30S Subunit Research Articles

Functions of block of proliferation 1 during anterior development in Xenopus laevis.

Block of proliferation 1 (Bop1) is a nucleolar protein known to be necessary for the assembly of the 60S subunit of ribosomes. Here, we show a specific bop1 expression in the developing anterior tissue of the South African clawed frog Xenopus laevis. Morpholino oligonucleotide-mediated knockdown approaches demonstrated that Bop1 is required for proper development of the cranial cartilage, brain, and the eyes. Furthermore, we show that bop1 knockdown leads to impaired retinal lamination with disorganized cell layers. Expression of neural crest-, brain-, and eye-specific marker genes was disturbed. Apoptotic and proliferative processes, which are known to be affected during ribosomal biogenesis defects, are not hindered upon bop1 knockdown. Because early Xenopus embryos contain a large store of maternal ribosomes, we considered if Bop1 might have a role independent of de novo ribosomal biogenesis. At early embryonic stages, pax6 expression was strongly reduced in bop1 morphants and synergy experiments indicate a common signaling pathway of the two molecules, Bop1 and Pax6. Our studies imply a novel function of Bop1 independent of ribosomal biogenesis.

Wide mutational analysis to ascertain the functional roles of eL33 in ribosome biogenesis and translation initiation

An extensive mutational analysis of RPL33A, encoding the yeast ribosomal protein L33A (eL33) allowed us to identify several novel rpl33a mutants with different translational phenotypes. Most of the rpl33a mutants are defective in the processing of 35S and 27S pre-rRNA precursors and the production of mature rRNAs, exhibiting reductions in the amounts of ribosomal subunits and altered polysome profiles. Some of the rpl33a mutants exhibit a Gcd− phenotype of constitutive derepression of GCN4 translation and strong slow growth phenotypes at several temperatures. Interestingly, some of the later mutants also show a detectable increase in the UUG/AUG translation initiation ratio that can be suppressed by eIF1 overexpression, suggesting a requirement for eL33 and a correct 60S/40S subunit ratio for the proper recognition of the AUG start codon. In addition to producing differential reductions in the rates of pre-rRNA maturation and perhaps in r-protein assembly, most of the point rpl33a mutations alter specific molecular interactions of eL33 with the rRNAs and other r-proteins in the 60S structure. Thus, rpl33a mutations cause distinctive effects on the abundance and/or functionality of 60S subunits, leading to more or less pronounced defects in the rates and fidelity of mRNA translation.

The Bacillus subtilis open reading frame ysgA encodes the SPOUT methyltransferase RlmP forming 2'-O-methylguanosine at position 2553 in the A-loop of 23S rRNA.

A previous bioinformatic analysis predicted that the ysgA open reading frame of Bacillus subtilis encodes an RNA methyltransferase of the SPOUT superfamily. Here we show that YsgA is the 2'-O-methyltransferase that targets position G2553 (Escherichia coli numbering) of the A-loop of 23S rRNA. This was shown by a combination of biochemical and mass spectrometry approaches using both rRNA extracted from B. subtilis wild-type or ΔysgA cells and in vitro synthesized rRNA. When the target G2553 is mutated, YsgA is able to methylate the ribose of adenosine. However, it cannot methylate cytidine nor uridine. The enzyme modifies free 23S rRNA but not the fully assembled ribosome nor the 50S subunit, suggesting that the modification occurs early during ribosome biogenesis. Nevertheless, ribosome subunits assembly is unaffected in a B. subtilis ΔysgA mutant strain. The crystal structure of the recombinant YsgA protein, combined with mutagenesis data, outlined in this article highlights a typical SPOUT fold preceded by an L7Ae/L30 (eL8/eL30 in a new nomenclature) amino-terminal domain.

Changes in the Transcriptome Caused by Mutations in the Ribosomal Protein uS10 Associated with a Predisposition to Colorectal Cancer.

