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Transfer RNA Synthesis02:36

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One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Ribosome synthesis is a highly complex and coordinated process involving more than 200 assembly factors. The synthesis and processing of ribosomal components occurs not only in the nucleolus but also in the nucleoplasm and the cytoplasm of eukaryotic cells.
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NASC-seq monitors RNA synthesis in single cells.

Gert-Jan Hendriks1, Lisa A Jung2, Anton J M Larsson1

  • 1Department of Cell and Molecular Biology, Karolinska Instiutet, Biomedicum, Solnavägen 9, 171 65, Solna, Sweden.

Nature Communications
|July 19, 2019
PubMed
Summary

We developed NASC-seq, a new method to track newly synthesized and existing RNA in single cells. This powerful technique precisely monitors gene expression dynamics during cellular processes like immune response and differentiation.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Cell Biology

Background:

  • Current RNA sequencing methods often analyze cell populations, limiting insights into individual cell transcriptional dynamics.
  • High temporal resolution is crucial for understanding dynamic gene expression changes in response to stimuli.

Purpose of the Study:

  • To develop a single-cell RNA sequencing method capable of simultaneously monitoring newly synthesized and pre-existing RNA.
  • To enable precise temporal resolution of transcriptional dynamics at the single-cell level.

Main Methods:

  • Developed transcriptome alkylation-dependent single-cell RNA sequencing (NASC-seq).
  • Validated NASC-seq using pre-labelled RNA and by assessing mRNA turnover rates.
  • Applied NASC-seq to monitor Jurkat T-cell activation.

Main Results:

  • NASC-seq successfully distinguishes newly synthesized from pre-existing RNA within single cells.
  • Validated method showed higher detection of newly synthesized RNA for high mRNA turnover genes.
  • Observed distinct newly synthesized and pre-existing transcriptomes post-T-cell activation, reflecting two time points.
  • Identified rapidly up- and down-regulated genes during T-cell activation, with induced genes predominantly newly transcribed.

Conclusions:

  • NASC-seq provides simultaneous measurement of gene expression at two distinct time points within single cells.
  • This method offers precise temporal monitoring of RNA synthesis at single-cell resolution.
  • NASC-seq is applicable to studying homeostasis, perturbation responses, and cellular differentiation.