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Abstract
Both RNA synthesis and decay must be balanced within a cell to achieve proper gene
expression. Additionally, modulation of RNA decay specifically offers the cell an
opportunity to rapidly reshape the transcriptome in response to specific stimuli or
cues. Therefore, it is critical to understand the underlying mechanisms through which
RNA decay contribute to gene expression homeostasis. Cell-free reconstitution approaches
have been used successfully to reveal mechanisms associated with numerous post-transcriptional
RNA processes. Historically, it has been difficult to examine all aspects of RNA decay
in such an in vitro setting due, in part, to limitations on the ability to resolve
larger RNAs through denaturing polyacrylamide gels. Thus, in vitro systems to study
RNA decay rely on smaller, less biologically relevant RNA fragments. Herein, we present
an approach to more confidently examine RNA decay parameters of large mRNA size transcripts
through the inclusion of an engineered XRN1-resistant reporter RNA (xrRNA). By placing
a 67 nucleotide xrRNA near the 3' end of any in vitro transcribed RNA with variable
size or sequence context, investigators can observe the accumulation of the xrRNA
as a readout of exoribonuclease-mediated 5'-3' decay. This approach may allow in vitro
RNA decay assays to include full biologically relevant mRNA/mRNPs, extending their
utility and allow improved experimental design considerations to promote biologically
relevant outcomes.