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iPSC-Reprogramming Keeps it Nice and Splice-y!

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Review of “Multiphasic and Dynamic Changes in Alternative Splicing during Induction of Pluripotency Are Coordinated by Numerous RNA-Binding Proteins” from Cell Reports by Stuart P. Atkinson

Alternative splicing (AS) is a post-transcriptional process which allows for a single gene to code for multiple proteins. AS is vitally important for processes such as cell fate transition, development, disease [1], and as we have discovered recently, for pluripotency and for the generation of induced pluripotent stem cells (iPSCs).

To gain a broader understanding of AS during the entire reprogramming process, researchers from the laboratories of Yi Xing and Russ P. Carstens have analyzed reprogramming using RNA sequencing (RNA-seq) in a temporal manner. Using the discovered AS patterns and the differential expression of RNA-binding proteins (RBPs) and known splicing factors the authors have identified influential splicing regulators whose expression can improve the reprogramming process [2].

To allow for synchronized reprogramming, the authors utilized mouse embryonic fibroblasts (MEFs) carrying a doxycycline (Dox)-inducible polycistronic OKSM cassette [3]. This system allows the simple and synchronized production of transgene-independent iPSCs following doxycycline treatment. At specific points during the process, the authors selected Ssea1-positive cells to select cells at an intermediate stage of reprogramming [4] and subjected them to RNA-based analysis, with a special focus on skipped exons (SEs).

This suggested the relative importance of different splicing factors at defined stages, and so the study attempted to correlate these findings to gene expression values for 226 genes encoding RBPs with known or inferred functions in splicing regulation. Subsequently applied criteria then reduced this number down to 95 RBPs which were likely to be vitally important to reprogramming.

So what important factors did the paper uncover?

  • Mbnl1/2 downregulated throughout reprogramming
  • Zcchc24 inactivated at early stages
  • Rbm47 upregulated at very late stages
  • Esrp1 and Esrp2 upregulation important for the mesenchymal-to-epithelial transition (MET) phase of reprogramming.
    • Ectopic expression of Esrp1 substantially enhanced reprogramming
    • This correlates to the introduction of exon 5 in transcription factor Grainyhead-like 1 (Grhl1) and a subsequent increase in transcriptional activity

The next stage of this interesting study looked at RBP-binding motifs to implicate specific factors in the regulation of splicing during reprogramming. This highlighted an enrichment of Esrp1-binding sites upstream and Mbnl1-binding sites downstream of exons undergoing skipping during reprogramming, and also provided evidence for the importance of Rbfox2, Ptbp1, Rbm24, and Rbm38 binding sites.

Overall, the study highlights that different phases of AS controlled by multiple RNA-binding proteins are vitally important throughout the reprogramming process and also, that artificially affecting splicing can improve reprogramming efficiency. The authors hope that their studies will serve to identify even more splicing factors important to both pluripotency and lineage-specific differentiation and to then harness these to improve reprogramming and differentiation techniques.

References

  1. Kalsotra A and Cooper TA Functional consequences of developmentally regulated alternative splicing. Nat Rev Genet 2011;12:715-729.
  2. Cieply B, Park JW, Nakauka-Ddamba A, et al. Multiphasic and Dynamic Changes in Alternative Splicing during Induction of Pluripotency Are Coordinated by Numerous RNA-Binding Proteins. Cell Rep 2016;15:247-255.
  3. Stadtfeld M, Maherali N, Borkent M, et al. A reprogrammable mouse strain from gene-targeted embryonic stem cells. Nat Methods 2010;7:53-55.
  4. Brambrink T, Foreman R, Welstead GG, et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2008;2:151-159.