mRNA Turnover Research Articles

Proteomic analysis identifies endoribouclease EhL-PSP and EhRRP41 exosome protein as novel interactors of EhCAF1 deadenylase.

This study provides evidences for the functional deadenylase activity of EhCAF1 and shows a link between different mRNA degradation proteins in E. histolytica. By proteomic tools and pull down assays, we evidenced that EhCAF1 interacts with the putative endoribonuclease EhL-PSP, which in turn interacts with exosome EhRRP41 protein. Our data suggest for the first time the presence of two complexes, one containing the endoribonuclease EhL-PSP and the deadenylase EhCAF1 in P-bodies; and another containing the endoribonuclease EhL-PSP and the exosome EhRRP41 exoribonuclease. Overall, these results provide novel data that may help to understand mRNA decay mechanisms in this parasite.

A novel posttranscriptional mechanism for dietary cholesterol-mediated suppression of liver LDL receptor expression

It is well-established that over-accumulation of dietary cholesterol in the liver inhibits sterol-regulatory element binding protein (SREBP)-mediated LDL receptor (LDLR) gene transcription leading to a reduced hepatic LDLR mRNA level in hypercholesterolemic animals. However, it is unknown whether elevated cholesterol levels can elicit a cellular response to increase LDLR mRNA turnover to further repress LDLR expression in liver tissue. In the current study, we examined the effect of a high cholesterol diet on the hepatic expression of LDLR mRNA binding proteins in three different animal models and in cultured hepatic cells. Our results demonstrate that high cholesterol feeding specifically elevates the hepatic expression of LDLR mRNA decay promoting factor heterogeneous nuclear ribonucleoprotein (HNRNP)D without affecting expressions of other LDLR mRNA binding proteins in vivo and in vitro. Employing the approach of adenovirus-mediated gene knockdown, we further show that depletion of HNRNPD in the liver results in a marked reduction of serum LDL-cholesterol and a substantial increase in liver LDLR expression in hyperlipidemic mice. Additional studies of gene knockdown in albumin-luciferase-untranslated region (UTR) transgenic mice provide strong evidence supporting the essential role of 3'UTR in HNRNPD-mediated LDLR mRNA degradation in liver tissue. Altogether, this work identifies a novel posttranscriptional regulatory mechanism by which dietary cholesterol inhibits liver LDLR expression via inducing HNRNPD to accelerate LDLR mRNA degradation.

Ubiquitination-mediated interaction among domains is responsible for inhibition of RNA endonuclease activity of mRNA cycling sequence binding protein from L. donovani (LdCSBP)

In nearly complete absence of transcriptional regulation, messenger RNA (mRNA) turnover mediated through specific cis-elements plays a predominant role in the control of differential gene expression for the disease causing trypanosomatid parasites. In these organisms, the periodic accumulation of S-phase messages during cell cycle is determined by the presence of one or more copies of a conserved CAUAGAAG octanucleotide motif in the untranslated regions of mRNAs. In our previous studies, a multi-domain cycling sequence binding protein LdCSBP from Leishmania donovani was characterized, which binds specifically to the octamer-containing RNAs via its uniquely arranged CCCH-type Zn fingers and degrades them through its small MutS-related (Smr) endonuclease domain, indicative of its potential role in the turnover of the S-phase mRNAs. Interestingly, the protein is modified by the incorporation of a monoubiquitin residue, and the posttranslational modification inhibits its riboendonuclease activity. However, the mechanism of such inhibition was previously unknown. Here, we establish that the CCCH-type Zn finger domain is the site of ubiquitination in LdCSBP and the interaction of CUE domain of the protein with the ubiquitinated Zn finger domain is responsible for inhibition of its riboendonuclease activity. The findings elucidate an inhibitory mechanism of RNA cleavage through ubiquitination-mediated intramolecular interaction among domains of the enzyme. Furthermore, the riboendonuclease activity is inhibited by anti-leishmanial drug paromomycin suggesting that the regulation of RNA metabolism could be a target of the drug.

