Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

Těšina, Petr; Lessen, Laura N.; Buschauer, Robert; Cheng, Jingdong; Wu, Colin Chih-Chien; Berninghausen, Otto; Buskirk, Allen R.; Becker, Thomas; Beckmann, Roland; Green, Rachel

doi:10.1101/755652

Cited by 19 publications

(33 citation statements)

References 75 publications

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“…The hairpin prevents binding of the release factor 1 (RF1), thus inhibiting translation termination and inducing translational bypassing (Agirrezabala et al, 2017). Another short two-basepair-long hairpin was observed in the A site of the ribosome in which the P and A sites were occupied by an inhibitory codon pair CGA-CCG that is known to cause ribosome stalling (Gamble et al, 2016;Tesina et al, 2020). These observations suggest that occlusion of the A site by RNA stem-loops may be a common strategy shared by many regulatory stem-loops.…”

Section: Discussionmentioning

confidence: 99%

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Bao

Loerch

Ling

et al. 2020

Preprint

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Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.

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Section: Discussionmentioning

confidence: 99%

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Bao

Loerch

Ling

et al. 2020

Preprint

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“…Such mRNAs can be generated by premature cleavage and polyadenylation, a process that is enhanced during neuronal stimulation and in cancer cells (8,9). In the absence of a stop codon, the ribosome translates the poly(A) tail, where interactions with both the nascent poly-lysine peptide and poly(A) sequence cause the ribosome to stall (10)(11)(12).…”

Section: Main Textmentioning

confidence: 99%

Dynamic regulation of translation quality control associated with ribosome stalling

Goldman

Livingston

Movsik

et al. 2020

Preprint

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Translation of problematic mRNA sequences induces ribosome stalling. Collided ribosomes at the stall site are recognized by cellular quality control machinery, resulting in dissociation of the ribosome from the mRNA and subsequent degradation of the nascent polypeptide and in some organisms, decay of the mRNA. However, the timing and regulation of these processes are unclear. We developed a SunTag-based reporter to monitor translation in real time on single mRNAs harboring difficult-to-translate poly(A) stretches. This reporter recapitulates previous findings in human cells that an internal poly(A) stretch reduces protein output ~10-fold, while mRNA levels are relatively unaffected. Long-term imaging of translation indicates that poly(A)-containing mRNAs are robustly translated in the absence of detectable mRNA cleavage. However, quantification of ribosome density reveals a ~3-fold increase in the number of ribosomes on poly(A)-containing mRNAs compared to a control mRNA, consistent with queues of many stalled ribosomes. Using single-molecule harringtonine runoff experiments, we observe the resolution of these queues in real-time by the cellular quality control machinery, and find that rescue is very slow compared to both elongation and termination. We propose that the very slow clearance of stalled ribosomes provides the basis for the cell to distinguish between

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“…S. cerevisiae ribosomes lacking the RACK1 homolog Asc1 are able to translate through the CGA-CGA stalling sequences and increase normally attenuated protein output (Wolf and Grayhack, 2015). The increase in amount of synthesized protein from CGA-CGA sequences is a consequence of overall reduced elongation rates of yeast ribosomes that lack Asc1 (Tesina et al, 2019). Slower elongation rates may also influence cellular responses to ribosome pausing.…”

Section: Plasmodium Ribosomes Polya and Poly-lysine Sequencesmentioning

confidence: 99%

“…Slower elongation rates may also influence cellular responses to ribosome pausing. The position of RACK1/Asc1 near the mRNA exit channel on the ribosome could be important in sensing ribosome collisions that lead to activation of ribosome rescue and mRNA surveillance pathways (Kim et al, 2014; Simms and Zaher, 2016; Tesina et al, 2019). The fact that Plasmodium ribosomes lack interaction with PfRACK1 could be beneficial for translation of polyA tracks into poly-lysine runs.…”

Section: Plasmodium Ribosomes Polya and Poly-lysine Sequencesmentioning

confidence: 99%

Adaptation of Translational Machinery in Malaria Parasites to Accommodate Translation of Poly-Adenosine Stretches Throughout Its Life Cycle

2019

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Malaria is caused by unicellular apicomplexan parasites of the genus Plasmodium, which includes the major human parasite Plasmodium falciparum. The complex cycle of the malaria parasite in both mosquito and human hosts has been studied extensively. There is tight control of gene expression in each developmental stage, and at every level of gene synthesis: from RNA transcription, to its subsequent translation, and finally post-translational modifications of the resulting protein. Whole-genome sequencing of P. falciparum has laid the foundation for significant biological advances by revealing surprising genomic information. The P. falciparum genome is extremely AT-rich (∼80%), with a substantial portion of genes encoding intragenic polyadenosine (polyA) tracks being expressed throughout the entire parasite life cycle. In most eukaryotes, intragenic polyA runs act as negative regulators of gene expression. Recent studies have shown that translation of mRNAs containing 12 or more consecutive adenosines results in ribosomal stalling and frameshifting; activating mRNA surveillance mechanisms. In contrast, P. falciparum translational machinery can efficiently and accurately translate polyA tracks without activating mRNA surveillance pathways. This unique feature of P. falciparum raises interesting questions: (1) How is P. falciparum able to efficiently and correctly translate polyA track transcripts, and (2) What are the specifics of the translational machinery and mRNA surveillance mechanisms that separate P. falciparum from other organisms? In this review, we analyze possible evolutionary shifts in P. falciparum protein synthesis machinery that allow efficient translation of an AU rich-transcriptome. We focus on physiological and structural differences of P. falciparum stage specific ribosomes, ribosome-associated proteins, and changes in mRNA surveillance mechanisms throughout the complete parasite life cycle, with an emphasis on the mosquito and liver stages.

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Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

Cited by 19 publications

References 75 publications

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Dynamic regulation of translation quality control associated with ribosome stalling

Adaptation of Translational Machinery in Malaria Parasites to Accommodate Translation of Poly-Adenosine Stretches Throughout Its Life Cycle

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