Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly

Numerous ribosomal proteins have a striking bipartite architecture: a globular body positioned on the ribosomal exterior and an internal loop buried deep into the rRNA core. In eukaryotes, a significant number of conserved r-proteins have evolved extra amino- or carboxy-terminal tail sequences, which thread across the solvent-exposed surface. The biological importance of these extended domains remains to be established. In this study, we have investigated the universally conserved internal loop and the eukaryote-specific extensions of yeast L4. We show that in contrast to findings with bacterial L4, deleting the internal loop of yeast L4 causes severely impaired growth and reduced levels of large ribosomal subunits. We further report that while depleting the entire L4 protein blocks early assembly steps in yeast, deletion of only its extended internal loop affects later steps in assembly, revealing a second role for L4 during ribosome biogenesis. Surprisingly, deletion of the entire eukaryote-specific carboxy-terminal tail of L4 has no effect on viability, production of 60S subunits, or translation. These unexpected observations provide impetus to further investigate the functions of ribosomal protein extensions, especially eukaryote-specific examples, in ribosome assembly and function.     

The C-terminal fragment of ribosomal protein S15 is located in the decoding site of the human ribosome

Protein S15 is a characteristic component of the mammalian 80S ribosome that neighbors the mRNA codon at the decoding site and the downstream triplets. The S15 fragment juxtaposed in the human ribosome to mRNA nucleotides +4 to +12 relative to the first nucleotide of the P-site codon was determined. S15 was modified using a set of mRNA analogs containing the triplet UUU/UUC at the 5′ end and a perfluorophenyl azide-carrying uridine at various positions downstream of this triplet. The mRNA analogs were positioned on the ribosome with the use of tRNA^Phe, cognate to the UUU/UUC triplet, targeted to the P site. Modified S15 was isolated from complexes of 80S ribosomes with tRNA^Phe and the mRNA analogs after irradiation with mild UV light and hydrolyzed with cyanogen bromide, cleaving the polypeptide chain after Met residues. Analysis of the modified oligopeptides resulting from hydrolysis demonstrated that the crosslinking site was in C-terminal fragment 111–145 of S15 in all cases, suggesting the involvement of this fragment in the decoding site of the eukaryotic ribosome.     

Protein S3 fragments neighboring mRNA during elongation and termination of translation on the human ribosome

Protein S3 fragments were determined that crosslink to modified mRNA analogues in positions +5 to +12 relative to the first nucleotide in the P-site bound codon in model complexes mimicking states of ribosomes at the elongation and translation termination steps. The mRNA analogues contained a Phe codon UUU/UUC at the 5′-termini that could predetermine the position of the tRNA^Phe on the ribosome by the P-site binding and perfluorophenylazidobenzoyl group at a nucleotide in various positions 3′ of the UUU/UUC codon. The crosslinked S3 protein was isolated from 80S ribosomal complexes irradiated with mild UV light and subjected to cyanogen bromide—induced cleavage at methionine residues with subsequent identification of the crosslinked oligopeptides. An analysis of the positions of modified oligopeptides resulting from the cleavage showed that, in dependence on the positions of modified nucleotides in the mRNA analogue, the crosslinking sites were found in the N-terminal half of the protein (fragment 2–217) and/or in the C-terminal fragment 190–236; the latter reflects a new peculiarity in the structure of the mRNA binding center in the ribosome, unknown to date. The results of crosslinking did not depend on the type of A-site codon or on the presence of translation termination factor eRF1.     

A cluster of methylations in the domain IV of 25S rRNA is required for ribosome stability

In all three domains of life ribosomal RNAs are extensively modified at functionally important sites of the ribosome. These modifications are believed to fine-tune the ribosome structure for optimal translation. However, the precise mechanistic effect of modifications on ribosome function remains largely unknown. Here we show that a cluster of methylated nucleotides in domain IV of 25S rRNA is critical for integrity of the large ribosomal subunit. We identified the elusive cytosine-5 methyltransferase for C2278 in yeast as Rcm1 and found that a combined loss of cytosine-5 methylation at C2278 and ribose methylation at G2288 caused dramatic ribosome instability, resulting in loss of 60S ribosomal subunits. Structural and biochemical analyses revealed that this instability was caused by changes in the structure of 25S rRNA and a consequent loss of multiple ribosomal proteins from the large ribosomal subunit. Our data demonstrate that individual RNA modifications can strongly affect structure of large ribonucleoprotein complexes.     

Identification of an Escherichia coli S1-like protein in the spinach chloroplast ribosome

Antibodies directed against E. coli ribosomal protein S1 were used in immunoblotting assays to search for an S1-like protein in the ribosome of spinach chloroplast. An immunological cross-reaction was reproducibly detected on the blots and inhibition experiments have demonstrated its specificity. The chloroplastic ribosomal protein which has epitopes common to antigenic determinants of the E. coli protein S1 was identified as being protein S2/S3.     

The role of human ribosomal proteins in the maturation of rRNA and ribosome production

Production of ribosomes is a fundamental process that occurs in all dividing cells. It is a complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNA) with about 80 ribosomal proteins (r-proteins) involving more than 150 nonribosomal proteins and other factors. Diamond Blackfan anemia (DBA) is an inherited red cell aplasia caused by mutations in one of several r-proteins. How defects in r-proteins, essential for proliferation in all cells, lead to a human disease with a specific defect in red cell development is unknown. Here, we investigated the role of r-proteins in ribosome biogenesis in order to find out whether those mutated in DBA have any similarities. We depleted HeLa cells using siRNA for several individual r-proteins of the small (RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, RPS28) or large subunit (RPL5, RPL7, RPL11, RPL14, RPL26, RPL35a) and studied the effect on rRNA processing and ribosome production. Depleting r-proteins in one of the subunits caused, with a few exceptions, a decrease in all r-proteins of the same subunit and a decrease in the corresponding subunit, fully assembled ribosomes, and polysomes. R-protein depletion, with a few exceptions, led to the accumulation of specific rRNA precursors, highlighting their individual roles in rRNA processing. Depletion of r-proteins mutated in DBA always compromised ribosome biogenesis while affecting either subunit and disturbing rRNA processing at different levels, indicating that the rate of ribosome production rather than a specific step in ribosome biogenesis is critical in patients with DBA.     

Interaction of trigger factor with the ribosome

The trigger factor of Escherichia coli is a prolyl isomerase and a chaperone. It interacts with the ribosome and affects the folding of newly formed protein chains. Therefore, the dynamics of the interactions of trigger factor with the ribosome and with unfolded protein chains should be tailored for this function. Previously, we had found that binding of unfolded proteins to trigger factor is fast and that the lifetime of the complex between these two components is only about 100 ms. Here, we have labeled the trigger factor in its amino-terminal, ribosome-binding domain with a fluorescent dye and investigated how it interacts with the ribosome. We found that this association, as well as the dissociation of the complex, are rather slow processes. The average lifetime of the complex is about 30 seconds (at 20 degrees C). The strong differences in the dynamics of the interactions of trigger factor with the ribosome and with protein substrates might ensure that, on the one hand, trigger factor remains bound to the ribosome while a protein chain is being synthesized, and, on the other hand, allows it to scan the newly formed protein for prolyl bonds that need catalysis of isomerization.     

Distribution of rRNA introns in the three-dimensional structure of the ribosome

More than 1200 introns have been documented at over 150 unique sites in the small and large subunit ribosomal RNA genes (as of February 2002). Nearly all of these introns are assigned to one of four main types: group I, group II, archaeal and spliceosomal. This sequence information has been organized into a relational database that is accessible through the Comparative RNA Web Site (http://www.rna.icmb.utexas.edu/) While the rRNA introns are distributed across the entire tree of life, the majority of introns occur within a few phylogenetic groups. We analyzed the distributions of rRNA introns within the three-dimensional structures of the 30S and 50S ribosomes. Most sites in rRNA genes that contain introns contain only one type of intron. While the intron insertion sites occur at many different coordinates, the majority are clustered near conserved residues that form tRNA binding sites and the subunit interface. Contrary to our expectations, many of these positions are not accessible to solvent in the mature ribosome. The correlation between the frequency of intron insertions and proximity of the insertion site to functionally important residues suggests an association between intron evolution and rRNA function.     

The landscape of human mutually exclusive splicing

Mutually exclusive splicing of exons is a mechanism of functional gene and protein diversification with pivotal roles in organismal development and diseases such as Timothy syndrome, cardiomyopathy and cancer in humans. In order to obtain a first genomewide estimate of the extent and biological role of mutually exclusive splicing in humans, we predicted and subsequently validated mutually exclusive exons (MXEs) using 515 publically available RNA‐Seq datasets. Here, we provide evidence for the expression of over 855 MXEs, 42% of which represent novel exons, increasing the annotated human mutually exclusive exome more than fivefold. The data provide strong evidence for the existence of large and multi‐cluster MXEs in higher vertebrates and offer new insights into MXE evolution. More than 82% of the MXE clusters are conserved in mammals, and five clusters have homologous clusters in Drosophila. Finally, MXEs are significantly enriched in pathogenic mutations and their spatio‐temporal expression might predict human disease pathology.     

Alternative splicing mechanisms for the modulation of protein function: conservation between human and other species

Alternative splicing (AS) is an important process in eukaryotic organisms by which a given gene may express a set of different protein isoforms depending on the tissue, or the developmental stage of the individual. In the present work, we have compared AS among species, focusing on the conservation of AS mechanisms for the modulation of protein function. For this purpose, we first analysed the frequency with which different species, human, mouse, rat and fruitfly, utilise them. Second, we focused more directly on the conservation among species of the mechanisms themselves. To this end, we compared biologically equivalent AS events between human and mouse, or rat. Our results indicate only minor differences in the frequency of use of these mechanisms, as well as a high degree of conservation among the species studied.     

SR蛋白家族在RNA剪接中的调控作用

SR蛋白家族成员都具有一个富含丝氨酸/精氨酸(S/R)重复序列的RS结构域,在RNA剪接体的组装和选择性剪接的调控过程中具有重要的作用。绝大多数SR蛋白是生存的必需因子,通过其RS结构域和特有的其他结构域,实现与前体mRNA的特异性序列或其他剪接因子的相互作用,协同完成剪接位点的正确选择或促进剪接体的形成。深入研究SR蛋白家族在RNA选择性剪接中的调控机制,可以促进以疾病治疗或害虫防治为目的的应用研究。该文总结了SR蛋白家族在基础研究和应用方面的进展。     

The ribosome collision sensor Hel2 functions as preventive quality control in the secretory pathway

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Changes in the protein composition of cytoplasmic ribsomes during the greening of etiolated barley leaves

We examined changes in the protein composition of cytoplasmic ribosomes in etiolated barley leaves following illumination. Cytoplasmic ribosomes were isolated from greening barley leaves by sucrose density gradient centrifugation, and were analyzed by radical-free highly reducing polyacrylamide gel electrophoresis (RFHR-PAGE). Eighty-nine proteins were resolved from the ribosomal fraction; among them, 8 proteins changed their copy numbers depending on the stage of greening. We designated these as phase dependent ribosomal proteins (PD1–PD8). Two of the proteins (PD1 and 5) present in the ribosomes of etiolated leaves showed a decrease in level during greening. In contrast, the levels of 6 ribosomal proteins (PD2, 3, 4, 6, 7 and 8) increased as greening proceeded. N-terminal amino acid sequence of PD8 showed high homology to rat ribosomal protein L34. The ribosomal proteins that appeared after illumination were not found in any fraction of the etiolated leaves, suggesting that they were synthesized after the onset of illumination. Copy numbers of other ribosomal proteins did not change during greening.     

Structural changes in the ribosome during the elongation cycle

Ample data on structural changes that arise in the ribosome during translation have been accumulated. The most interesting information on such changes has been obtained by cryoelectron microscopy of ribosome complexes with various ligands and by rRNA site-directed mutagenesis combined with a structural analysis of the ribosome by a chemical modification technique (chemical probing). The review considers the best-known structural changes that arise in the translating ribosome upon its interactions with tRNA and the elongation factors. The changes are discussed in the context of interactions between the functional centers of the ribosome. A universal system of rRNA helices and proteins is described in detail. The system integrates the functional centers of the ribosome and allows transduction of allosteric conformational signals. Biochemical data are considered in terms of the structures and interactions of ribosomal elements, and a hypothesis is advanced that the position of the GTPase-associated center in the ribosome regulates the binding of the elongation factors.     

RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis

RsgA is a 30S ribosomal subunit-binding GTPase with an unknown function, shortage of which impairs maturation of the 30S subunit. We identified multiple gain-of-function mutants of Escherichia coli rbfA, the gene for a ribosome-binding factor, that suppress defects in growth and maturation of the 30S subunit of an rsgA-null strain. These mutations promote spontaneous release of RbfA from the 30S subunit, indicating that cellular disorders upon depletion of RsgA are due to prolonged retention of RbfA on the 30S subunit. We also found that RsgA enhances release of RbfA from the mature 30S subunit in a GTP-dependent manner but not from a precursor form of the 30S subunit. These findings indicate that the function of RsgA is to release RbfA from the 30S subunit during a late stage of ribosome biosynthesis. This is the first example of the action of a GTPase on the bacterial ribosome assembly described at the molecular level.