A number of mutations in the RPS20 gene encoding the ribosomal protein uS10 have been found to be associated with a predisposition to hereditary non-polyposis colorectal carcinoma (CRC). We transfected HEK293T cells with constructs carrying the uS10 minigene with mutations identical to those mentioned above and examined the effects of the produced proteins on the cellular transcriptome. We showed that uS10 with mutations p.V50SfsX23 or p.L61EfsX11 cannot be incorporated into 40S ribosomal subunits, while the protein with the missense mutation p.V54L functionally replaces the respective endogenous protein in the 40S subunit assembly and the translation process. The comparison of RNA-seq data obtained from cells producing aberrant forms of uS10 with data for those producing the wild-type protein revealed overlapping sets of upregulated and downregulated differently expressed genes (DEGs) related to several pathways. Among the limited number of upregulated DEGs, there were genes directly associated with the progression of CRC, e.g., PPM1D and PIGN. Our findings indicate that the accumulation of the mutant forms of uS10 triggers a cascade of cellular events, similar to that which is triggered when the cell responds to a large number of erroneous proteins, suggesting that this may increase the risk of cancer.

Ribosomal protein eL39 is important for maturation of the nascent polypeptide exit tunnel and proper protein folding during translation.

During translation, nascent polypeptide chains travel from the peptidyl transferase center through the nascent polypeptide exit tunnel (NPET) to emerge from 60S subunits. The NPET includes portions of five of the six 25S/5.8S rRNA domains and ribosomal proteins uL4, uL22, and eL39. Internal loops of uL4 and uL22 form the constriction sites of the NPET and are important for both assembly and function of ribosomes. Here, we investigated the roles of eL39 in tunnel construction, 60S biogenesis, and protein synthesis. We show that eL39 is important for proper protein folding during translation. Consistent with a delay in processing of 27S and 7S pre-rRNAs, eL39 functions in pre-60S assembly during middle nucleolar stages. Our biochemical assays suggest the presence of eL39 in particles at these stages, although it is not visualized in them by cryo-electron microscopy. This indicates that eL39 takes part in assembly even when it is not fully accommodated into the body of pre-60S particles. eL39 is also important for later steps of assembly, rotation of the 5S ribonucleoprotein complex, likely through long range rRNA interactions. Finally, our data strongly suggest the presence of alternative pathways of ribosome assembly, previously observed in the biogenesis of bacterial ribosomal subunits.

The structure–function analysis of Obg‐like GTPase proteins along the evolutionary tree from bacteria to humans

Obg proteins belong to P‐loop guanine triphosphatase (GTPase) that are conserved from bacteria to humans. Like other GTPases, Obg cycles between guanine triphosphate (GTP) bound “on” state and guanine diphosphate (GDP)‐bound “off” state, thereby controlling various cellular processes. Different members of this group have unique structural characteristics; a conserved glycine‐rich N‐terminal domain known as obg fold, a central conserved nucleotide binding domain, and a less conserved C‐terminal domain of other functions. Obg is a ribosome dependent GTPase helps in ribosome maturation by interacting with several proteins of the 50S subunit of the ribosome. Obg proteins have been widely considered as a regulator of cellular functions, helping in DNA replication, cell division. Apart from that, this protein also takes part in various stress adaptation pathways like a stringent response, sporulation, and general stress response. In this particular review, the structural features of ObgE have been highlighted and how the structure plays important role in interacting with regulators like GTP, ppGpp that are crucial for executing biological function has been orchestrated. In particular, we believe that Obg‐like proteins can provide a link between different global pathways that are necessary for fine‐tuning cellular processes to maintain the cellular energy status.

