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An internally normalized approach to comparing RNA levels between samples using nucleoside recoding chemistry

ABSTRACT: An internally normalized approach to comparing RNA levels between samples using nucleoside recoding chemistry

PROVIDER: PRJNA713771 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13934567_1.fastq.gz	Fastqsanger.gz
	SRR13934567_2.fastq.gz	Fastqsanger.gz
	SRR13934568_1.fastq.gz	Fastqsanger.gz
	SRR13934568_2.fastq.gz	Fastqsanger.gz
	SRR13934569_1.fastq.gz	Fastqsanger.gz

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An internally normalized approach to comparing RNA levels between samples using nucleoside recoding chemistry

Project description:RNA sequencing allows for transcriptome-wide comparative studies of RNA levels, including gene expression, localization in subcellular compartments, or content of ribonucleoprotein complexes. It is often challenging, however, to separate real biological variation from technical artifacts arising from variable sample preparation, uneven contamination and difficulties normalizing sequencing counts between samples. These challenges are magnified in complex biochemical preparations, such as isolating polysomes to study translation. To address these challenges, we developed TILAC, an approach to compare RNA content between samples with internal controls and normalization. TILAC uses two different metabolic labels (4-thiouridine, s4U, and 6-thioguanisine, s6G) to differentially label RNA from each condition, allowing the samples to be pooled prior to downstream processing. TILAC uses nucleotide-recoding chemistry and sequencing to determine which RNAs are enriched in each sample. TILAC accurately identifies known changes in the transcriptome during RNA polymerase II inhibition and heat shock response. Using TILAC, we discovered a set of transcripts that are enriched in actively-translating ribosome complexes during stress, including MCM2 and DDX5, and verified their translational upregulation. These results demonstrate the power of TILAC to uncover differences between samples revealing new biology.

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TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding

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Expanding the Nucleoside Recoding Toolkit: Revealing RNA Population Dynamics with 6-thioguanisine

Project description:Expanding the Nucleoside Recoding Toolkit: Revealing RNA Population Dynamics with 6-thioguanisine

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Expanding the Nucleoside Recoding Toolkit: Revealing RNA Population Dynamics with 6-thioguanisine

Project description:RNA-sequencing (RNA-seq) measures RNA abundance in a biological sample but does not provide temporal information about the sequenced RNAs. Metabolic labeling can be used to distinguish newly made RNAs from pre-existing RNAs. Mutations induced from chemical recoding of the hydrogen bonding pattern of the metabolic label can reveal which RNAs are new in the context of a sequencing experiment. These nucleotide recoding strategies have been developed for a single uridine analogue, 4-thiouridine (s4U), limiting the scope of these experiments. Here we report expansion of TimeLapse sequencing (TimeLapse-seq) to the guanosine analogue, 6-thioguanosine (s6G), which can be recoded under RNA-friendly nucleophilic-aromatic substitution conditions to produce adenine analogues (substituted 2-aminoadenosines). We demonstrate the first use of s6G recoding experiments to reveal transcriptome-wide RNA population dynamics.

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Internally controlled RNA sequencing comparisons using nucleoside recoding chemistry.

Project description:Quantitative comparisons of RNA levels from different samples can lead to new biological understanding if they are able to distinguish biological variation from variable sample preparation. These challenges are pronounced in comparisons that require complex biochemical manipulations (e.g. isolating polysomes to study translation). Here, we present Transcript Regulation Identified by Labeling with Nucleoside Analogues in Cell Culture (TILAC), an internally controlled approach for quantitative comparisons of RNA content. TILAC uses two metabolic labels, 4-thiouridine (s4U) and 6-thioguanosine (s6G), to differentially label RNAs in cells, allowing experimental and control samples to be pooled prior to downstream biochemical manipulations. TILAC leverages nucleoside recoding chemistry to generate characteristic sequencing signatures for each label and uses statistical modeling to compare the abundance of RNA transcripts between samples. We verified the performance of TILAC in transcriptome-scale experiments involving RNA polymerase II inhibition and heat shock. We then applied TILAC to quantify changes in mRNA association with actively translating ribosomes during sodium arsenite stress and discovered a set of transcripts that are translationally upregulated, including MCM2 and DDX5. TILAC is broadly applicable to uncover differences between samples leading to improved biological insights.

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ENVIRONMENT MODULATES PROTEIN HETEROGENEITY THROUGH TRANSCRIPTIONAL AND TRANSLATIONAL STOP CODON RECODING

Project description:In order to systematically assess the frequency and origin of stop codon recoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon recoding may occur with a rate as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon recoding events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon recoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon recoding by mass spectrometry revealed that temperature regulates the expression of cryptic sequences generated by stop codon recoding in E. coli. Overall, our findings suggest that the environment influences the accuracy of protein production which increases protein heterogeneity when the organisms need to adapt to new conditions.

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Project description:Genetic recoding, a process that modifies genome templated protein sequence, plays an important role especially in the small genomes of viruses and bacteria. Chloroplasts have evolved from cyanobacterial ancestry and mainly retain the prokaryotic gene expression machinery. I-iota, a plastome mutant of Oenothera (the evening primrose), carries a single adenine insertion in a polyA stretch near to the 5’-end of the atpB gene, encoding the β-CF1 subunit of the ATP synthase This causes formation of a truncated protein. Surprisingly, a full-length AtpB protein is detectable in I-iota leaves, suggesting the presence of genetic recoding operating in I-iota. In order to analyze this phenomenon in more detail, several tobacco transplastomic lines were generated inducing and destroying genetic recoding of atpB. We identified that a two adenine (+2A) insertion was more efficiently compensated in plants than +1A, -1A or -2A mutations at the same location. Furthermore, a homopolymeric composition of the polyA stretch is essential for successful recoding: plants carrying a destroyed polyA stretch have an albino-phenotype, indicating the absence of genetic recoding. Here we show evidence for genetic recoding acting in the chloroplast that plays presumablya compensatory function and enables correction of indels in an essential gene for autotrophic growth.

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Environment Modulates Protein Heterogeneity Through Transcriptional and Translational Stop Codon Recoding

Project description:Stop codon recoding events give rise to longer proteins, which may alter the proteins function and thereby generate short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of recoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors in E.coli using fluorescent microscopy. We found that under stress conditions, stop codon recoding may occur as high as 80 percent of the time, depending on the genetic context, suggesting that evolution frequently samples stop codon recoding events. Targeted mass spectrometry and RNA-seq analyses showed that not only translational but also transcriptional errors contribute to stop codon recoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide mass -spectrometry revealed that temperature regulates the expression of cryptic peptides generated by stop codon recoding in E.coli. Overall, our findings suggest that the environment influences the accuracy of protein production which increases protein heterogeneity when the organisms need to adapt to new conditions.

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