Related Experiment Video
Updated: Nov 7, 2025
Using the E1A Minigene Tool to Study mRNA Splicing Changes
Published on: April 22, 2021
Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved alternative splicing
Carrie Kovalak1,2, Scott Donovan3, Alicia A Bicknell3
1RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
Background:
Alternative splicing, which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing splicing pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses.
Results:
RNA immunoprecipitation in tandem (RIPiT) of exon junction complex components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compare exon junction complex RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identify thousands of previously unannotated splicing events; while many can be attributed to splicing noise, others are evolutionarily conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis.
Conclusions:
Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the nonsense-mediated decay-linked alternative splicing pathway predominates. Exon junction complex RIPiT-Seq also revealed numerous conserved but previously unannotated AS-NMD events.
Insights
Alternative splicing generates diverse mRNA isoforms, but RNA-Seq misses short-lived ones. Exon junction complex RIPiT-Seq captures these, revealing new AS-NMD events crucial for protein homeostasis and potentially cancer.
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- Alternative splicing is key for eukaryotic gene expression, producing multiple mRNA isoforms from single genes.
- Traditional RNA-Seq struggles to quantify splicing pathways due to varying mRNA decay rates, potentially missing short-lived isoforms like those in AS-NMD.
- Some mRNA isoforms with short half-lives, subject to translation-dependent nonsense-mediated decay (AS-NMD), may be entirely missed by standard RNA-Seq.
Purpose of the Study:
- To compare exon junction complex RIPiT-Seq with whole cell RNA-Seq for quantifying alternative splicing events.
- To investigate the flux through alternative splicing, nonsense-mediated decay (AS-NMD) pathways, and identify previously unannotated splicing events.
- To assess the role of splicing machinery in reading frame detection and identify conserved AS-NMD isoforms involved in protein homeostasis.
Main Methods:
- RNA immunoprecipitation in tandem (RIPiT) of exon junction complex components to purify pre-translational mRNPs.
- Deep sequencing of RNAs from purified pre-translational mRNPs (RIPiT-Seq).
- Comparison of RIPiT-Seq data with whole cell RNA-Seq data from HEK293 cells.
Main Results:
- RIPiT-Seq revealed substantially higher flux through known AS-NMD pathways compared to RNA-Seq.
- The study definitively showed that the splicing machinery does not detect reading frame.
- Thousands of unannotated splicing events were identified, including conserved AS-NMD isoforms potentially involved in protein homeostasis, with some linked to cancer prognosis.
Conclusions:
- Deep sequencing of RNAs in post-splicing, pre-translational mRNPs (RIPiT-Seq) quantifies splicing events without decay influence.
- The nonsense-mediated decay-linked alternative splicing pathway predominates for many known AS-NMD targets.
- Exon junction complex RIPiT-Seq identified numerous conserved, unannotated AS-NMD events, offering new insights into gene regulation and disease.
Related Concept Videos
02:27Nonsense-mediated mRNA Decay
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
02:27Nonsense-mediated mRNA Decay
02:31Nuclear Export of mRNA
02:31Nuclear Export of mRNA
02:18Alternative RNA Splicing
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...
02:18Alternative RNA Splicing