RNA regulons are essential in intestinal homeostasis

Abstract

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.

INTRODUCTION

The intestinal epithelium is one of the most rapidly proliferating organs in the human body and renews itself every 3–5 days (19). Within the epithelial layer, there are several different subtypes of cells, each with unique and/or overlapping genetic markers identified in recent years (13–17). The intestinal epithelium is highly dynamic as a function of its requirement to rapidly adapt to environmental changes. The ability of the epithelium to respond to acute or chronic environmental changes may be disturbed during prolonged intestinal injury, inflammation, irradiation, toxin exposure, or cancer. Therefore, developing a more comprehensive understanding of how key pathways are regulated during homeostasis will lay a foundation for defining new therapeutic strategies in the future.

RNA-binding proteins (RBPs) have long been studied for their roles in fundamental biological processes, including transcription and translation of target RNAs, leading to regulation of various cellular functions. RBPs bind their target RNAs on specific domains and binding motifs within ribonucleoprotein (RNP) complexes and can facilitate mRNA stability, degradation, translation, splicing, polyadenylation, localization, and transport in different contexts (reviewed in Ref. 37). As such, RBPs represent a rapid and efficient mechanism to regulate gene expression and often do so through regulating multiple transcripts within the same pathway, representing posttranscriptional “regulons” (37). From a teleological perspective, RBPs may be particularly critical in rapidly proliferating organs to facilitate cellular adaptation to normal homeostatic stressors as well as during pathological states. It is therefore critical to understand both the basic biology of RBPs and how they may contribute to disease. In this review, we will provide a summary of intestinal epithelial RBPs and how they contribute to normal intestinal epithelial cell dynamics, with a focus on intestinal stem cells and homeostasis.

INTESTINAL RBPs REGULATE CRYPT CELL DYNAMICS

The intestinal epithelial layer functions as conveyor belt of cells that allows for rapid turnover. This phenomenon is dependent on the presence of stem cells residing in the base of intestinal and colonic crypts (1, 62). Two distinct populations of stem cells have been described in the intestinal epithelium. Active stem cells or crypt base columnar cells (CBCs), as their name suggests, reside in the crypt bottoms intermingled with Paneth cells, a differentiated cell type contributing to intestinal immune response and the stem cell niche. CBCs are rapidly cycling (approximately every 24 h), self-renewing, and responsible for maintaining homeostasis during tissue turnover (1). The canonical Wnt target gene Lgr5 was the first protein shown to mark this population, and thus far it has been used extensively to study these cells along with newly discovered markers such as Olfm4 and Ascl2 (1, 71, 72). Reserve intestinal stem cells (rISCs) comprise a functionally distinct population of stem cells in intestinal epithelium, cycling less frequently than CBCs and residing roughly four cell positions from the crypt base (62). Commonly used markers for studying rISCs are Bmi1, Hopx, Lrig1, mTert, among others (25, 50, 56, 62, 65). Studies have shown that during conditions of stress such as whole body irradiation or inflammation, which largely ablate CBCs, rISCs are able to exit their slow-cycling state and acquire stem-like activity, making them essential for intestinal regeneration (58, 67, 79). To regulate stem cell proliferation and differentiation during homeostasis, damage, and disease, intestinal stem cells must be capable of efficient regulation of gene expression. RBPs provide a mechanism by which stem cells can fine-tune or amplify gene expression at the posttranscriptional level via sequestration of newly transcribed RNA molecules into RNP particles where they can be triaged based on context-dependent cellular needs.

