Louis Parham

Summary: The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic activity predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineageagnostic marker for the prospective identification of facultative stem cells.

Background and methods: : Our lab demonstrated previously that IGF2 mRNA binding protein 1 (IMP1) deletion in the intestinal epithelium increases autophagy, indicating IMP1 can act as an autophagy suppressor. Our objective is to elucidate the molecular mechanisms underlying IMP1's regulation of autophagy in the intestine. Based on our prior work suggesting that IMP1 may suppress autophagy translation, we hypothesized that IMP1 binds directly to and represses translation of MAP1LC3 (LC3) mRNA dependent on phosphorylation of IMP1 by mTORC2. To address our hypothesis, we used transformed cell lines and primary mouse intestinal enteroids with IMP1 knockout (KO) for western, qPCR, luciferase assays, immunofluorescence, and mass spectrometry to evaluate how phosphorylation of IMP1 alters LC3 translation. Results: IMP1 KO led to an increase in total LC3 protein in contrast to other autophagy genes such as ATG3 and ATG5, suggesting selective regulation of LC3 by IMP1. We did not observe an increase in LC3 mRNA with IMP1 KO. To evaluate a direct effect of IMP1 KO on LC3 translation, we utilized a dual‐luciferase reporter assay with the 3′UTR of LC3, identified as a possible IMP1 binding region using computational prediction. We found that IMP1 KO leads to an increase in luciferase activity corresponding to increased translation of the luciferase‐LC3 3′UTR fusion construct compared to controls. Next, we observed that we can disrupt translation enhancement via introducing a mutation into IMP1 serine 181 to create a non‐phosphorylatable alanine. This phosphorylation, which we confirmed by mass spectrometry, is suggested to be critical for mRNA binding and is co‐translationally added to IMP1 by mTORC2. We found that luciferase activity with the mutant is similar to KO cells, demonstrating the requirement of phosphorylation at S181 for IMP1's regulation of LC3 translation. Finally, previous work from others demonstrate IMP1 localization to cytoplasmic granules including processing bodies (PBs). We confirmed that IMP1 localizes to PBs, however, cell expressing IMP1 S181A revealed re‐localization of IMP1 from PBs to the nucleus. Conclusions: Our results support the hypothesis that IMP1 regulates directly LC3 translation in a manner dependent upon IMP1's phosphorylation at S181. IMP1 localization to PBs is also dependent on phosphorylation at S181. Ongoing studies will evaluate the dependence of IMP1 phosphorylation of LC3 on co‐localization of IMP1 with LC3 transcript in PBs or other cytoplasmic granules. Taken together, our study identifies a new layer of autophagy regulation which may be important in both homeostatic and disease processes in the intestine.

Intestinal epithelial cells are among the most rapidly proliferating cell types in the human body. There are several different subtypes of epithelial cells, each with unique functional roles in responding to the ever-changing environment. The epithelium’s ability for rapid and customized responses to environmental changes requires multitiered levels of gene regulation. An emerging paradigm in gastrointestinal epithelial cells is the regulation of functionally related mRNA families, or regulons, via RNA-binding proteins (RBPs). RBPs represent a rapid and efficient mechanism to regulate gene expression and cell function. In this review, we will provide an overview of intestinal epithelial RBPs and how they contribute specifically to intestinal epithelial stem cell dynamics. In addition, we will highlight key gaps in knowledge in the global understanding of RBPs in gastrointestinal physiology as an opportunity for future studies.