Translation initiation site of mRNA is selected through dynamic interaction with the ribosome

Initiation of protein synthesis from the correct start codon of messenger RNA (mRNA) is crucial to translation fidelity. In bacteria, the start codon is usually preceded by a 4- to 6-mer adenosine/guanosine-rich Shine–Dalgarno (SD) sequence. Both the SD sequence and the start codon comprise the core ribosome-binding site (RBS), to which the 30S ribosomal subunit binds to initiate translation. How the rather short and degenerate information inside the RBS can be correctly accommodated by the ribosome is not well understood. Here, we used single-molecule techniques to tackle this long-standing issue. We found that the 30S subunit initially binds to mRNA through the SD sequence, whereas the downstream RBS undergoes dynamic motions, especially when it forms structures. The mRNA is either dissociated or stabilized by initiation factors, such as initiation factor 3 (IF3). The initiator transfer RNA (tRNA) further helps the 30S subunit accommodate mRNA and unwind up to 3 base pairs of the RBS structure. Meanwhile, the formed complex of the 30S subunit with structured mRNA is not stable and tends to disassociate. IF3 promotes dissociation by dismissing the bound initiator tRNA. Thus, initiation factors may accelerate the dynamic assembly–disassembly process of 30S–mRNA complexes such that the correct RBS can be preferentially selected. Our study provides insights into how the bacterial ribosome identifies a typical translation initiation site from mRNA.

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex.

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2–GTP–Met-tRNAiMet and eIF3. The ‘open’ 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The ‘closed’ form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

Direct measurements of mRNA translation kinetics in living cells

Ribosome mediated mRNA translation is central to life. The cycle of translation, however, has been characterized mostly using reconstituted systems, with only few techniques applicable for studies in the living cell. Here we describe a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding and translating an mRNA, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in translation at any particular time, and that the 30S and 50S ribosomal subunits spend the same average time bound to an mRNA, revealing that 30S re-initiation on poly-cistronic mRNAs is not prevalent in E. coli. Instead, our results are best explained by substantial 70S re-initiation of translation of poly-cistronic mRNAs, which is further corroborated by experiments with translation initiation inhibitors. Finally, we find that a variety of previously described orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs.

Emergence of the primordial pre-60S from the 90S pre-ribosome.

SummarySynthesis of ribosomes begins in the nucleolus with formation of the 90S pre-ribosome, during which the pre-40S and pre-60S pathways diverge by pre-rRNA cleavage. However, it remains unclear how, after this uncoupling, the earliest pre-60S subunit continues to develop. Here, we reveal a large-subunit intermediate at the beginning of its construction when still linked to the 90S, the precursor to the 40S subunit. This primordial pre-60S is characterized by the SPOUT domain methyltransferase Upa1-Upa2, large α-solenoid scaffolds, Mak5, one of several RNA helicases, and two small nucleolar RNA (snoRNAs), C/D box snR190 and H/ACA box snR37. The emerging pre-60S does not efficiently disconnect from the 90S pre-ribosome in a dominant mak5 helicase mutant, allowing a 70-nm 90S-pre-60S bipartite particle to be visualized by electron microscopy. Our study provides insight into the assembly pathway when the still-connected nascent 40S and 60S subunits are beginning to separate.

50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA

Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2′-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.

Knockout of ribosomal genes bS22 and bL37 increases the sensitivity of mycobacteria to antibiotics

In recent years, two novel proteins in the ribosomes of mycobacteria have been discovered by cryo-electron microscopy. The protein bS22 is located near the decoding center of the 30S subunit, and the protein bL37 is located near the peptidyl transferase center of the 50S subunit. Since these two proteins bind to conserved regions of the ribosome targeted by antibiotics, it is speculated that they might affect the binding of related drugs to these targets. Therefore, we knocked out the genes encoding these two proteins in wild-type Mycolicibacterium smegmatis mc2155 through homologous recombination, and then determined the growth curves of these mutants and their sensitivity to related antibiotics. The results showed that compared with the wild-type strain, the growth rate of these two mutants did not change significantly. However, mutant ΔbS22 showed increased sensitivity to capreomycin, kanamycin, amikacin, streptomycin, gentamicin, paromomycin, and hygromycin B, while mutant ΔbL37 showed increased sensitivity to linezolid. These changes in antibiotics sensitivity were restored by gene complementation. This study hints at the possibility of using ribosomal proteins bS22 and bL37 as targets for drug design.