In recent years, several RBPs have been identified with key roles in intestinal homeostasis and response to injury (Fig. 1). Among the most studied RBPs in the intestine are the Musashi (MSI) family of proteins, with particular focus on the highly conserved mammalian isoforms, MSI1 and MSI2. It has been previously reported that the MSI proteins are highly expressed throughout intestinal crypts; however, ablation of these proteins in the intestine using the Villin promoter fails to produce any overt phenotype. Moreover, Msi deletion does not affect stem cell activity or canonical WNT pathway signaling in CBCs. Conversely, Msi ablation specifically in Bmi1/Hopx+ “reserve” stem cells was sufficient to induce G₁ arrest during homeostasis and abrogate epithelial regeneration following gamma-irradiation. Msi proteins are further necessary and sufficient to activate rISCs and induce cell cycle reentry, highlighting the critical role of MSI proteins in reserve stem cell dynamics (84). MSI1 and MSI2 are functionally redundant and overexpression of either is sufficient for epithelial transformation. Moreover, loss of both proteins is required to inhibit proliferation in a carcinogen-induced colitis-associated model of intestinal tumorigenesis (40). Together, these studies suggest that despite the dispensability of MSI proteins during homeostasis, their expression in rISCs is critical in regulating crypt dynamics during stress.

Fig. 1.

Schematic of RNA-binding protein (RBP) expression during homeostasis and disease. RBPs are preferentially expressed in intestine and colon crypts. Brackets indicate general area where expression has been observed. Arrows indicate expression described in a specific cell type. Font size indicates relative expression levels. During homeostasis, yellow box indicates that overexpression can lead to deregulated crypt proliferation and tumorigenesis, green box indicates that altered expression of the RBP does not affect homeostasis, and blue indicates that deregulated expression of the RBP affects homeostasis but has not been shown to induce tumorigenesis. During injury or disease, a red box indicates expression of the RBP is increased during intestinal regeneration and cancer, while orange indicates that expression is associated with cancer. CBC, crypt base columnar cell.

LIN28A and LIN28B are RBPs expressed during embryonic development in the intestine. LIN28B is expressed in crypt cells in the adult mouse intestine, with lower expression in villus tip epithelia (48). LIN28B modulates several key pathways in the intestine, both directly by binding to target transcripts in the pathways (26, 68) and indirectly via inhibiting maturation of members of the let-7 miRNA family. LIN28B in turn is regulated by the let-7 microRNA family (30, 31, 55, 59, 75). Intestinal Lin28b overexpression via the murine Vil1 promoter is sufficient to induce intestinal hypertrophy, extensive crypt proliferation, and transformation. This phenotype was attributed mainly to repression of Let7 miRNA through LIN28B, which inhibits intestinal epithelial growth by repressing crypt fission and cell cycle progression (47, 48). Following this study, an independent group also demonstrated enhanced crypt proliferation as well as loss of various differentiated cells following global LIN28A and LIN28B overexpression (68). The same group found that LIN28 overexpression in Apc^Min/+ mice accelerated tumor progression and increased invasiveness (68). Finally, studies in Drosophila intestine demonstrate that Lin28 loss leads to decreased intestinal stem cell numbers and slower intestinal stem cell rates of division. Furthermore, Lin28 deficiency significantly decreased symmetric intestinal stem cell division after feeding via reduced insulin-like receptor levels (12). Given the role that LIN28 RBPs play in regulating crypt proliferation, their expression specifically in the stem cell compartment is likely critical to maintain intestinal homeostasis.

Similar to LIN28B, IGF2 mRNA-binding protein 1 (IMP1) is expressed highly during embryonic development in the intestine and its expression is low and limited to the crypts in adult mouse intestine (10, 21). IMP1 has been shown to regulate several developmental signaling pathways such as the IGF2 and WNT/β-CATENIN pathways. IMP1 hypomorphic mice exhibit dwarfism and compromised intestinal development (29). Recent studies demonstrated that IMP1 is upregulated in mice following whole body irradiation, suggesting a possible role during intestinal regeneration. The same study demonstrated that IMP1 is limited or absent in the intestinal epithelium except for crypt cells and is upregulated in reserve stem cells following irradiation (10). Loss of Imp1 in reserve stem cells was associated with reduced weight loss and increased crypt proliferation following whole body irradiation. Furthermore, this study demonstrated that intestinal Imp1 deletion in the context of LIN28B overexpression promoted increased expression of stem cell markers and WNT target genes while increasing crypt proliferation, underscoring a role for IMP1 in regulating stem cell homeostasis (10).

