Estimating mRNA 5' degradation from RNA-seq illumina strand-specific reads
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11 weeks ago
Adrian Pelin ★ 2.6k

I am interested in seeing whether one of my conditions has more 5' mRNA decay compared to another condition.

This may be happening to only some genes or all genes, so I wish to test them all.

I am expressing a 5' RNA decapping (like Dcp2) enzyme with or without an inhibitor in human cells.

I tried this tool (https://github.com/csglab/REMBRANDTS) which based on paper (https://www.nature.com/articles/s41467-017-00867-z) postulates that it can estimate mRNA half-life by comparing exon and intron expression levels. I do not understand the rational completely but the paper and software are 5 years old so I thought I would reach out here to see if there are better tools.

There must be a more direct way of comparing coverage of 5' to 3' portions of transcripts to estimate mRNA degradation.

mRNA decay RNA-seq • 205 views
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Entering edit mode
11 weeks ago

It seems like a very difficult problem to address, you are probably better off comparing the counts at the TSS compared to some range downstream... (e.g. +1 to +5000) I would take a look at the data to get an idea for where to set this range. Maybe you could calculate this ratio for every gene in each condition and then compare it between conditions.

Decay rates:

The rationale for decay-rates is that the intron-exon coverage should be relatively constant (imagine some kind of steady state) but when that system is perturbed the rate of transcription in the nucleus will decrease and by proxy the amount of intronic reads. The same goes for mRNA decay. This oddly intrinsicly linked to the idea of RNA-velocity.

  • [introns] + [exons] = total concentration
  • if [introns] goes up and [exons] goes up or is constant it suggests transcription is upregulated
  • if [introns] stay constant and [exons] go down it suggests increased decay rate
  • if [introns] go down and [exons] stay constant it suggests transcription is downregulated
  • if [introns] stay constant and [exons] increase it suggests decreased decay rate

All of these pathways are intertwined so the assumption is actually not ideal... and probably not as thoroughly investigated as it should be.

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Interesting idea with TSS sites. The Decay rate seems like a more straightforward way of looking at this. Decapped mRNA should decay faster, correct? The paper claims to "estimate the transcription rate-dependent bias from RNA-seq data and subtracts it from Δexon–Δintron, providing unbiased estimates of differential mRNA half-life."

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