Contributions of transcription and mRNA decay to gene expression dynamics of fission yeast in response to oxidative stress

The cooperation of transcriptional and post-transcriptional levels of control to shape gene regulation is only partially understood. Here we show that a combination of two simple and non-invasive genomic techniques, coupled with kinetic mathematical modeling, affords insight into the intricate dynamics of RNA regulation in response to oxidative stress in the fission yeast Schizosaccharomyces pombe. This study reveals a dominant role of transcriptional regulation in response to stress, but also points to the first minutes after stress induction as a critical time when the coordinated control of mRNA turnover can support the control of transcription for rapid gene regulation. In addition, we uncover specialized gene expression strategies associated with distinct functional gene groups, such as simultaneous transcriptional repression and mRNA destabilization for genes encoding ribosomal proteins, delayed mRNA destabilization with varying contribution of transcription for ribosome biogenesis genes, dominant roles of mRNA stabilization for genes functioning in protein degradation, and adjustment of both transcription and mRNA turnover during the adaptation to stress. We also show that genes regulated independently of the bZIP transcription factor Atf1p are predominantly controlled by mRNA turnover, and identify putative cis-regulatory sequences that are associated with different gene expression strategies during the stress response. This study highlights the intricate and multi-faceted interplay between transcription and RNA turnover during the dynamic regulatory response to stress.

PIWI proteins and their interactors in piRNA biogenesis, germline development and gene expression

PIWI-interacting RNAs (piRNAs) are a complex class of small non-coding RNAs that are mostly 24-32 nucleotides in length and composed of at least hundreds of thousands of species that specifically interact with the PIWI protein subfamily of the ARGONAUTE family. Recent studies revealed that PIWI proteins interact with a number of proteins, especially the TUDOR-domain-containing proteins, to regulate piRNA biogenesis and regulatory function. Current research also provides evidence that PIWI proteins and piRNAs are not only crucial for transposon silencing in the germline, but also mediate novel mechanisms of epigenetic programming, DNA rearrangements, mRNA turnover, and translational control both in the germline and in the soma. These new discoveries begin to reveal an exciting new dimension of gene regulation in the cell.

Special Topic: Frontiers in RNA Research: The ever-expanding RNA world.

RNA is perhaps the most mystical molecule in life. As a single molecule endowed with both informational and catalytic capacities, it was proposed by Alexander Rich 52 years ago to be the first molecule that self-replicated at the origin of life, to be taken over by DNA only later. Although this ‘RNA world’ (vividly phrased by Walter Gilbert in 1986) remains an enticing yet unproven hypothesis, the crucial roles of messenger RNAs (mRNAs), transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs) in transcribing and translating genetic information into proteins and, in turn, life processes have been indisputably established and extensively illustrated in the past half-century, as elegantly simplified in the central dogma of molecular biology. The discovery of microRNAs (miRNAs) and small interfering RNAs (siRNAs) since early 1993 further enriched the dogma by revealing that these two classes of small non-coding RNAs mediate new mechanisms that regulate the turnover and translation of mRNAs. In the past 10 years, the advent of the DNA deep-sequencing technology further allowed the discovery of an even larger RNA world full of various types of novel non-coding RNAs, such asmillions of Piwi-interacting RNAs (piRNAs) and thousands of long non-coding RNAs (lncRNAs).

The structure of the Pan2-Pan3 core complex reveals cross-talk between deadenylase and pseudokinase.