The RBP ELAV-like protein 1 or HuR (human antigen R) is highly expressed in the gut mucosa and has been implicated in intestinal proliferation and regeneration after injury (46). Mice with intestine-specific HuR deletion exhibit increased injury with doxorubicin-induced acute intestinal damage (24). In a separate study, loss of HuR in the intestine was associated with mucosal atrophy as well a robust decrease in crypt proliferation during homeostasis. Additionally, HuR-deficient mice exhibited decreased regenerative potential after exposure to irradiation (43). Interestingly, colonic mucosal growth was unaffected by HuR loss. HuR directly binds to and enhances translation of the WNT coreceptor LRP6 and the WNT target gene MYC. Furthermore, HuR silencing is sufficient to inhibit intestinal epithelial cell proliferation in WNT3A-transfected IEC-6 cells, suggesting an intestinal epithelial cell intrinsic mechanism by which HuR regulates the WNT pathway and intestinal homeostasis (43). Although HuR expression in specific intestinal epithelial cell populations has not yet been addressed, the reliance of the intestinal stem cell compartment on appropriate WNT-signaling regulation to maintain tissue homeostasis suggests HuR may be a key factor in stem cell dynamics. Additionally, it has been shown that deletion of HuR decreases proliferation and tumor burden in multiple models of intestinal tumorigenesis (24).

A member of the CUG-binding protein ELAV-like family of RBPs recently shown to regulate intestinal homeostasis is CELF1 or CUGBP1. CUGBP1 has been associated with upregulation of inhibitor of apoptosis 1 and inhibitor of apoptosis 2 in intestinal epithelial cells, making them more resistant to TNF-α and cyclohexamide-induced apoptosis (20). Furthermore, CUGBP1 regulates tight junction expression and gut barrier function by inhibiting occludin expression at the translational level (85). In the intestine, CUGBP1 is found to compete with HuR for binding to MYC mRNA to repress its translation at homeostasis (44). Moreover, in vitro ectopic CUGBP1 expression in intestinal epithelial cells leads to G₁-phase growth arrest and conversely CUGBP1 silencing enhances cell proliferation (44). A transposon-based genetic screen in mice identified CELF1, as a potential promoter of colorectal cancer (64). Beyond the intestine, CUGBP1 effects mRNA stability and is associated with increased proliferation and cell motility and decreased apoptosis in nonsmall cell lung cancer cell lines (23).

Finally, the MEX3 family of RBPs is evolutionarily conserved and differentially recruited to processing bodies in the cytoplasm, suggesting a role in RNA turnover (7). In the intestine, single-cell transcriptome analysis revealed that Mex3a is expressed in a subset of Lgr5+ stem cells that are slow dividing (2). These Mex3a+ cells converted to fast-dividing cell following chemotherapy or radiation-induced toxicity, thus promoting intestinal regeneration. In CaCo2 cells, MEX3A overexpression was associated with enhanced expression of stem cell markers including LGR5, MSI, and BMI1. Furthermore, intestinal MEX3A represses the function of the homeobox transcription factor CDX2 and affects intestinal reprogramming (53). In summary, there are multiple RBPs with emerging roles in intestinal stem cells and the broader crypt compartment, suggesting an essential role for posttranscriptional regulation in stem cell dynamics and plasticity.

RBPs MODULATE WNT AND OTHER KEY SIGNALING PATHWAYS IN INTESTINAL EPITHELIAL CELLS

WNT signaling is essential in intestinal crypt stem cell maintenance. Proliferation and survival of CBC stem cells rely on WNT ligands (54, 70, 88), which are produced in part by Paneth cells, among other nonepithelial cells in the small intestine (63). Indeed, intestinal stem cell growth is enhanced upon culture with Paneth cells, and this can be reversed with WNT inhibition. Inducible β-catenin deletion results in terminal differentiation of intestinal stem cells and subsequent lethality, emphasizing the importance of WNT/β-CATENIN signaling in stem cell maintenance (22).

RBPs play an important role in the regulation of the WNT/β-CATENIN-signaling cascade, emphasizing a significant role of these posttranscriptional regulators in the intestinal stem cell niche (Table 1). The LIN28 family of RBPs has been shown to cooperate with the WNT-signaling axis in tumor growth and progression (8) whereas recent studies demonstrate opposing roles for LIN28B and IMP1 in regulation of WNT signaling during intestinal tumorigenesis (10). During homeostasis, Imp1 deletion is associated with upregulation of WNT target genes including Lgr5, Sox9, and Wnt3, suggesting that IMP1 may normally suppress these genes and enhance intestinal stem cell differentiation in this context (10).

Table 1.

Intestinal epithelial pathways associated with RBP regulation

Pathway/Function/RBPs	Targets Evaluated*	Reference
WNT/β-CATENIN
HuR	LRP6	(41)
KSRP	DSVL3	(4)
RBM3	β-CATENIN	(74)
MSI1	FRAT1, MYC, CCND, CCND2	(57)
CIRP	LGR5	(60)
LIN28B	Global changes	(10, 68)
IMP1	C-MYC** LGR5, SOX9, WNT3	(77) (10)
NOTCH
MSI1	NICD, HES1	(57)
mTOR
MSI1/2	PTEN, BMPR1A, LRIG1	(84)
Intestinal stem cells
IMP1	LGR5, BMI1, SOX9, WNT3	(10)
HUR	OLFM4	(24)
MEX3A	LGR5, MSI, BMI1	(53)
CIRP	BMI1, LGR5, DCLK1	(60)
RBM3	DCLK1, LGR5, CD44	(74)

RBP, RNA-binding protein.

^*Targets are not necessarily direct binding targets.

^**Not shown in intestine or colon cells specifically.

While studies evaluating LIN28 and IMP1 suggest global regulation of WNT, studies evaluating mRNA binding are needed to ascertain direct or indirect regulation. The RBP HuR binds to LRP6 mRNA, which is essential for FRIZZLED receptor function and response to WNT (41, 87). HuR can also participate in cell cycle regulation through binding and enhancing translation of CDC42 mRNA, a GTPase that controls cell morphology, migration, and cell cycle progression (45). The RBP K-homology splicing regulator protein (KSRP) has been shown to interact with COOH-terminus of Dishevelled-3 (DVL3), a key component of the FRIZZLED1-LRP-DISHEVELLED pathway. The interaction of KSRP and DVL3 correlated with decreased β-CATENIN mRNA levels, suggesting that KSRP is important for the stability of β-CATENIN mRNA, although the mechanism of this still remains elusive (4).

RBM3, a cold-inducible RBP that is important for cell cycle progression and stress response, increases stability and translation of cyclooxygenase-2, IL-8, and VEGF mRNA. It has also been shown to influence the stability of β-CATENIN. When RBM3 was overexpressed in colon cancer cell lines, both β-CATENIN signaling and expression of LGR5 were increased. Furthermore, overexpression of RBM3 resulted in phosphorylation and attenuation of GSK3B activity, which promotes increased stability and accumulation of β-CATENIN (74). A related protein, Cold-inducible RNA-binding protein (CIRP or hnRNP A18), is also involved in the regulation of β-CATENIN. CIRP has been shown to be upregulated in colitis-associated cancer and inflammatory bowel disease and CIRP levels correlated with levels of BMI1, LGR5, and DCLK1 implicating this protein in stem cell dynamics (60). Although a direct mechanism remains to be demonstrated, in several cell types GSK-3β, which inhibits β-CATENIN, upregulates CIRP transcription and phosphorylates CIRP protein to promote its cytosolic translocation (42, 80, 81). Furthermore, with the use of immunoprecipitation and PCR, it was demonstrated that CIRP maintains expression of β-CATENIN in Xenopus laevis, demonstrating that this protein binds and/or modulates expression of this mRNA (52).

In addition to modulation of WNT signaling, RBPs may also regulate the NOTCH pathway, which is important for postmitotic cell fate decisions in intestinal epithelium (73, 86). Treatment of intestinal epithelial primary cultures with the agonist WNT3A led to an upregulation of Msi1 mRNA, which subsequently increased β-CATENIN expression and nuclear translocation (57). Similarly, overexpression of Msi1 led to an upregulation of the β-CATENIN stabilizer Frat1 and Notch-1 receptor intracellular domain (NICD). Upregulation of Msi1 was found to be due to the binding of TCF/LEF to the Msi1 promoter (57). Finally, the mammalian target of rapamycin (mTOR) pathway is emerging as a key player in intestinal stem cell dynamics (61, 83). Msi proteins have been shown to bind to and inhibit negative regulators of mTORC1 including PTEN, BMPR1A, and LRIG1, leading to induction of this nutrient-sensing protein and ultimately proliferation and differentiation.

PLEIOTROPIC ROLES FOR RBPs

RBPs are rapid and effective regulators since they bind already transcribed targets. Therefore, the functional roles of RBPs depend on the transcripts present within a specific cell type. Even in the same cell type, changes in the transcriptome, due to environmental changes or injury, can lead to different functional outcomes for RBPs. This is also true for different tissues or organs where transcriptomes vary. Furthermore, since RBP binding to target transcripts can result in a number of functional outcomes, it may be difficult to elucidate the functional role from one context to another. For example, although IMP1 is recognized as an oncofetal protein due to its overexpression in colorectal cancers and increased tumor burden with IMP1 expression in xenograft models (28), IMP1 deletion in intestinal mesenchymal cells increased tumor burden in mice (27). Similar, opposing roles for IMP1 have also been reported in breast cancer (51) (76) (66). IMP1 therefore can exhibit both tumor-initiating or tumor suppressive functions. Another example of this paradigm is HuR, which stabilizes and increases translation of several key cancer related pathways (49). In a recent study in pancreatic ductal adenocarcinoma cells, HuR deletion resulted in a decrease in proliferation and an increase in cell death, possibly via regulation of KRAS pathway genes (39). In contrast, other studies have shown that HuR knockdown in myeloid lineage cells in mice caused an enhanced susceptibility to colitis-associated cancer and increases inflammatory mRNAs (82).

Similarly, while CELF1 deletion in several cancer cell lines decreased proliferation and migration (23, 32), and Celf1-deficient mice exhibited growth retardation (38), CELF1 has also been reported to promote apoptotic resistance in esophageal cancer (9). CELF1 exhibits opposing effects on cell proliferation in primary versus malignant T cells due to transcriptomic differences (6). In yet another example, Mex3a is expressed in slow dividing intestinal stem cells (2), but in CRC cell lines the effect of MEX3A on differentiation depends on cell confluence. At low confluency, MEX3A inhibition increased CDX2 expression, but this effect was reverted when the cells were highly confluent (53). Taken together, these studies demonstrate that RBPs represent a complex regulatory network and their functional roles depend on the tissue type, microenvironment, and the transcriptome of cells in which they are expressed. Therefore, understanding the precise, context-dependent mechanisms by which RBPs regulate protein expression in combination with other factors will be essential to understanding their complex roles in gastrointestinal cancers.

EMERGING TECHNIQUES TO EVALUATE RBPs IN THE INTESTINE

There are multiple experimental methods to determine the landscape of RBP biology and their roles in disease. As illustrated above, the functional role of RBPs can vary depending on the cellular context. Therefore, integration of multiple methodologies is required to understand fully the role of these posttranscriptional regulons in the gut. One method for identifying direct roles of RNA-binding proteins has been to determine their target transcripts using crosslinking immunoprecipitation (CLIP). The RBP under investigation is crosslinked in vivo to its target transcripts via a crosslinking agent, such as ultraviolet light, forming covalent bonds between RNA and protein. Cells are then lysed and the target RBP is precipitated using an antibody. Proteinase K is subsequently used to digest the RBP, leaving the bound transcripts intact. After ligating adapters and linkers to RNA, the transcripts are reverse transcribed to cDNA and identified via high-throughput sequencing (69). CLIP techniques have been refined over the years to increase precision and sensitivity as reviewed in Ref. 11. CLIP-sequencing libraries have been generated for several RBPs, including LIN28B (48), IMP1 (18), and MSI1 (3); however, one must consider differences in steady-state transcriptomes in the cell lines in which these experiments were performed before generalizing binding targets from one cell type to another.

In addition to identifying binding targets using CLIP-sequencing, functional effects of RBPs can be studied by overexpressing or deleting RBP genes followed by high-throughput RNA sequencing. This generates a static snapshot of total RNA abundance in the presence or absence of a given RBP. Since RBPs can regulate the stabilization, degradation, localization, and posttranscriptional modification (for example, splicing) of their targets, RNA sequencing provides a robust tool to study these changes. However, as proteins are the functional effectors in cells, RNA sequencing may not serve as a direct indication of the functional role of RBPs. For example, some RBPs may bind to their target transcripts and cage them in RNP granules, rendering these targets inaccessible to the translation machinery (5). In these cases, mRNA abundance does not correlate with translation/functional effects.

In recent years, ribosome footprint profiling has emerged as a powerful tool to obtain a global snapshot of actively translating transcripts within a cell (33–35). This ribosome-centric technique uses binding and density of ribosomes on a transcript as an indicator of active translation. Profiling is done by deep sequencing of ribosome-bound transcripts as well as sequencing total RNA abundance. Polysomes (a cluster of ribosomes held together by a strand of messenger RNA that each ribosome is translating) are stabilized by blocking translation elongation using cycloheximide before cell lysis (36). This technique detects relative changes and therefore relies on cells or tissues with deletion or overexpression of the desired RBP. This technique can help distinguish between genes regulated at the transcriptome and “translatome” levels. The ratio of ribosome footprint profiling to total mRNA abundance for each transcript reveals the translation efficiency for that gene that can be compared across genotypes or conditions (88).

In summary, a comprehensive understanding of RBP functions requires evaluating 1) target transcripts, 2) the context in which binding occurs, 3) downstream effects on transcription and translation of a given transcript or family of transcripts, and 4) the “net effect” of the RBP expression. To address the latter, in vivo models modulating RBP expression are key to understanding the role of RBPs at the whole organisms level. Genetic mouse models of RBP deletion or overexpression in specific cell types have and will continue to be instrumental in understanding RBP roles at homeostasis and injury or disease models.

CONCLUSIONS

Regulation of cellular dynamics via RBPs is becoming recognized increasingly as a key feature in tissue homeostasis and disease. While existing studies evaluating RBPs in intestinal epithelial cell biology are compelling, several questions remain to be addressed in this growing field. For example, how do RBPs cooperate or act in opposition to one another? Our recent study evaluating the LIN28B-IMP1 axis is one example in which genetic mouse models may reveal new and unexpected roles that may be opposing (10). Similarly, miRNAs may also augment the functional roles of RBPs, as reviewed in Ref. 78, and therefore must be accounted for when determining the relative role of posttranscriptional regulation in a given process. In addition, RNA modifications or the “epi-transcriptome,” the outcome of which is largely governed by RBPs, is an emerging field that will shed new light on how RNA regulons determine cellular functions. Finally, the importance of RBPs and posttranscriptional regulation in other gastrointestinal epithelial stem or progenitor cells (e.g., esophageal, stomach) is largely unexplored. Studies defining how RBPs contribute to stem cell dynamics and disease will not only uncover new paradigms in basic gastrointestinal biology but may also hold the promise for new therapeutics as well.

GRANTS

This study was supported by a grant from the University of Pennsylvania Institute for Translational Medicine and Therapeutics (to K. E. Hamilton); National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants K01-DK-100485 and R03-DK-11446301 (to K. E. Hamilton) and K01-DK-103953 and R03-DK-118304 (to K. A. Whelan); University of Pennsylvania NIDDK Center for Molecular Studies in Digestive and Liver Diseases Grant P30-DK-050306), and Institutional Development Funds from Children’s Hospital of Philadelphia Research Institute.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

L.R.P., P.A.W., and K.E.H. conceived and designed research; L.R.P., P.C., and K.E.H. prepared figures; L.R.P., P.A.W., P.C., K.A.W., and K.E.H. drafted manuscript; P.A.W., P.C., K.A.W., and K.E.H. edited and revised manuscript; P.C. and K.E.H. approved final version of manuscript.