Stereorandomized Oncocins with Preserved Ribosome Binding and Antibacterial Activity.

We recently showed that solid-phase peptide synthesis using racemic amino acids yields stereorandomized peptides comprising all possible diastereomers as homogeneous, single-mass products that can be purified by HPLC and that stereorandomization modulates activity, toxicity, and stability of membrane-disruptive cyclic and linear antimicrobial peptides (AMPs) and dendrimers. Here, we tested if stereorandomization might be compatible with target binding peptides with the example of the proline-rich AMP oncocin, which inhibits the bacterial ribosome. Stereorandomization of up to nine C-terminal residues preserved ribosome binding and antibacterial effects including activities against drug-resistant bacteria and protected against serum degradation. Surprisingly, fully stereorandomized oncocin was as active as L-oncocin in dilute growth media stimulating peptide uptake, although it did not bind the ribosome, indicative of an alternative mechanism of action. These experiments show that stereorandomization can be compatible with target binding peptides and can help understand their mechanism of action.

Ribosomal RNA expansion segments and their role in ribosome biology.

Ribosomes are universally conserved cellular machines that catalyze protein biosynthesis. The active sites underly immense evolutionary conservation resulting in virtually identical core structures of ribosomes in all domains of life including organellar ribosomes. However, more peripheral structures of cytosolic ribosomes changed during evolution accommodating new functions and regulatory options. The expansion occurred at the riboprotein level, including more and larger ribosomal proteins and at the RNA level increasing the length of ribosomal RNA. Expansions within the ribosomal RNA occur as clusters at conserved sites that face toward the periphery of the cytosolic ribosome. Recent biochemical and structural work has shed light on how rRNA-specific expansion segments (ESs) recruit factors during translation and how they modulate translation dynamics in the cytosol. Here we focus on recent work on yeast, human and trypanosomal cytosolic ribosomes that explores the role of two specific rRNA ESs within the small and large subunit respectively. While no single regulatory strategy exists, the absence of ESs has consequences for proteomic stability and cellular fitness, rendering them fascinating evolutionary tools for tailored protein biosynthesis.

Evolving precision: rRNA expansion segment 7S modulates translation velocity and accuracy in eukaryal ribosomes.

Ribosome-enhanced translational miscoding of the genetic code causes protein dysfunction and loss of cellular fitness. During evolution, open reading frame length increased, necessitating mechanisms for enhanced translation fidelity. Indeed, eukaryal ribosomes are more accurate than bacterial counterparts, despite their virtually identical, conserved active centers. During the evolution of eukaryotic organisms ribosome expansions at the rRNA and protein level occurred, which potentially increases the options for translation regulation and cotranslational events. Here we tested the hypothesis that ribosomal RNA expansions can modulate the core function of the ribosome, faithful protein synthesis. We demonstrate that a short expansion segment present in all eukaryotes' small subunit, ES7S, is crucial for accurate protein synthesis as its presence adjusts codon-specific velocities and guarantees high levels of cognate tRNA selection. Deletion of ES7S in yeast enhances mistranslation and causes protein destabilization and aggregation, dramatically reducing cellular fitness. Removal of ES7S did not alter ribosome architecture but altered the structural dynamics of inter-subunit bridges thus affecting A-tRNA selection. Exchanging the yeast ES7S sequence with the human ES7S increases accuracy whereas shortening causes the opposite effect. Our study demonstrates that ES7S provided eukaryal ribosomes with higher accuracy without perturbing the structurally conserved decoding center.

tRNA Synthetases Are Recruited to Yeast Ribosomes by rRNA Expansion Segment 7L but Do Not Require Association for Functionality.

Protein biosynthesis is essential for any organism, yet how this process is regulated is not fully understood at the molecular level. During evolution, ribosomal RNA expanded in specific regions, referred to as rRNA expansion segments (ES). First functional roles of these expansions have only recently been discovered. Here we address the role of ES7La located in the large ribosomal subunit for factor recruitment to the yeast ribosome and the potential consequences for translation. Truncation of ES7La has only minor effects on ribosome biogenesis, translation efficiency and cell doubling. Using yeast rRNA deletion strains coupled with ribosome-specific mass spectrometry we analyzed the interactome of ribosomes lacking ES7La. Three aminoacyl-tRNA synthetases showed reduced ribosome association. Synthetase activities however remained unaltered suggesting that the pool of aminoacylated tRNAs is unaffected by the ES deletion. These results demonstrated that aminoacylation activities of tRNA synthetases per se do not rely on ribosome association. These findings suggest a role of ribosome-associated aminoacyl-tRNA synthetase beyond their core enzymatic functions.

Positive epistasis between disease-causing missense mutations and silent polymorphism with effect on mRNA translation velocity.

Epistasis refers to the dependence of a mutation on other mutation(s) and the genetic context in general. In the context of human disorders, epistasis complicates the spectrum of disease symptoms and has been proposed as a major contributor to variations in disease outcome. The nonadditive relationship between mutations and the lack of complete understanding of the underlying physiological effects limit our ability to predict phenotypic outcome. Here, we report positive epistasis between intragenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene responsible for cystic fibrosis (CF) pathology. We identified a synonymous single-nucleotide polymorphism (sSNP) that is invariant for the CFTR amino acid sequence but inverts translation speed at the affected codon. This sSNP in cis exhibits positive epistatic effects on some CF disease-causing missense mutations. Individually, both mutations alter CFTR structure and function, yet when combined, they lead to enhanced protein expression and activity. The most robust effect was observed when the sSNP was present in combination with missense mutations that, along with the primary amino acid change, also alter the speed of translation at the affected codon. Functional studies revealed that synergistic alteration in ribosomal velocity is the underlying mechanism; alteration of translation speed likely increases the time window for establishing crucial domain-domain interactions that are otherwise perturbed by each individual mutation.

