The xrFrag method


All cellular transcripts are generally degraded by the contribution of at least one of the typical decay pathways depicted below, namely exonucleolytic or endonucleolytic degradation.

Overview

Image from Boehm et al. 2016 Nat Comm

In order to identify the pathway that is responsible for the turnover of a certain transcript of interest, we developed the xrFrag method (xrFrag = XRN1-resistant decay fragment).

Using the virus-derived xrRNA element, decay intermediates that arise due to the turnover of RNAs can be visualized.

We have successfully used the xrFrag method to analyze multiple mRNA decay pathways such as nonsense-mediated mRNA decay (NMD), miRNA-induced decay and degradation caused by AU-rich elements or cytokine 3' UTRs

If you are interested, here you can find the original publication: PubMed

Workflow

The full protocol enabling the detection of XRN1-resistant decay intermediates with molecular biology and imaging methods was developed together with the Chao lab in Basel (Link) and is now published in Nature Protocols:

Protocol

Image from Voigt et al. 2019 Nat Protoc.

Workflow

Here is a simplified overview of the xrFrag method:

Overview


Sequences

This is the standard xrRNA element consisting of two xrRNA sequences, which is the minimal element to block cellular XRN1:

Overview

Image from Boehm et al. 2016 Nat Comm

This is one of the plasmids used for transient transfection of mammalian cells and expression of the globin reporter mRNA harboring the xrRNA element in the 3' UTR:

Overview


The fully annotated vector sequence can be downloaded here


If you would like to use the xrFrag method and require assistance with the experimental setup or would like us to share plasmids, primer sequence, etc. just contact either Jenny or Volker


Addgene

Several different xrRNA-containing reporter plasmids are available by following this link to Addgene: