Exploring Ribosome-Positioning on Translating Transcripts with Ribosome Profiling

Cope, Alexander L; Vellappan, Sangeevan; Favate, John; Skalenko, Kyle S.; Yadavalli, Srujana S.; Shah, Premal

doi:10.1007/978-1-0716-1851-6_5

Cited by 9 publications

(11 citation statements)

References 27 publications

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“…The samples were sequentially depleted of human then Toxoplasma rRNA sequences by species specific riboPOOLs (siTOOLs Biotech) as per manufacturer’s specifications. Subsequent Illumina library preparation was performed as previously described (53, 54). Sequencing libraries were read at 2 x 150 bp and can be accessed at GSE243206.…”

Section: Methodsmentioning

confidence: 99%

mRNA cap-binding protein eIF4E1 is a novel regulator ofToxoplasma gondiilatency

Holmes,

Bastos,

Dey

et al. 2023

Preprint

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The protozoan parasiteToxoplasma gondiicauses serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and dormant cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5’-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogenous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence.SignificanceToxoplasma gondiiis an opportunistic pathogen of importance to global human and animal health. There are currently no chemotherapies targeting the encysted form of the parasite. Consequently, a better understanding of the mechanisms controlling encystation are required. Here we show that the mRNA cap-binding protein, eIF4E1, is involved in regulating the encystation process. Encysted parasites reduce eIF4E1 levels and depletion of eIF4E1 decreases the translation of ribosome-associated machinery and drivesToxoplasmaencystation. Together, these data reveal a new layer of mRNA translational control that regulates parasite encystation and latency.

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Section: Methodsmentioning

confidence: 99%

mRNA cap-binding protein eIF4E1 is a novel regulator ofToxoplasma gondiilatency

Holmes,

Bastos,

Dey

et al. 2023

Preprint

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“…Comparing the approximately 25,000 transcripts detected in both data sets, METTL3 deletion impacted more transcripts at the level of RNA translation than abundance; we found 6,101 transcripts exclusively altered at the level of ribosome protected fragments, 3,004 with changes only in RNA abundance, and 1,374 with changes in both (Figure 6A and Data S1). We next compared the ratio of ribosome protected fragments to mRNA abundance for each transcript to produce the translational efficiency ("TE") of each transcript in METTL3 deleted and control enteroids (16,41). Averaging all 2,124 transcripts with significant changes in TE, the predominant effect of METTL3 deletion was a reduction in TE.…”

Section: Mettl3 Deletion Leads To Downregulation Of Global Mrna Trans...mentioning

confidence: 99%

“…Frozen enteroid pellets were resuspended in ice-cold lysis buffer containing 20mM Tris-HCl pH 7.4, 150mM MgCl2, 150mM NaCl, 100ug/ml cycloheximide, 1% v/v Triton X-100, 1mM DTT, 1U/ul SUPERase•IN RNase inhibitor (ThermoFisher), 25U/ml Turbo DNase1 (ThermoFisher), and 1X EDTA-free protease inhibitor cocktail (Roche). The cells were then lysed by trituration through a 26-gauge needle 10 times for efficient cell lysis.The samples were then processed, and the libraries were prepared as previously described(41) with the following modifications. First, we performed sucrose cushion to pellet the ribosome-associated mRNAs and proceeded with RNAse I digestion (10 U/μl by the Epicentre definition) for 30ug of RNA.…”

mentioning

confidence: 99%

Intestinal transit amplifying cells require METTL3 for growth factor signaling, KRAS expression, and cell survival

Danan

Naughton

Hayer

et al. 2023

Preprint

Self Cite

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The METTL3-METTL14 methyltransferase complex adds N6-methyladenosine (m6A) to mRNA with profound impacts on cell fate. Studies delete METTL3 or METTL14 interchangeably to define the role of m6A in target tissues despite a lack of data confirming that these deletions are equivalent. Intestinal epithelial METTL14 deletion triggers stem cell death in the colon with no overt phenotype in the small intestine. The effect of METTL3 deletion in the same tissues remains unknown. We report that intestinal epithelial METTL3 deletion caused unexpected severe defects in the small intestine, including crypt and villus atrophy associated with cellular senescence and death in the crypt transit amplifying zone. Ribosome profiling and m6A-sequencing demonstrated downregulated translation of hundreds of unique methylated transcripts, including genes essential to growth factor signal transduction, such as Kras. Our study suggests that METTL3 is essential for small intestinal homeostasis via enhanced translation of growth factor signaling in crypt transit amplifying cells.

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“…Translation of the genetic information encoded in messenger RNA (mRNA) by ribosomes is an extremely rapid biological process currently estimated to be about ~16.8 nucleotides (nt) being read per second, with an incorporation and elongation rate of the growing polypeptide chain of about ~5.6 amino acids per second [ 1 , 2 , 3 ]. Polyribosome-based reading of single mRNAs involving both circular and linear mRNA–ribosome configurations can considerably amplify the rate of this process [ 4 , 5 ].…”

Section: Introductionmentioning

confidence: 99%

“…Polyribosome-based reading of single mRNAs involving both circular and linear mRNA–ribosome configurations can considerably amplify the rate of this process [ 4 , 5 ]. The rapid primary, secondary, tertiary and other three-dimensional folding of amino acids into nascent polypeptide chains occurs simultaneously with amino acid polymerization within the ribosomal cleft immediately after their biosynthesis [ 2 , 3 , 5 , 6 ]. The overall process of rapid translation involving the formation of biologically active protein structures has emerged and evolved to maintain cellular protein homeostasis, sometimes referred to as proteostasis, by diminishing the possibility of both polypeptide misfolding and the self-aggregation of newly formed polypeptides, thus ensuring the maintenance and functionality of a healthy and optimally performing cellular proteome [ 5 , 6 , 7 , 8 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

Recent Advances in Our Molecular and Mechanistic Understanding of Misfolded Cellular Proteins in Alzheimer’s Disease (AD) and Prion Disease (PrD)

Lukiw

2022

Biomolecules

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Naturally occurring neuron-abundant proteins including amyloid Aβ42 peptide and the microtubule-associated protein tau (MAPT) can, over time and under pathological situations, assume atypical conformations, altering their normal biological structure and function, and causing them to aggregate into insoluble and neurotoxic intracellular inclusions. These misfolded proteins ultimately contribute to the pathogenesis of several progressive, age-related and ultimately lethal human neurodegenerative disorders. The molecular mechanism of this pathological phenomenon of neuronal protein misfolding lends support to the ‘prion hypothesis’, which predicts that the aberrant folding of endogenous natural protein structures into unusual pathogenic isoforms can induce the atypical folding of other similar brain-abundant proteins, underscoring the age-related, progressive nature and potential transmissible and spreading capabilities of the aberrant protein isoforms that drive these invariably fatal neurological syndromes. The abnormal folding and aggregation of host proteins is a consistent feature of both amyloidopathies and tauopathies that encompass a continuous spectrum of brain diseases that include Alzheimer’s disease (AD), prion disorders (PrD) such as scrapie in sheep and goats (Bovidae), experimental prion infection of rodents (Muridae), Creutzfeldt–Jakob disease (CJD) and Gerstmann–Sträussler–Scheinker syndrome (GSS) in humans (Hominidae), and other fatal prion-driven neurological disorders. Because AD patients accumulate both misfolded tau and Aβ peptides, AD may be somewhat unique as the first example of a ‘double prion disorder’. This commentary will examine current research trends in this fascinating research area, with a special emphasis on AD and PrD, and the novel pathological misfolded protein processes common to both intractable neurological disorders.

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Exploring Ribosome-Positioning on Translating Transcripts with Ribosome Profiling

Cited by 9 publications

References 27 publications

mRNA cap-binding protein eIF4E1 is a novel regulator ofToxoplasma gondiilatency

mRNA cap-binding protein eIF4E1 is a novel regulator ofToxoplasma gondiilatency

Intestinal transit amplifying cells require METTL3 for growth factor signaling, KRAS expression, and cell survival

Recent Advances in Our Molecular and Mechanistic Understanding of Misfolded Cellular Proteins in Alzheimer’s Disease (AD) and Prion Disease (PrD)

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