strands segments and adopt an overall topology that is an alphabetaalpha three-layer sandwich, representing a unique fold. Three loops on one end of the alphabetaalpha sandwich have been mapped as the RNA binding site on the basis of structural comparison, chemical shift perturbation and the inter-molecular nuclear Overhauser effects to the RNA. The structural and dynamic comparison of f L30 and b L30 reveals that local dynamics may play an important role in the RNA binding. The fourth helix in b L30 is longer than in f L30, and is stabilized by RNA binding. The exposed hydrophobic surface that is buried upon RNA binding may provide the energy necessary to drive secondary structure formation, and may account for the increased stability of b L30.     

Translation of CGA codon repeats in yeast involves quality control components and ribosomal protein L1

Translation of CGA codon repeats in the yeast Saccharomyces cerevisiae is inefficient, resulting in dose-dependent reduction in expression and in production of an mRNA cleavage product, indicative of a stalled ribosome. Here, we use genetics and translation inhibitors to understand how ribosomes respond to CGA repeats. We find that CGA codon repeats result in a truncated polypeptide that is targeted for degradation by Ltn1, an E3 ubiquitin ligase involved in nonstop decay, although deletion of LTN1 does not improve expression downstream from CGA repeats. Expression downstream from CGA codons at residue 318, but not at residue 4, is improved by deletion of either ASC1 or HEL2, previously implicated in inhibition of translation by polybasic sequences. Thus, translation of CGA repeats likely causes ribosomes to stall and exploits known quality control systems. Expression downstream from CGA repeats at amino acid 4 is improved by paromomycin, an aminoglycoside that relaxes decoding specificity. Paromomycin has no effect if native tRNA^Arg(ICG) is highly expressed, consistent with the idea that failure to efficiently decode CGA codons might occur in part due to rejection of the cognate tRNA^Arg(ICG). Furthermore, expression downstream from CGA repeats is improved by inactivation of RPL1B, one of two genes encoding the universally conserved ribosomal protein L1. The effects of rpl1b-Δ and of either paromomycin or tRNA^Arg(ICG) on CGA decoding are additive, suggesting that the rpl1b-Δ mutant suppresses CGA inhibition by means other than increased acceptance of tRNA^Arg(ICG). Thus, inefficient decoding of CGA likely involves at least two independent defects in translation.     

rRNA binding domain of yeast ribosomal protein L25. Identification of its borders and a key leucine residue

We have delineated the region of yeast ribosomal protein L25 responsible for its specific binding to 26 S rRNA by a novel approach using in vitro synthesized, [35S]methionine-labeled fragments as well as point mutants of the L25 protein. The rRNA binding capacity of these mutant polypeptides was tested by incubation with an in vitro transcribed, biotinylated fragment of yeast 26 S rRNA that contains the complete L25 binding site. Protein-rRNA interaction was assayed by binding of the rRNA-r-protein complex to streptavidin-agarose followed either by analysis of the bound polypeptide by SDS/polyacrylamide gel electrophoresis or by precipitation with trichloroacetic acid. Our results show that the structural elements necessary and sufficient for specific interaction of L25 with 26 S rRNA are contained in the region bordered by amino acids 62 and 126. The remaining parts of the protein, in particular the C-terminal 16 residues, while not essential for binding, do enhance its affinity for 26 S rRNA. To test whether, as suggested by the results of the deletion experiments, the evolutionarily conserved sequence motif K120KAYVRL126 is involved in rRNA binding, we replaced the leucine residue at position 126 by either isoleucine or lysine. The first substitution did not affect binding. The second, however, completely abolished the specific rRNA binding capacity of the protein. Thus, Leu126, and possibly the whole conserved sequence motif, plays a key role in binding of L25 to 26 S rRNA.     

Characterization of nuclear localizing sequences derived from yeast ribosomal protein L29.

Two particular seven-amino-acid segments from yeast ribosomal protein L29 caused a non-nuclear reporter protein to associate almost exclusively with the yeast nucleus. The two L29-derived nuclear localizing sequences were identical in five of the seven residues, many of which were basic amino acids. Generally, localization of the reporter protein was most impaired by replacement of the basic residues. A particular Arg residue was unique; substitution by any amino acid including Lys diminished nuclear localization of the reporter protein. In L29 the corresponding Arg 25----Lys substitution within the nuclear localizing sequence distal to the N-terminus was without effect, as evidence by normal rates of ribosome assembly and cell growth. However, the analogous Arg 8----Lys substitution within the localizing sequence proximal to the N-terminus led to greatly reduced rates of ribosome assembly and cell growth. Finally, when both localizing sequences contained the Arg----Lys substitution a still greater decrease in ribosome assembly and cell growth was observed. These results were as expected if the two short peptide sequences functioned in nuclear localization and/or assembly of yeast ribosomal protein L29.     

