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A developmentally programmed splicing failure attenuates the DNA damage response during mammalian zygotic genome activation

Christopher D. R. Wyatt, Barbara Pernaute, André Gohr, Marta Miret-Cuesta, Lucia Goyeneche, Quirze Rovira, Ozren Bogdanovic, Sophie Bonnal, Manuel Irimia

Preprint posted on November 27, 2020 https://www.biorxiv.org/content/10.1101/2020.11.25.397794v2

Splice of life: learn how RNA splicing regulates genome stability during mammalian zygotic genome activation.

Selected by Ram

Context1,2

After fertilization, the zygotic genome is transcriptionally activated (also called zygotic genome activation, or ZGA) to kick-start the gene expression program of the new organism. ZGA is a complex process that constitutes a plethora of events like chromatin organization, cell cycle, nuclear-to-cytoplasm ratios, degradation of maternal mRNA, and initiation of zygotic transcription. The principles of ZGA are common among different species, but the time at which it happens could be different.

While earlier studies investigated the transcriptional changes during ZGA, here the authors investigated the alternative splicing (AS) events or the transcriptomic diversity that occurs during ZGA in three different species.

Key findings

  1. The authors of the current preprint re-analyzed publicly available steady-state RNA levels (RNA-seq) generated at different stages of development in three different species – human, mouse, and cow (fig a). They curated the AS and gene expression profiles and provided them for the research community (http://vastdb.crg.eu).

    (a) Summary of datasets used in this study for each species. Golden lines indicate the ZGA for each species. Schematic view of the methodological steps taken to process the RNA-Seq data and obtain AS quantifications with vast-tools (right side). (b) A working hypothesis for alternative exon regulation during ZGA in the mouse. Taken and modified directly from Wyatt CDR B et. al., 2020 under a CC-BY 4.0 international license.
  2. They found increased AS events at the stage of ZGA in all the three studied species (fig a). For example, in humans, 41.7% of differentially regulated AS events occurred at ZGA. Interestingly, the majority of the AS events are transient and recouped to earlier levels after the embryos passed the ZGA in all three species revealing the temporal nature of AS events in embryo development.
  3. Based on their analysis, they found that most AS events that occur transiently during ZGA produce non-functional or cryptic-like mRNA isoforms leading to disruptive open-reading frames. Notably, in all three species, the disruptive AS events significantly belong to the genes involved in DNA damage response pathways (DDR). This suggests that the DDR pathway is challenged in embryos especially during ZGA. To test this, the authors treated mouse embryos with etoposide (DNA damaging agent and topoisomerase II inhibitor) and evaluated the cell death (TUNEL assay) and DNA damage response (p53 phosphorylation). They found that embryos manifested positive for TUNEL assay and activated p53 at morula stage after the ZGA but not during ZGA reinforcing their prediction DDR is attenuated during  ZGA.
  4. Furthermore in their analysis, they found that two splicing proteins – Snrpb and Snrpd2 – could impact the AS events during ZGA among the three species (fig b). They found a decrease in Snrpb and Snrpd2 levels right before the ZGA stage followed by an increase after the ZGA (using western blot and RNA-seq). To test the negative correlation between ZGA and these splicing proteins, they injected the Snrpb and Snrpd2 mRNAs into mouse embryos at the 1C stage (mCherry mRNA was used as control) and evaluated the 2C transcriptome via RNA-seq. They found that ectopic expression of Snrpb and Snrpd2 before or at ZGA incurred reversion of AS events (but not gene expression changes) usually detected at the ZGA. Additionally, ectopic expression of Snrpb and Snrpd2 sensitized the embryos to activation of DDR (p53)  when exposed to etoposide. Thus, they suggest that Snrpb and Snrpd2 mediated changes in AS events could impact DDR during embryo development.

Conclusion and perspective

The past two decades of research propound an important role of RNA processing proteins in maintaining genome stability3. Here, the authors report a role of two developmental regulated RNA splicing proteins that could negatively regulate ZGA. They suggest that these splicing proteins could impact the genome stability by modulating the AS patterns of genes involved in DDR during embryonic development.

However, it is very intriguing to think why nature keeps embryos vulnerable to genotoxicity, exactly at a time when they need to be protected.

Acknowledgments: Thanks to Manuel Irimia, Barbara Pernaute, and all the authors of this work for their support.

(Note: I only highlighted the key findings of the preprint without commenting on the computational analysis. Please feel free to do so: the authors are exceptionally open to discuss their work.)

References:

  1. https://doi.org/10.1016/j.semcdb.2017.12.006
  2. https://doi.org/10.1016/j.devcel.2017.07.026
  3. https://doi.org/10.1016/j.molcel.2009.06.021
  4. https://www.biorxiv.org/content/10.1101/2020.11.05.367888v2

Tags: dna damage response, rna splicing, zga

Posted on: 5th February 2021

doi: https://doi.org/10.1242/prelights.27229

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Author's response

Manuel Irimia (MI), Barbara Pernaute (BP), and others shared

1. Do the AS peaks at ZGA correlate with transcription activity?

MI, BP, and others: Genes with AS peaks are not significantly enriched or depleted among genes with changes in gene expression (Extended Data Figure 3). More precisely, this figure analyzes all AS events that change at each transition, but it will likely be the same for AS peaks alone. However, it is worth noting that in our Random Forest analysis, the fold change in gene expression at ZGA was a relevant feature, so there is probably some association. This is perhaps not surprising, since genes with AS peaks must have quite an active transcription so they have enough mRNA turnover for us to detect isoform changes.

2. Metazoan RNA splicing is predominantly co-transcriptional. Hence, do the authors think using a nascent RNA-seq profile that can help reinforce their analysis (for example4)?

MI, BP, and others: That would be very interesting to have, indeed, but it’s very far from trivial for these stages. Our guess, though, is that the associated introns are likely “slow” post-transcriptional introns.

3. Is the lower response to etoposide during ZGA, somehow impacted by the cell cycling times at the ZGA?

MI, BP, and others: That’s a very good question as this is the case for other vertebrates like Xenopus or zebrafish, in which prior to the mid blastula transition (ZGA) the cell cycle is extremely short, gap phases are eliminated, and DDR and checkpoints are abolished. However, in mammals, the cell cycle during pre-implantation development is not particularly short (in mouse and human the first and second cell divisions take 18-20h each and the following divisions up to the blastocyst stage take 12-14h each) (2). Moreover, G1 and G2 phases are present and checkpoints are active at all stages; in fact, a high dose of etoposide treatment will trigger DDR at ZGA, suggesting that checkpoints are functional but the threshold for etoposide-induced DDR is higher at this developmental stage.

4. Do the authors know if ectopic expression of Snrpb or Snrpd2 impacts DDR differently in cultured somatic cells and embryonic stem cells? In other words, if their role is specific to the development stage?

MI, BP, and others: The short answer is that we do not know. But it is important to note here that what is quite specific to pre-ZGA stages is the fact the basal levels of Srnpb/d2 are very low. This is in contrast to most other cells. Therefore, overexpression in cultured somatic cells and embryonic stem cells will result in an increase of already high levels of Srnpb/d2, which is unlikely to have a similar effect to the one we report for early embryos.

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