EIF6 rebinding dynamically couples ribosome maturation and translation

Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1—both mutated in the leukemia predisposition disorder Shwachman-Diamond syndrome — license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. We find that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. These data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukemia predisposition disorder.

Metabolomic profiles in a green alga (Raphidocelis subcapitata) following erythromycin treatment: ABC transporters and energy metabolism

A recent study showed that erythromycin (ERY) exposure caused hormesis in a model alga (Raphidocelis subcapitata) where the growth was promoted at an environmentally realistic concentration (4 µg/L) but inhibited at two higher concentrations (80 and 120 µg/L), associated with opposite actions of certain signaling pathways (e.g., xenobiotic metabolism, DNA replication). However, these transcriptional alterations remain to be investigated and verified at the metabolomic level. This study uncovered metabolomic profiles and detailed toxic mechanisms of ERY in R. subcapitata using untargeted metabolomics. The metabolomic analysis showed that metabolomic pathways including ABC transporters, fatty acid biosynthesis and purine metabolism were associated with growth promotion in algae treated with 4 µg/L ERY. An overcompensation was possibly activated by the low level of ERY in algae where more resources were reallocated to efficiently restore the temporary impairments, ultimately leading to the outperformance of growth. By contrast, algal growth inhibition in the 80 and 120 µg/L ERY treatments was likely attributed to the dysfunction of metabolomic pathways related to ABC transporters, energy metabolism and metabolism of nucleosides. Apart from binding of ERY to the 50S subunit of ribosomes to inhibit protein translation as in bacteria, the data presented here indicate that inhibition of protein translation and growth performance of algae by ERY may also result from the suppression of amino acid biosynthesis and aminoacyl-tRNA biosynthesis. This study provides novel insights into the dose-dependent toxicity of ERY on R. subcapitata.

Annealing synchronizes the 70S ribosome into a minimum-energy conformation

Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70S ribosome from Escherichia coli as a model, wherein the 30S subunit undergoes a thermally driven intersubunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70S ribosome homogeneity and improves local resolution on the 30S subunit. After annealing, the 70S ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes. Manifold-based analysis further indicates that the annealed 70S ribosome takes a narrow conformational distribution and exhibits a minimum-energy state in the free-energy landscape. Our experimental results offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing shows great potential for synchronizing proteins on a single-molecule level and can be extended to study protein folding and explore conformational and energy landscapes.

Genome-wide RNAi screen identifies novel players in human 60S subunit biogenesis including key enzymes of polyamine metabolism.

Ribosome assembly is an essential process that is linked to human congenital diseases and tumorigenesis. While great progress has been made in deciphering mechanisms governing ribosome biogenesis in eukaryotes, an inventory of factors that support ribosome synthesis in human cells is still missing, in particular regarding the maturation of the large 60S subunit. Here, we performed a genome-wide RNAi screen using an imaging-based, single cell assay to unravel the cellular machinery promoting 60S subunit assembly in human cells. Our screen identified a group of 310 high confidence factors. These highlight the conservation of the process across eukaryotes and reveal the intricate connectivity of 60S subunit maturation with other key cellular processes, including splicing, translation, protein degradation, chromatin organization and transcription. Intriguingly, we also identified a cluster of hits comprising metabolic enzymes of the polyamine synthesis pathway. We demonstrate that polyamines, which have long been used as buffer additives to support ribosome assembly in vitro, are required for 60S maturation in living cells. Perturbation of polyamine metabolism results in early defects in 60S but not 40S subunit maturation. Collectively, our data reveal a novel function for polyamines in living cells and provide a rich source for future studies on ribosome synthesis.

Ribosome Associated Protein Quality Control: Mechanism and Function

Due to numerous reasons, including faulty m RNA, insufficient availability of charged t RNA, genetic errors, ribosomes are failed to synthesize protein sometimes. All organisms develop their machinery to recognize stalled ribosomes. Stalled ribosomes, results in the production of a truncated polypeptide which can affect cells. So, they must be eliminated, by mechanisms known as Ribosome-associated protein quality control (RQC). E3 ubiquitin ligase Ltn1 in RQC promotes clearance of 60S subunit and targets aberrant nascent polypeptides for proteasomal degradation. In eukaryotes, RQC facilitates the ribosomal rescue, where staled m RNAs release and allow to degrade and ribosomal subunits are to be recycled for further use. Ribosome-associated protein quality control in yeast is accomplished by Hel2-dependent ubiquitination of uS10 and RQC-trigger (RQT) complex. RQC in a mammal is done by ZNF598-dependent ubiquitination of collided ribosomes, which also activates signal integrator 3, a component of the ASCC complex. Human RQT (h RQT) is made up of ASCC3, ASCC2, TRIP4, which are orthologs of RNA helicase Slh1, ubiquitin-binding protein Cue3, and ykR023W protein respectively. Ubiquitin-binding activity and ATPase activity of ASCC2 and ASCC3 respectively, are important for RQC. So, it is obvious that the h RQT complex recognizes the ubiquitinated defective ribosome and induces subunit dissociation for RQC. Biogenesis of new polypeptide, folding, correct localization are the fundamental processes to maintain proteostasis, which involve various factors directly attached with ribosomes and chaperones. Ribosome-associated protein biogenesis factors mediate the cellular proteostasis network to form integrity.

RbgA ensures the correct timing in the maturation of the 50S subunits functional sites.

RbgA is an essential protein for the assembly of the 50S subunit in Bacillus subtilis. Depletion of RbgA leads to the accumulation of the 45S intermediate. A strain expressing a RbgA variant with reduced GTPase activity generates spontaneous suppressor mutations in uL6. Each suppressor strain accumulates a unique 44S intermediate. We reasoned that characterizing the structure of these mutant 44S intermediates may explain why RbgA is required to catalyze the folding of the 50S functional sites. We found that in the 44S particles, rRNA helices H42 and H97, near the binding site of uL6, adopt a flexible conformation and allow the central protuberance and functional sites in the mutant 44S particles to mature in any order. Instead, the wild-type 45S particles exhibit a stable H42-H97 interaction and their functional sites always mature last. The dependence on RbgA was also less pronounced in the 44S particles. We concluded that the binding of uL6 pauses the maturation of the functional sites, but the central protuberance continues to fold. RbgA exclusively binds intermediates with a formed central protuberance and licenses the folding of the functional sites. Through this mechanism, RbgA ensures that the functional sites of the 50S mature last.

40S scanning unveiled by smFRET

The longstanding scanning model of eukaryotic translation initiation states that the small (40S) ribosomal subunit is recruited to the 5′ end of the messenger RNA (mRNA) and then moves towards the 3′ end to locate the first start codon within a favorable sequence context, followed by recruitment of the large 60S subunit to begin protein synthesis. This process is orchestrated by numerous eukaryotic translation initiation factors (eIFs). Despite extensive biochemical, structural, and genetics analyses, scanning by 40S subunits has not been observed directly, hindering our mechanistic understanding of initiation.

RPS3 enhances full-length NFκB-DNA binding through ternary complex formation

Full-length NFκB (RelA-p50 heterodimer) is a transcription factor that responds to inflammatory and immune stress in human cells and is involved in cancer, inflammation, and autoimmune disorders. To better comprehend the control of the transcription of genes driven by NFκB, it is important to understand the roles played by other proteins in its kinetic regulation. Ribosomal Protein S3 (RPS3) is a protein component of the 40S subunit of the ribosome. It has been previously reported that NFκB gene expression is downregulated by RPS3 knockdown, indicating that NFκB has a dependence on RPS3.