Functional features of the C-terminal region of yeast ribosomal protein L5

The aim of this study was to analyze the functional importance of the C-terminus of the essential yeast ribosomal protein L5 (YrpL5). Previous studies have indicated that the C-terminal region of YrpL5 forms an alpha-helix with a positively charged surface that is involved in protein-5S rRNA interaction. Formation of an YrpL5.5S rRNA complex is a prerequisite for nuclear import of YrpL5. Here we have tested the importance of the alpha-helix and the positively charged surface for YrpL5 function in Saccharomyces cerevisiae using site directed mutagenesis in combination with functional complementation. Alterations in the sequence forming the putative alpha-helix affected the functional capacity of YrpL5. However, the effect did not correlate with a decreased ability of the protein to bind to 5S rRNA as all rpL5 mutants tested were imported to the nucleus whether or not the alpha-helix or the positively charged surface were intact. The alterations introduced in the C-terminal sequence affected the growth rate of cells expressing mutant but functional forms of YrpL5. The reduced growth rate was correlated with a reduced ribosomal content per cell indicating that the alterations introduced in the C-terminus interfered with ribosome assembly.     

Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16.

Temperature-sensitive mutants defective in 60S ribosomal subunit protein L16 of Saccharomyces cerevisiae were isolated through hydroxylamine mutagenesis of the RPL16B gene and plasmid shuffling. Two heat-sensitive and two cold-sensitive isolates were characterized. The growth of the four mutants is inhibited at their restrictive temperatures. However, many of the cells remain viable if returned to their permissive temperatures. All of the mutants are deficient in 60S ribosomal subunits and therefore accumulate translational preinitiation complexes. Three of the mutants exhibit a shortage of mature 25S rRNA, and one accumulates rRNA precursors. The accumulation of rRNA precursors suggests that ribosome assembly may be slowed in this mutant. These phenotypes lead us to propose that mutants containing the rpl16b alleles are defective for 60S subunit assembly rather than function. In the mutant carrying the rpl16b-1 allele, ribosomes initiate translation at the noncanonical codon AUA, at least on the rpl16b-1 mRNA, bringing to light a possible connection between the rate and the fidelity of translation initiation.     

The rna2 mutation of yeast affects the processing of actin mRNA as well as ribosomal protein mRNAs

The temperature sensitive rna2 mutation of Saccharomyces cerevisiae causes a rapid and dramatic decrease in the abundance of most ribosomal protein mRNAs We and others have recently shown that the processing of ribosomal protein mRNAs is defective at the nonpermissive temperature, suggesting that inefficient mRNA processing might be responsible for the decline in ribosomal protein mRNA levels. Actin is the only known intron-containing non-ribosomal protein yeast nuclear gene We show here that the processing of actin mRNA is also defective at the nonpermissive temperature in rna2-containing strains. The observation supports the notion that all intron-containing genes are affected in a similar fashion by the rna2 mutation.     

The primary structure of rat liver ribosomal protein L37. Homology with yeast and bacterial ribosomal proteins

The covalent structure of the rat liver 60 S ribosomal subunit protein L37 was determined. Twenty-four tryptic peptides were purified and the sequence of each was established; they accounted for all 111 residues of L37. The sequence of the first 30 residues of L37, obtained previously by automated Edman degradation of the intact protein, provided the alignment of the first 9 tryptic peptides. Three peptides (CN1, CN2, and CN3) were produced by cleavage of protein L37 with cyanogen bromide. The sequence of CN1 (65 residues) was established from the sequence of secondary peptides resulting from cleavage with trypsin and chymotrypsin. The sequence of CN1 in turn served to order tryptic peptides 1 through 14. The sequence of CN2 (15 residues) was determined entirely by a micromanual procedure and allowed the alignment of tryptic peptides 14 through 18. The sequence of the NH2-terminal 28 amino acids of CN3 (31 residues) was determined; in addition the complete sequences of the secondary tryptic and chymotryptic peptides were done. The sequence of CN3 provided the order of tryptic peptides 18 through 24. Thus the sequence of the three cyanogen bromide peptides also accounted for the 111 residues of protein L37. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. There is a disulfide bridge between half-cystinyl residues at positions 40 and 69. Rat liver ribosomal protein L37 is homologous with yeast YP55 and with Escherichia coli L34. Moreover, there is a segment of 17 residues in rat L37 that occurs, albeit with modifications, in yeast YP55 and in E. coli S4, L20, and L34.