Induced pluripotent stem cells (iPSCs) could be a panacea for a wide range of human diseases. Both the generation of iPSCs and their differentiation into various lineages require massive and coordinated changes in gene expression. When cells are pushed from the adult state into pluripotency a large scale induction of pluripotency genes is required, but also a large scale down-regulation of genes expressed in the differentiated parent cells. Similarly, the more natural process of differentiation requires down-regulation of pluripotency factors with concomitant increased expression of tissue-specific genes. These changes occur at the transcriptional level, but also post-transcriptionally, particularly at the level of mRNA decay. Importantly, the contributions of post-transcriptional events to the achievement and maintenance of pluripotency are poorly characterized. We propose to study three post- transcriptional RNA regulons that likely contribute to the achievement and/or maintenance of pluripotency. First, we determined that a large number of Zinc Finger Protein (ZNF) mRNAs are stabilized in iPSCs. As these ZNF transcription factors have vital roles in development, and perhaps in controlling retrotransposition, understanding their regulation is essential. We will examine a potential mechanism by which miRNAs target repeated domains in the ORF of ZNF mRNAs to induce decay. We will also investigate why these ZNF mRNAs have unusually short poly(A) tails and uncover how this contributes to their expression. Second, we found that methylation of mRNAs, and particularly ZNF mRNAs, is correlated with increased stability in iPSCs. We will uncover the mechanisms by which this modification influences gene expression in pluripotent cells. Finally, we have shown that many mRNAs encoding transcription factors important for embryonic development contain C-rich elements and are significantly destabilized in iPSCs. We will identify the factors responsible for this regulation and decipher their mechanism of action. Overall, these experiments will give much needed insights into post- transcriptional control in stem cells that may lead to improved reprogramming and a better understanding of the differences between pluripotent cells and their fully differentiated relatives.

Public Health Relevance

Induced pluripotent stem cells (iPSCs) are unique in that they are able to divide indefinitely while retaining the ability to turn into any cell type in the body. Recent advances mean that we can generate these cells by reprogramming cells from adult tissue. However, we do not fully understand the differences between iPSCs and adult tissues which limits their applications. Our results will increase our understanding of the pathways involved in reprogramming and perhaps lead to safer stem cells for therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM114247-04
Application #
9438547
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
2015-03-01
Project End
2019-02-28
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
4
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Colorado State University-Fort Collins
Department
Microbiology/Immun/Virology
Type
Schools of Veterinary Medicine
DUNS #
785979618
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
Russo, Joseph; Heck, Adam M; Wilusz, Jeffrey et al. (2017) Metabolic labeling and recovery of nascent RNA to accurately quantify mRNA stability. Methods 120:39-48