Slowing ribosome velocity restores folding and function of mutant CFTR

Oliver, Kathryn E.; Rauscher, Robert; Mijnders, Marjolein; Wang, Wei; Wolpert, Matthew J; Maya, Jessica; Kesterson, Robert A.; Kirk, Kevin L.; Rab, András; Braakman, Ineke; Hong, Jeong S.; Hartman, John L.; Ignatova, Zoya; Sorscher, Eric J.

doi:10.1172/jci124282

Cited by 39 publications

(60 citation statements)

References 61 publications

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“…Some of these features include rare codons, charged amino acids and hydrophobic residues which can induce ribosome pausing. Indeed, subdomains of proteins have been found to require optimal synthesis rates that promote efficient folding, as is the case for CFTR ( 56 , 59 , 68 , 69 ). Elongation rates that are either faster or slower than normal can prevent certain proteins from folding normally and may lead to aggregation ( 70 , 71 ).…”

Section: Discussionmentioning

confidence: 99%

Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins

Campos

Wijeratne

Shah

et al. 2020

Nucleic Acids Research

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The ribosomal stalk proteins, RPLP1 and RPLP2 (RPLP1/2), which form the ancient ribosomal stalk, were discovered decades ago but their functions remain mysterious. We had previously shown that RPLP1/2 are exquisitely required for replication of dengue virus (DENV) and other mosquito-borne flaviviruses. Here, we show that RPLP1/2 function to relieve ribosome pausing within the DENV envelope coding sequence, leading to enhanced protein stability. We evaluated viral and cellular translation in RPLP1/2-depleted cells using ribosome profiling and found that ribosomes pause in the sequence coding for the N-terminus of the envelope protein, immediately downstream of sequences encoding two adjacent transmembrane domains (TMDs). We also find that RPLP1/2 depletion impacts a ribosome density for a small subset of cellular mRNAs. Importantly, the polarity of ribosomes on mRNAs encoding multiple TMDs was disproportionately affected by RPLP1/2 knockdown, implying a role for RPLP1/2 in multi-pass transmembrane protein biogenesis. These analyses of viral and host RNAs converge to implicate RPLP1/2 as functionally important for ribosomes to elongate through ORFs encoding multiple TMDs. We suggest that the effect of RPLP1/2 at TMD associated pauses is mediated by improving the efficiency of co-translational folding and subsequent protein stability.

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Section: Discussionmentioning

confidence: 99%

Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins

Campos

Wijeratne

Shah

et al. 2020

Nucleic Acids Research

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“…Suppression of RPL12, a component of 60S subunit P stalk, was also demonstrated to rescue folding and function of F508del and other CFTR mutants by modulating ribosome velocity. CFTR rescue was increased when lumacaftor was concurrently administered (Veit et al, 2016b;Oliver et al, 2019).…”

Section: Correctors: Rescuing the Protein Folding Processing And Trmentioning

confidence: 99%

CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine

2020

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Cystic fibrosis (CF) is a lethal inherited disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which result in impairment of CFTR mRNA and protein expression, function, stability or a combination of these. Although CF leads to multifaceted clinical manifestations, the respiratory disorder represents the major cause of morbidity and mortality of these patients. The life expectancy of CF patients has substantially lengthened due to early diagnosis and improvements in symptomatic therapeutic regimens. Quality of life remains nevertheless limited, as these individuals are subjected to considerable clinical, psychosocial and economic burdens. Since the discovery of the CFTR gene in 1989, tremendous efforts have been made to develop therapies acting more upstream on the pathogenesis cascade, thereby overcoming the underlying dysfunctions caused by CFTR mutations. In this line, the advances in cell-based high-throughput screenings have been facilitating the fast-tracking of CFTR modulators. These modulator drugs have the ability to enhance or even restore the functional expression of specific CF-causing mutations, and they have been classified into five main groups depending on their effects on CFTR mutations: potentiators, correctors, stabilizers, read-through agents, and amplifiers. To date, four CFTR modulators have reached the market, and these pharmaceutical therapies are transforming patients' lives with short-and long-term improvements in clinical outcomes. Such breakthroughs have paved the way for the development of novel CFTR modulators, which are currently under experimental and clinical investigations. Furthermore, recent insights into the CFTR structure will be useful for the rational design of next-generation modulator drugs. This review aims to provide a summary of recent developments in CFTR-directed therapeutics. Barriers and future directions are also discussed in order to optimize treatment adherence, identify feasible and sustainable solutions for equitable access to these therapies, and continue to expand the pipeline of novel modulators that may result in effective precision medicine for all individuals with CF.

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“…Blackfan–Diamond anemia ( 1 , 2 ). In other instances, potential treatments for disorders such as cystic fibrosis and fragile X syndrome, were designed to intentionally decrease accuracy or the rate of protein synthesis ( 3 ). Slowing ribosome translocation along the mRNA could partially restore folding of the mutant cystic fibrosis transmembrane conductance regulator protein in vitro as well as in a mouse model ( 3 ).…”

Section: Introductionmentioning

confidence: 99%

“…In other instances, potential treatments for disorders such as cystic fibrosis and fragile X syndrome, were designed to intentionally decrease accuracy or the rate of protein synthesis ( 3 ). Slowing ribosome translocation along the mRNA could partially restore folding of the mutant cystic fibrosis transmembrane conductance regulator protein in vitro as well as in a mouse model ( 3 ). Therefore, it is important to be able to monitor and manipulate translation in real-time, preferably with the single codon resolution in specific transcripts or even all transcripts at once.…”

Section: Introductionmentioning

confidence: 99%

Translation elongation rate varies among organs and decreases with age

Gerashchenko

Péterfi

Yim

et al. 2020

Nucleic Acids Research

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There has been a surge of interest towards targeting protein synthesis to treat diseases and extend lifespan. Despite the progress, few options are available to assess translation in live animals, as their complexity limits the repertoire of experimental tools to monitor and manipulate processes within organs and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type-specific translation elongation rates in vivo. It is based on time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ more than 50% among mouse organs and determined them to be 6.8, 5.0 and 4.3 amino acids per second for liver, kidney, and skeletal muscle, respectively. We further found that the elongation rate is reduced by 20% between young adulthood and mid-life. Thus, translation, a major metabolic process in cells, is tightly regulated at the level of elongation of nascent polypeptide chains.

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Slowing ribosome velocity restores folding and function of mutant CFTR

Abstract: Conflict of interest: JLH is founder of and holds equity in Spectrum PhenomX LLC, which aims to commercialize quantitative high-throughput cell array phenotyping (Q-HTCP) technology.

Cited by 39 publications

References 61 publications

Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins

Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins

CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine

Translation elongation rate varies among organs and decreases with age

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