Pan2-Pan3 is a conserved complex involved in the shortening of mRNA poly(A) tails, the initial step in eukaryotic mRNA turnover. We show that recombinant Saccharomyces cerevisiae Pan2-Pan3 can deadenylate RNAs in vitro without needing the poly(A)-binding protein Pab1. The crystal structure of an active ~200-kDa core complex reveals that Pan2 and Pan3 interact with an unusual 1:2 stoichiometry imparted by the asymmetric nature of the Pan3 homodimer. An extended region of Pan2 wraps around Pan3 and provides a major anchoring point for complex assembly. A Pan2 module formed by the pseudoubiquitin-hydrolase and RNase domains latches onto the Pan3 pseudokinase with intertwined interactions that orient the deadenylase active site toward the A-binding site of the interacting Pan3. The molecular architecture of Pan2-Pan3 suggests how the nuclease and its pseudokinase regulator act in synergy to promote deadenylation.

A dual luciferase system for analysis of post-transcriptional regulation of gene expression in Leishmania

Gene expression in kinetoplastid parasites is regulated via post-transcriptional mechanisms that modulate mRNA turnover, translation rate, and/or post-translational protein stability. To facilitate the analysis of post-transcriptional regulation, a dual luciferase system was developed in which firefly and Renilla luciferase reporters genetically fused to compatible drug resistance genes are integrated in place of one allele of the gene of interest and of an internal control gene, respectively, in a manner that preserves the cognate pre-mRNA processing signals. The sensitivity and reproducibility of the assay coupled with the ability to rapidly assemble reporter integration constructs render the dual luciferase system suitable for analysis of multiple candidates derived from global expression analysis platforms. To demonstrate the utility of the system, regulation of three genes in response to purine starvation was examined in Leishmania donovani promastigotes. This dual luciferase system should be directly applicable to the analysis of post-transcriptional regulation in other kinetoplastids.

Post-Transcriptional Regulation of Interferons and Their Signaling Pathways

Interferons (IFNs) are low molecular weight cell-derived proteins that include the type I, II, and III IFN families. IFNs are critical for an optimal immune response during microbial infections while dysregulated expression can lead to autoimmune diseases. Given its role in disease, it is important to understand cellular mechanisms of IFN regulation. 3' untranslated regions (3' UTRs) have emerged as potent regulators of mRNA and protein dosage and are controlled through multiple regulatory elements including adenylate uridylate (AU)-rich elements (AREs) and microRNA (miRNA) recognition elements. These AREs are targeted by RNA-binding proteins (ARE-BPs) for degradation and/or stabilization through an ARE-mediated decay process. miRNA are endogenous, single-stranded RNA molecules ~22 nucleotides in length that regulate mRNA translation through the miRNA-induced silencing complex. IFN transcripts, like other labile mRNAs, harbor AREs in their 3' UTRs that dictate the turnover of mRNA. This review is a survey of the literature related to IFN regulation by miRNA, ARE-BPs, and how these complexes interact dynamically on the 3' UTR. Additionally, downstream effects of these post-transcriptional regulators on the immune response will be discussed. Review topics include past studies, current understanding, and future challenges in the study of post-transcriptional regulation affecting IFN responses.

Hypertonicity Compromises Renal Mineralocorticoid Receptor Signaling through Tis11b-Mediated Post-Transcriptional Control

The mineralocorticoid receptor (MR) mediates the Na(+)-retaining action of aldosterone. MR is highly expressed in the distal nephron, which is submitted to intense variations in extracellular fluid tonicity generated by the corticopapillary gradient. We previously showed that post-transcriptional events control renal MR abundance. Here, we report that hypertonicity increases expression of the mRNA-destabilizing protein Tis11b, a member of the tristetraprolin/ZFP36 family, and thereby, decreases MR expression in renal KC3AC1 cells. The 3'-untranslated regions (3'-UTRs) of human and mouse MR mRNA, containing several highly conserved adenylate/uridylate-rich elements (AREs), were cloned downstream of a reporter gene. Luciferase activities of full-length or truncated MR Luc-3'-UTR mutants decreased drastically when cotransfected with Tis11b plasmid, correlating with an approximately 50% shorter half-life of ARE-containing transcripts. Using site-directed mutagenesis and RNA immunoprecipitation, we identified a crucial ARE motif within the MR 3'-UTR, to which Tis11b must bind for destabilizing activity. Coimmunoprecipitation experiments suggested that endogenous Tis11b physically interacts with MR mRNA in KC3AC1 cells, and Tis11b knockdown prevented hypertonicity-elicited repression of MR. Moreover, hypertonicity blunted aldosterone-stimulated expression of glucocorticoid-induced leucine-zipper protein and the α-subunit of the epithelial Na(+) channel, supporting impaired MR signaling. Challenging the renal osmotic gradient by submitting mice to water deprivation, diuretic administration, or high-Na(+) diet increased renal Tis11b and decreased MR expression, particularly in the cortex, thus establishing a mechanistic pathway for osmotic regulation of MR expression in vivo. Altogether, we uncovered a mechanism by which renal MR expression is regulated through mRNA turnover, a post-transcriptional control that seems physiologically relevant.

Phosphorylation‐independent recruitment of 14‐3‐3 alters tristetraprolin function (748.3)

Cell signaling pathways influence mRNA turnover rates through reversible post‐translational modifications that alter the activity of a number of mRNA binding proteins. Tristetraprolin (TTP) is a zinc‐finger RNA binding protein that binds to AU‐rich elements (AREs) in the 3’ untranslated regions (UTRs) of transiently expressed messages encoding cytokines and pro‐inflammatory proteins. Unphosphorylated TTP causes rapid mRNA turnover by recruiting mRNA decay enzymes, but phosphorylation of TTP at serines 52 and 178 by the p38 MAPK activated kinase MK2 prevents its ability to do so. Phosphorylation also leads to the recruitment of 14‐3‐3, is associated with alterations in the localization of TTP, and may protect TTP from dephosphorylation and destruction by the proteasome. However, because mutations that destroy 14‐3‐3 binding sites in TTP also prevent its phosphorylation, it is unclear whether 14‐3‐3 binding is required to inhibit TTP function. To better understand the role of 14‐3‐3 in the inhibition of TTP we replaced serines 52 and 178 in TTP via site‐directed mutagenesis with either a peptide sequence known to bind 14‐3‐3 independently of phosphorylation or with an altered version of this peptide sequence that does not. We tested the ability of these mutant forms of TTP to recruit 14‐3‐3 using co‐immunoprecipitation assays from transiently transfected Human Embryonic Kidney (HEK) 293T cell lysates. We observed that protein levels for mutant forms of TTP that failed to associate with 14‐3‐3 were consistently lower than versions of TTP that retained the ability to do so, supporting the hypothesis that 14‐3‐3 may protect TTP from destruction by the proteasome. We also observed that recruitment of 14‐3‐3 was sufficient to alter localization of TTP from the nucleus to the cytoplasm. Experiments are currently underway to determine whether 14‐3‐3 association is sufficient to alter the recruitment of mRNA decay enzymes and the protein phosphatase PP2A.

Regulation of long noncoding RNA by RCK/p54 (LB205)

RCK/p54, or DDX6, is a member of the conserved DEAD‐box RNA helicase family, and is encoded by the DDX6 gene in human. Early studies of Dhh1, the yeast homolog of RCK/p54, have shown that that Dhh1 is a core component of cellular decapping machinery and a general translational repressor. In human cells, it has been shown that that RCK/p54 is essential for both miRNA‐mediated and AU‐Rich‐Element‐dependent translational repression. In addition, depletion of RCK/p54 disrupts the formation of P‐bodies, the putative foci of mRNA decay and storage. These results suggest that RCK/p54 is required for translational repression and mRNA turnover. Studies in C.elegans, Drosophila, and Xenopus have also shown that DDX6 homologs function in storage of maternal messages during early development. These findings implicate that RCK/p54 participates in multiple post‐transcriptional gene modulation mechanisms.Long noncoding RNAs (lncRNAs) has recently emerged as essential players in the control of cell fate and the regulation cellular physiology. Extensive studies have been launched to decode the functions of lncRNAs, but fewer studies described the regulation of lncRNAs to date. In this study, we tried to investigate whether lncRNAs, like mRNAs, are subjects to regulation by RCK/p54. Here, we combine RCK/p54 RIP‐seq and RCK/p54‐depletion RNA‐seq for an integrative analysis. Our result provides evidences that RCK/p54 directly interacts with lncRNAs and regulates their function.

Characterizing the interaction between protein phosphatase 2A and the mRNA decay protein Tristetraprolin (748.4)

Post‐transcriptional and post‐translational processes play key roles in regulating rapid changes in gene expression by altering the activity of mRNA binding proteins on their target transcripts. The zinc‐finger mRNA binding protein TTP activates the destruction of mRNAs encoding cytokines, growth factors, and pro‐inflammatory proteins by binding to AU‐rich elements (AREs) located within the 3’ untranslated region (UTR) and recruiting mRNA decay enzymes to these targets. In response to stimuli such as a bacterial infection, the p38 MAPK activated kinase MK2 phosphorylates TTP and reduces its ability to stimulate mRNA turnover by blocking decay enzyme association. While phosphorylation of TTP has been well‐characterized, much less is known about how this process is reversed to ensure a timely but transient burst of gene expression. In order to better understand the mechanism by which TTP is dephosphorylated, we observed both the dephosphorylation status of TTP as well as PP2A heterotrimer protein levels in lipopolysaccharide (LPS) stimulated RAW 264.7 murine macrophage cells via Western blot analysis. TTP levels were highest 12 hours after LPS exposure, with dephosphorylation beginning approximately16 hours after LPS induction. Interestingly, PP2A‐C levels increased approximately five‐fold during TTP dephosphorylation. These results suggest that dephoshporylation may be regulated in part by modulation of PP2A levels. In order to better understand the mechanism by which PP2A gains access to dephosphorylate and reactivate TTP, we are currently testing the ability of TTP to associate with PP2A in both macrophages as well as in human embryonic kidney (HEK 293) cells stably transfected with wild‐type or deletion mutant forms of TTP. The results of these experiments will provide a greater understanding of the sequence of events that attenuate inflammatory responses by dephosphorylating TTP and reactivating mRNA decay.

Posttranscriptional regulation of tristetraprolin and tumor necrosis factor through a two‐compartment model (902.12)

TNF is a pro‐inflammatory cytokine that influences a broad range of immunological responses, including chronic inflammation of the gastrointestinal tract. Prolonged inflammation of gastrointestinal tract is linked with inflammatory bowel disease. TNF biosynthesis is regulated both at transcription and posttranscriptional levels. However, the stimulation‐induced increase in translation rate is much larger. This might indicate the possibility of a posttranscriptional regulatory mechanism. How, during basal conditions, is the free concentration of TNF tightly regulated at low levels in cytosol? The stability and translational efficiency of TNF transcript are regulated by an AU‐rich element (ARE) in the 3′‐UTR of messenger RNA. A transacting protein, TTP, binds to ARE and enhances the mRNA turnover. Here, we examine a proposal that TNF homeostasis is regulated by a two‐compartment interaction loop between TTP and TNF. We propose a computational framework of this regulatory loop by modeling the shuttling of TTP between nuclear and cytosolic compartments. This two‐compartment regulatory loop between TTP and TNF is based on activation, deactivation, and translocation/re‐translocation cycle of TTP protein. The mutual interaction of these two proteins regulates the biosynthesis response of both TTP and TNF during basal and inflammatory conditions.

Physiological networks and disease functions of RNA‐binding protein AUF1

Regulated messenger RNA (mRNA) decay is an essential mechanism that governs proper control of gene expression. In fact, many of the most physiologically potent proteins are encoded by short-lived mRNAs, many of which contain AU-rich elements (AREs) in their 3'-untranslated region (3'-UTR). AREs target mRNAs for post-transcriptional regulation, generally rapid decay, but also stabilization and translation inhibition. AREs control mRNA turnover and translation activities through association with trans-acting RNA-binding proteins that display high affinity for these AU-rich regulatory elements. AU-rich element RNA-binding protein (AUF1), also known as heterogeneous nuclear ribonucleoprotein D (HNRNPD), is an extensively studied AU-rich binding protein (AUBP). AUF1 has been shown to regulate ARE-mRNA turnover, primarily functioning to promote rapid ARE-mRNA degradation. In certain cellular contexts, AUF1 has also been shown to regulate gene expression at the translational and even the transcriptional level. AUF1 comprises a family of four related protein isoforms derived from a common pre-mRNA by differential exon splicing. AUF1 isoforms have been shown to display multiple and distinct functions that include the ability to target ARE-mRNA stability or decay, and transcriptional activation of certain genes that is controlled by their differential subcellular locations, expression levels, and post-translational modifications. AUF1 has been implicated in controlling a variety of physiological functions through its ability to regulate the expression of numerous mRNAs containing 3'-UTR AREs, thereby coordinating functionally related pathways. This review highlights the physiological functions of AUF1-mediated regulation of mRNA and gene expression, and the consequences of deficient AUF1 levels in different physiological settings.

A eukaryotic-like 3′ untranslated region in Salmonella enterica hilD mRNA

Long 3′ untranslated regions (3′UTRs) are common in eukaryotic mRNAs. In contrast, long 3′UTRs are rare in bacteria, and have not been characterized in detail. We describe a 3′UTR of 310 nucleotides in hilD mRNA, a transcript that encodes a transcriptional activator of Salmonella enterica pathogenicity island 1 (SPI-1). Deletion of the hilD 3′UTR increases the hilD mRNA level, suggesting that the hilD 3′UTR may play a role in hilD mRNA turnover. Cloning of the hilD 3′UTR downstream of the green fluorescent protein (gfp) gene decreases green fluorescent protein (GFP) activity in both Escherichia coli and S. enterica, indicating that the hilD 3′UTR can act as an independent module. S. enterica mutants lacking either ribonuclease E or polynucleotide phosphorylase contain similar amounts of hilD and hilD Δ3′UTR mRNAs, suggesting that the hilD 3′UTR is a target for hilD mRNA degradation by the degradosome. The hilD 3′UTR is also necessary for modulation of hilD and SPI-1 expression by the RNA chaperone Hfq. Overexpression of SPI-1 in the absence of the hilD 3′UTR retards Salmonella growth and causes uncontrolled invasion of epithelial cells. Based on these observations, we propose that the S. enterica hilD 3′UTR is a cis-acting element that contributes to cellular homeostasis by promoting hilD mRNA turnover.

Is there quality control of localized mRNAs?

In eukaryotic cells many mRNAs are localized to specific regions of the cytosol, thereby allowing the local production of proteins. The process of mRNA localization can be coordinated with mRNA turnover, which can also be spatially controlled to increase the degree of mRNA localization. The coordination of mRNA localization, translation repression during transport, and mRNA degradation suggests the hypothesis that an additional layer of mRNA quality control exists in cells to degrade mRNAs that fail to be appropriately localized.

TAIL-seq: Genome-wide Determination of Poly(A) Tail Length and 3′ End Modifications

Global investigation of the 3' extremity of mRNA (3'-terminome), despite its importance in gene regulation, has not been feasible due to technical challenges associated with homopolymeric sequences and relative paucity of mRNA. We here develop a method, TAIL-seq, to sequence the very end of mRNA molecules. TAIL-seq allows us to measure poly(A) tail length at the genomic scale. Median poly(A) length is 50-100 nt in HeLa and NIH 3T3 cells.Poly(A) length correlates with mRNA half-life, but not with translational efficiency. Surprisingly, we discover widespread uridylation and guanylation at the downstream of poly(A) tail. The U tails are generally attached to short poly(A) tails (<25 nt), while the Gtails are found mainly on longer poly(A) tails (>40nt), implicating their generic roles in mRNA stability control. TAIL-seq is a potent tool to dissect dynamic control of mRNA turnover and translational control, and to discover unforeseen features of RNA cleavage and tailing.

Key Role of MicroRNA in the Regulation of Granulocyte Macrophage Colony-stimulating Factor Expression in Murine Alveolar Epithelial Cells during Oxidative Stress

GM-CSF is an endogenous pulmonary cytokine produced by normal alveolar epithelial cells (AEC) that is a key defender of the alveolar space. AEC GM-CSF expression is suppressed by oxidative stress through alternations in mRNA turnover, an effect that is reversed by treatment with recombinant GM-CSF. We hypothesized that specific microRNA (miRNA) would play a key role in AEC GM-CSF regulation. A genome-wide miRNA microarray identified 19 candidate miRNA altered in primary AEC during oxidative stress with reversal by treatment with GM-CSF. Three of these miRNA (miR 133a, miR 133a*, and miR 133b) are also predicted to bind the GM-CSF 3'-untranslated region (UTR). PCR for the mature miRNA confirmed induction during oxidative stress that was reversed by treatment with GM-CSF. Experiments using a GM-CSF 3'-UTR reporter construct demonstrated that miR133a and miR133b effects on GM-CSF expression are through interactions with the GM-CSF 3'-UTR. Using lentiviral transduction of specific mimics and inhibitors in primary murine AEC, we determined that miR133a and miR133b suppress GM-CSF expression and that their inhibition both reverses oxidant-induced suppression of GM-CSF expression and increases basal expression of GM-CSF in cells in normoxia. In contrast, these miRNAs are not active in regulation of GM-CSF expression in murine EL4 T cells. Thus, members of the miR133 family play key roles in regulation of GM-CSF expression through effects on mRNA turnover in AEC during oxidative stress. Increased understanding of GM-CSF gene regulation may provide novel miRNA-based interventions to augment pulmonary innate immune defense in lung injury.

Inhibition of Caspases Protects Mice from Radiation-induced Oral Mucositis and Abolishes the Cleavage of RNA-binding Protein HuR

The oral mucosal epithelium is typically insulted during chemotherapy and ionizing radiation (IR) therapy and disposed to mucositis, which creates painful inflammation and ulceration in the oral cavity. Oral mucositis alters gene expression patterns, inhibits cellular growth, and initiates cell death in the oral epithelial compartments. Such alterations are governed by several different factors, including transcription factors, RNA-binding proteins, and microRNAs. IR-induced post-transcriptional regulation of RNA-binding proteins exists but is poorly studied in clinically relevant settings. We herein report that the RNA-binding protein human antigen R (HuR) undergoes cleavage modification by caspase-3 following IR-induced oral mucositis and subsequently promotes the expression of the pro-apoptotic factor BAX (Bcl-2-associated X protein), as well as cell death. Further analyses revealed that the HuR cleavage product-1 (HuR-CP1) directly associates and stabilizes the BAX mRNA and concurrently activates the apoptotic pathway. On the other hand, a noncleavable isoform of HuR promotes the clonogenic capacity of primary oral keratinocytes and decreases the effect of IR-induced cell death. Additionally, specific inhibition of caspase-3 by a compound, NSC321205, increases the clonogenic capacity of primary oral keratinocytes and causes increased basal layer cellularity, thickened mucosa, and elevated epithelial cell growth in the tongues of mice with oral mucositis. This protective effect of NSC321205 is mediated by a decrease in caspase-3 activity and the consequent inhibition of HuR cleavage, which reduces the expression of BAX in mice with IR-induced oral mucositis. Thus, we have identified a new molecular mechanism of HuR in the regulation of mRNA turnover and apoptosis in oral mucositis, and our data suggest that blocking the cleavage of HuR enhances cellular growth in the oral epithelial compartment.