Global assessment of the integrated stress response in CF patient-derived airway and intestinal tissues.

BACKGROUND: Chronic inflammation is a hallmark among patients with cystic fibrosis (CF). We explored whether mutation-induced (F508del) misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR), and/or secondary colonization with opportunistic pathogens, activate tissue remodeling and innate immune response drivers. METHODS: Using RNA-seq to interrogate global gene expression profiles, we analyzed stress response signaling cascades in primary human bronchial epithelia (HBE) and intestinal organoids. RESULTS: Primary HBE acquired from CF patients with advanced disease and prolonged exposure to pathogenic microorganisms display a clear molecular signature of activated tissue remodeling pathways, unfolded protein response (UPR), and chronic inflammation. Furthermore, CFTR misfolding induces inflammatory signaling cascades in F508del patient-derived organoids from both the distal small intestine and colon. CONCLUSION: Despite the small patient cohort size, this proof-of-principle study supports the use of RNA-seq as a means to both identify CF-specific signaling profiles in various tissues and evaluate disease heterogeneity. Our global transcriptomic data is a useful resource for the CF research community for analyzing other gene expression sets influencing CF disease signature but also transcriptionally contributing to CF heterogeneity.

rRNA expansion segment 27Lb modulates the factor recruitment capacity of the yeast ribosome and shapes the proteome.

Fine-tuned regulation of protein biosynthesis is crucial for cellular fitness and became even more vital when cellular and organismal complexity increased during the course of evolution. In order to cope with this augmented demand for translation control, eukaryal ribosomes have gained extensions both at the ribosomal protein and rRNA levels. Here we analyze the functional role of ES27L, an rRNA expansion segment in the large ribosomal subunit of Saccharomyces cerevisiae. Deletion of the b-arm of this expansion segment, called ES27Lb, did not hamper growth during optimal conditions, thus demonstrating that this 25S rRNA segment is not inherently crucial for ribosome functioning. However, reductive stress results in retarded growth and rendered unique protein sets prone to aggregation. Lack of ES27Lb negatively affects ribosome-association of known co-translational N-terminal processing enzymes which in turn contributes to the observed protein aggregation. Likely as a compensatory response to these challenges, the truncated ribosomes showed re-adjusted translation of specific sets of mRNAs and thus fine-tune the translatome in order to re-establish proteostasis. Our study gives comprehensive insight into how a highly conserved eukaryal rRNA expansion segment defines ribosomal integrity, co-translational protein maturation events and consequently cellular fitness.

Slowing ribosome velocity restores folding and function of mutant CFTR.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response.

Timing during translation matters: synonymous mutations in human pathologies influence protein folding and function.

Ribosomes translate mRNAs with non-uniform speed. Translation velocity patterns are a conserved feature of mRNA and have evolved to fine-tune protein folding, expression and function. Synonymous single-nucleotide polymorphisms (sSNPs) that alter programmed translational speed affect expression and function of the encoded protein. Synergistic advances in next-generation sequencing have led to the identification of sSNPs associated with disease penetrance. Here, we draw on studies with disease-related proteins to enhance our understanding of mechanistic contributions of sSNPs to functional alterations of the encoded protein. We emphasize the importance of identification of sSNPs along with disease-causing mutations to understand genotype-phenotype relationships.

Alteration of protein function by a silent polymorphism linked to tRNA abundance.

Synonymous single nucleotide polymorphisms (sSNPs) are considered neutral for protein function, as by definition they exchange only codons, not amino acids. We identified an sSNP that modifies the local translation speed of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to detrimental changes to protein stability and function. This sSNP introduces a codon pairing to a low-abundance tRNA that is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting tissue-specific effects of this sSNP. Up-regulation of the tRNA cognate to the mutated codon counteracts the effects of the sSNP and rescues protein conformation and function. Our results highlight the wide-ranging impact of sSNPs, which invert the programmed local speed of mRNA translation and provide direct evidence for the central role of cellular tRNA levels in mediating the actions of sSNPs in a tissue-specific manner.

Tuning innate immunity by translation.

In multicellular organisms, the epithelia is a contact surface with the surrounding environment and is exposed to a variety of adverse biotic (pathogenic) and abiotic (chemical) factors. Multi-layered pathways that operate on different time scales have evolved to preserve cellular integrity and elicit stress-specific response. Several stress-response programs are activated until a complete elimination of the stress is achieved. The innate immune response, which is triggered by pathogenic invasion, is rather harmful when active over a prolonged time, thus the response follows characteristic oscillatory trajectories. Here, we review different translation programs that function to precisely fine-tune the time at which various components of the innate immune response dwell between active and inactive. We discuss how different pro-inflammatory pathways are co-ordinated to temporally offset single reactions and to achieve an optimal balance between fighting pathogens and being less harmful for healthy cells.

Efficient translation initiation dictates codon usage at gene start.

The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5-10 codons of protein-coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate.