Anota2seq Analysis for Transcriptome-Wide Studies of mRNA Translation

Oertlin, Christian; Watt, Kathleen; Ristau, Johannes; Larsson, Ola

doi:10.1007/978-1-0716-1920-9_15

Cited by 9 publications

(22 citation statements)

References 48 publications

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“…S2 ) (Ingolia et al 2009; Jensen et al 2014; Vasquez et al 2014; Smircich et al 2015). Analysis using the anota2seq package (Oertlin et al 2022) revealed that 102 transcripts were significantly altered in their translational efficiencies, resulting in either an increase (41 transcripts, TE UP) or a decrease (61 transcripts, TE DOWN) in translation efficiency ( Figure 3 , light and dark red, respectively; Tables S2 and S3 ). Note that because the transcript for DRBD18 was targeted via RNA interference using the 3’ UTR, many reads were counted for the gene in the induced samples, artificially marking DRBD18 as being reduced in translation.…”

Section: Resultsmentioning

confidence: 99%

“…Between 150 and 950 thousand reads per sample were mapped, low counts were due to heavy contamination of rRNA in the sequenced samples. To evaluate differential translation efficiency (TE), the anota2seq package (doi.org/10.1093/nar/gkz223, (Oertlin et al 2022) was applied with default parameters (log2FC > |1| and FDR less than 0.15). As before, experimental batch effects were introduced as a variable in the linear model.…”

Section: Library Preparation Sequencing and Analysismentioning

confidence: 99%

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Translational control byTrypanosoma bruceiDRBD18 contributes to the maintenance of the procyclic state

Ciganda

Sotelo‐Silveira

Smith

et al. 2023

Preprint

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Trypanosoma brucei occupies distinct niches throughout its life cycle, within both the mammalian and tsetse fly hosts. The immunological and biochemical complexity and variability of each of these environments require a reshaping of the protein landscape of the parasite both to evade surveillance and face changing metabolic demands. Whereas most well-studied organisms rely on transcriptional control as the main regulator of gene expression, post-transcriptional control mechanisms are particularly important in T. brucei, and these are often mediated by RNA-binding proteins. DRBD18 is a T. brucei RNA-binding protein that interacts with ribosomal proteins and translation factors. Here, we tested a role for DRBD18 in translational control. We show that DRBD18 depletion by RNA interference leads to altered polysomal profiles with a specific depletion of heavy polysomes. Ribosome profiling analysis reveals that 101 transcripts change in translational efficiency (TE) upon DRBD18 depletion: 41 exhibit decreased TE and 60 exhibit increased TE. A further 66 transcripts are buffered, i.e. changes in transcript abundance are compensated by changes in TE such that the total translational output is expected not to change. Proteomic analysis validates these data. In DRBD18-depleted cells, a cohort of transcripts that codes for procyclic form-specific proteins is translationally repressed while, conversely, transcripts that code for bloodstream form- and metacyclic form-specific proteins are translationally enhanced. These data suggest that DRBD18 contributes to the maintenance of the procyclic state through both positive and negative translational control of specific mRNAs.

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Section: Resultsmentioning

confidence: 99%

Section: Library Preparation Sequencing and Analysismentioning

confidence: 99%

Translational control byTrypanosoma bruceiDRBD18 contributes to the maintenance of the procyclic state

Ciganda

Sotelo‐Silveira

Smith

et al. 2023

Preprint

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“…1a). The resulting RNA pools from TSC1 -/and TSC1 +/+ NPCs were quantified using RNA sequencing followed by analysis using anota2seq 43,44 to identify three modes of TSC1associated gene expression alterations: (i) changes in polysome-associated mRNA not paralleled by corresponding alterations in total mRNA levels (denoted "translation" and, under conditions when translation elongation is unaffected, interpreted as changes in translational efficiency leading to modulation of protein levels); (ii) congruent changes of polysome-associated and total mRNA (denoted "abundance" representing alterations in mRNA levels impacting protein levels downstream of e.g. modulation of transcription and/or mRNA-stability); and (iii) alterations in total mRNA not paralleled by corresponding changes in polysome-associated mRNA (denoted "offsetting" and interpreted as instances where mRNA translation opposes alterations in protein levels imposed by modulation of mRNA levels [discussed in detail elsewhere] 45,46 ).…”

Section: Tsc1 Loss Leads To Widespread Alterations Of Mrna Translatio...mentioning

confidence: 99%

“…Similarly to above (Fig. 1b-c), we analyzed the resulting dataset using anota2seq 7 43,44 (Fig. 2c-d).…”

Section: Alterations Of Mrna Translation In Asd Patient Compared With...mentioning

confidence: 99%

“…Genes with 0 mapped RNA sequencing read in one or more samples were discarded resulting in analysis of 12950 and 11998 genes in post-mortem brain samples and NPCs, respectively. The data was TMM-log2 normalized and analyzed using anota2seq 44 (v. 1.14.0; parameters: minSlopeTranslation = -1, minSlopeBuffering = -2, maxSlopeTranslation = 2, maxSlopeBuffering = 1, deltaPT = deltaTP = deltaP = deltaT = log2(1.2)) 43,44 . During analysis of the NPC dataset, replicate was included in the model to correct for batch effects and three contrasts were assessed, namely (i) TSC1 -/vs TSC1 +/+ , (ii) TSC1 -/-+ rapamycin vs TSC1 -/and (iii) TSC1 +/+ + rapamycin vs TSC1 +/+ , with the following thresholds to identify differentially expressed genes: minEff = log2(1.5) and maxRvmPadj = 0.15 [i.e.…”

Section: Analysis Using Anota2seqmentioning

confidence: 99%

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Translatome analysis of Tuberous Sclerosis Complex-1 patient-derived neural progenitor cells reveal rapamycin-dependent and independent alterations

Aksoylu

Martín

Robert

et al. 2023

Preprint

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Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD). The hamartin-tuberin (TSC1-TSC2) protein complex inactivates mechanistic target of rapamycin complex 1 (mTORC1) signaling, leading to increased protein synthesis via inactivation of translational repressor eIF4E-binding proteins (4E-BPs). In TSC1-null neural progenitor cells (NPCs), we previously reported early ND phenotypic changes, including increased proliferation/altered neurite outgrowth, which were unaffected by mTORC1-inhibitor rapamycin. Here, using polysome-profiling to quantify translational efficiencies at a transcriptome-wide level, we observed numerous TSC1-dependent alterations in NPCs, largely recapitulated in post-mortem brains from ASD donors. Although rapamycin partially reversed TSC1-associated alterations, most neural activity/synaptic- or ASD-related genes remained insensitive but were inhibited by third-generation bi-steric, mTORC1-selective inhibitor RMC-6272, which also reversed altered ND phenotypes. Together these data reveal potential implications for treatment of TAND.

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