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Review
. 2021 Sep 27;1(5):100081.
doi: 10.1016/j.crmeth.2021.100081. Epub 2021 Sep 16.

Global approaches for profiling transcription initiation

Affiliations
Review

Global approaches for profiling transcription initiation

Robert A Policastro et al. Cell Rep Methods. .

Abstract

Transcription start site (TSS) selection influences transcript stability and translation as well as protein sequence. Alternative TSS usage is pervasive in organismal development, is a major contributor to transcript isoform diversity in humans, and is frequently observed in human diseases including cancer. In this review, we discuss the breadth of techniques that have been used to globally profile TSSs and the resulting insights into gene regulation, as well as future prospects in this area of inquiry.

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Conflict of interest statement

DECLARATION OF INTERESTS R.A.P. and G.E.Z. are employees of eGenesis, Inc. The topics covered in this review are not related to any current work at the company.

Figures

Figure 1
Figure 1
Effects of TSS selection on gene expression (A) Possible effects of 5′ TL lengthening on transcript stability and translation. Transcription factor (TF) 1 specifies use of a proximal promoter, leading to a transcript with a short 5′ TL, while TF2 activates an upstream promoter that produces a transcript with a long 5′ TL. The extended 5′ TL may contain a uORF, which can act as a “sponge” for ribosomes by preventing them from reaching the transcript's primary ORF and may also lead to destruction of the transcript via NMD if the uORF stop codon is recognized as premature. The 5′ TL may also contain an IRES, enabling cap-independent translation. We note that these 5′ TL features are not mutually exclusive and direct interested readers to a recent comprehensive review on the roles of 5′ TLs in gene regulation (Leppek et al., 2018). (B) Production of transcripts encoding distinct protein isoforms by TF-mediated activation of alternative promoters.
Figure 2
Figure 2
General approaches for TSS mapping In oligo-capping, total RNA is first treated enzymatically to dephosphorylate uncapped RNAs. Caps are then removed, leaving 5′ monophosphates compatible with ligation. The cap oligo is ligated to the decapped RNAs and reverse transcription is performed, yielding 5′-complete cDNA ready for further processing. In cap-trapping, RNA:cDNA hybrids are chemically treated to oxidize RNA caps, which are then biotinylated. Streptavidin purification is then used to selectively enrich capped hybrids for further processing. In TSRT, total RNA is reverse transcribed, and the cap stimulates the addition of nontemplated nucleotides to the 3′ end of the first-strand cDNA. A TSO then interacts with the additional nucleotides and reverse transcriptase incorporates the complement of the TSO sequence into the first-strand cDNA, resulting in 5′-complete cDNA ready for further processing. See Table 1 for advantages and disadvantages of each approach and Table 2 for RNA input requirements.
Figure 3
Figure 3
Computational processing of TSS mapping data A general workflow for processing and analysis of TSS mapping data is shown, with software that can be used for each step indicated. Asterisks indicate optional steps. More information on each piece of software listed here can be found at the following URLs: FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc); UMI-tools (https://github.com/CGATOxford/UMI-tools); Cutadapt (https://cutadapt.readthedocs.io/en/stable); STAR (https://github.com/alexdobin/STAR); Samtools (http://www.htslib.org); CAGEr (https://www.bioconductor.org/packages/release/bioc/html/CAGEr.html); icetea (https://www.bioconductor.org/packages/release/bioc/html/icetea.html); TSRchitect (https://www.bioconductor.org/packages/release/bioc/html/TSRchitect.html); TSRexploreR (https://zentnerlab.github.io/TSRexploreR/index.html); CAGEexploreR (https://github.com/edimont/CAGExploreR); CAGEd-oPPOSSUM (http://cagedop.cmmt.ubc.ca/CAGEd_oPOSSUM/); CAGEfightR (https://www.bioconductor.org/packages/release/bioc/html/CAGEfightR.html).

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