The extended loops of ribosomal proteins uL4 and uL22 of Escherichia coli contribute to ribosome assembly and protein translation

Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the only sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlAcrb pause peptide

Increasing Complexity of Ribosomes and Their Biogenesis

According to the classic ribosome model, developed in the 1960s and 1970s, its only function is to translate the four-letter nucleic acid code into the 20 amino acid peptide-code, while polymerizing amino acids into peptides with the help of a large complement of tRNAs and translation factors that cycle on and off the ribosome [...

Interaction between the assembly of the ribosomal subunits: Disruption of 40S ribosomal assembly causes accumulation of extra-ribosomal 60S ribosomal protein uL18/L5.

Inhibition of the synthesis of an essential ribosomal protein (r-protein) abrogates the assembly of its cognate subunit, while assembly of the other subunit continues. Ribosomal components that are not stably incorporated into ribosomal particles due to the disrupted assembly are rapidly degraded. The 60S protein uL18/L5 is an exception and this protein accumulates extra-ribosomally during inhibition of 60S assembly. Since the r-proteins in each ribosomal subunit are essential only for the formation of their cognate subunit, it would be predicted that accumulation of extra-ribosomal uL18/L5 is specific to restriction of 60S assembly and does not occur abolition of 40S assembly. Contrary to this prediction, we report here that repression of 40S r-protein genes does lead to accumulation of uL18/L5 outside of the ribosome. Furthermore, the effect varies depending on which 40S ribosomal protein is repressed. Our results also show extra-ribosomal uL18/L5 is formed during 60S assembly, not during degradation of mature cytoplasmic 60S subunits. Finally, we propose a model for the accumulation of extra-ribosomal uL18 in response to the abolition of 40S r-proteins

A Novel Model for the RNase MRP-Induced Switch between the Formation of Different Forms of 5.8S rRNA

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the “spacer” sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3′ end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5′ end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5′ end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5′ end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA

Co-Assembly of 40S and 60S Ribosomal Proteins in Early Steps of Eukaryotic Ribosome Assembly

In eukaryotes three of the four ribosomal RNA (rRNA) molecules are transcribed as a long precursor that is processed into mature rRNAs concurrently with the assembly of ribosomal subunits. However, the relative timing of association of ribosomal proteins with the ribosomal precursor particles and the cleavage of the precursor rRNA into the subunit-specific moieties is not known. To address this question, we searched for ribosomal precursors containing components from both subunits. Particles containing specific ribosomal proteins were targeted by inducing synthesis of epitope-tagged ribosomal proteins followed by pull-down with antibodies targeting the tagged protein. By identifying other ribosomal proteins and internal rRNA transcribed spacers (ITS1 and ITS2) in the immuno-purified ribosomal particles, we showed that eS7/S7 and uL4/L4 bind to nascent ribosomes prior to the separation of 40S and 60S specific segments, while uS4/S9, uL22, and eL13/L13 are bound after, or simultaneously with, the separation. Thus, the incorporation of ribosomal proteins from the two subunits begins as a co-assembly with a single rRNA molecule, but is finished as an assembly onto separate precursors for the two subunits

Regulation of Ribosomal Protein Synthesis in Vibrio cholerae

We have investigated the regulation of the S10 and spc ribosomal protein (r-protein) operons in Vibrio cholerae. Both operons are under autogenous control; they are mediated by r-proteins L4 and S8, respectively. Our results suggest that Escherichia coli-like strategies for regulating r-protein synthesis extend beyond the enteric members of the gamma subdivision of proteobacteria

Prediction of fracture nonunion leading to secondary surgery in patients with distal femur fractures

Aims Several previously identified patient-, injury-, and treatment-related factors are associated with the development of nonunion in distal femur fractures. However, the predictive value of these factors is not well defined. We aimed to assess the predictive ability of previously identified risk factors in the development of nonunion leading to secondary surgery in distal femur fractures.Methods We conducted a retrospective cohort study of adult patients with traumatic distal femur frac-ture treated with lateral locking plate between 2009 and 2018. The patients who underwent secondary surgery due to fracture healing problem or plate failure were considered having nonunion. Background knowledge of risk factors of distal femur fracture nonunion based on previous literature was used to form an initial set of variables. A logistic regression model was used with previously identified patient-and injury-related variables (age, sex, BMI, dia-betes, smoking, periprosthetic fracture, open fracture, trauma energy, fracture zone length, fracture comminution, medial side comminution) in the first analysis and with treatment-related variables (different surgeon-controlled factors, e.g. plate length, screw placement, and proximal fixation) in the second analysis to predict the nonunion leading to secondary surgery in distal femur fractures.Results We were able to include 299 fractures in 291 patients. Altogether, 31/299 fractures (10%) developed nonunion. In the first analysis, pseudo- R2 was 0.27 and area under the receiver operating characteristic curve (AUC) was 0.81. BMI was the most important variable in the prediction. In the second analysis, pseudo- R2 was 0.06 and AUC was 0.67. Plate length was the most important variable in the prediction.Conclusion The model including patient-and injury-related factors had moderate fit and predictive abil-ity in the prediction of distal femur fracture nonunion leading to secondary surgery. BMI was the most important variable in prediction of nonunion. Surgeon-controlled factors had a minor role in prediction of nonunion.Peer reviewe

Prediction of fracture nonunion leading to secondary surgery in patients with distal femur fractures

Aims: Several previously identified patient-, injury-, and treatment-related factors are associated with the development of nonunion in distal femur fractures. However, the predictive value of these factors is not well defined. We aimed to assess the predictive ability of previously identified risk factors in the development of nonunion leading to secondary surgery in distal femur fractures. Methods: We conducted a retrospective cohort study of adult patients with traumatic distal femur fracture treated with lateral locking plate between 2009 and 2018. The patients who underwent secondary surgery due to fracture healing problem or plate failure were considered having nonunion. Background knowledge of risk factors of distal femur fracture nonunion based on previous literature was used to form an initial set of variables. A logistic regression model was used with previously identified patient- and injury-related variables (age, sex, BMI, diabetes, smoking, periprosthetic fracture, open fracture, trauma energy, fracture zone length, fracture comminution, medial side comminution) in the first analysis and with treatment-related variables (different surgeon-controlled factors, e.g. plate length, screw placement, and proximal fixation) in the second analysis to predict the nonunion leading to secondary surgery in distal femur fractures. Results: We were able to include 299 fractures in 291 patients. Altogether, 31/299 fractures (10%) developed nonunion. In the first analysis, pseudo-R2 was 0.27 and area under the receiver operating characteristic curve (AUC) was 0.81. BMI was the most important variable in the prediction. In the second analysis, pseudo-R2 was 0.06 and AUC was 0.67. Plate length was the most important variable in the prediction. Conclusion: The model including patient- and injury-related factors had moderate fit and predictive ability in the prediction of distal femur fracture nonunion leading to secondary surgery. BMI was the most important variable in prediction of nonunion. Surgeon-controlled factors had a minor role in prediction of nonunion. Cite this article: Bone Jt Open 2023;4(8):584–593