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Total and newly transcribed (4sU-RNA) RNA of primary human fibroblasts infected with the vhs-null mutant of HSV-1

ABSTRACT: Primary human foreskin fibroblasts (HFF) were infected with vhs-null mutant of HSV-1 strain 17 at a multiplicity of infection (MOI) of 10. Newly transcribed RNA was labelled by adding 500µM 4-thiouridine (4sU) to the cell culture media for 1h. Total cellular RNA was isolated using Trizol. Newly transcribed RNA was purified following the protocol described in Raedle et al. JoVE 2013.

ORGANISM(S): Homo sapiens

PROVIDER: GSE129715 | GEO | 2020/10/26

REPOSITORIES: GEO

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Wide-spread disruption of transcription termination in HSV-1 infection: Next generation sequencing of total and newly transcribed (4sU-RNA) RNA

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Wide-spread disruption of transcription termination in HSV-1 infection: Next generation sequencing of total and newly transcribed (4sU-RNA) RNA

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Wide-spread disruption of transcription termination in HSV-1 infection: Next generation sequencing of total and newly transcribed (4sU-RNA) RNA from different virus strains and mutant viruses

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Total and newly transcribed (4sU-RNA) RNA of primary human fibroblasts infected with the vhs-null mutant of HSV-1

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Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution

Project description:RNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNA synthesis and decay rates. The kinetics of RNA processing, however, so far remained unresolved. Here, we show that ultra-short 4sU-tagging not only provides snap-shot pictures of eukaryotic gene expression but, when combined with progressive 4sU-tagging and RNA-seq, reveals global RNA processing kinetics at nucleotide resolution. Using this method, we identified classes of rapidly and slowly spliced/degraded introns. Interestingly, each class of splicing kinetics was characterized by a distinct association with intron length, gene length and splice site strength. For a large group of introns, we also observed long lasting retention in the primary transcript, but efficient secondary splicing or degradation at later time points. Finally, we show that processing of most, but not all small nucleolar (sno)RNA-containing introns is remarkably inefficient with the majority of introns being spliced and degraded rather than processed into mature snoRNAs. In summary, our study yields unparalleled insights into the kinetics of RNA processing and provides the tools to study molecular mechanisms of RNA processing and their contribution to the regulation of gene expression. 4sU-tagging was performed in human DG75 B-cells by adding 500 uM 4sU to cell culture medium for 5, 10, 15, 20 or 60 min. Following isolation of total cellular RNA, this was separated into nascent and untagged, pre-existing RNA. Nascent RNA as well as total and untagged RNA from 60 min 4sU-tagging were subjected to SOLiD sequencing (SOLiD II) obtaining 35 nt reads.

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