Along the Central Dogma-Controlling Gene Expression with Small Molecules.

The central dogma of molecular biology, that DNA is transcribed into RNA and RNA translated into protein, was coined in the early days of modern biology. Back in the 1950s and 1960s, bacterial genetics first opened the way toward understanding life as the genetically encoded interaction of macromolecules. As molecular biology progressed and our knowledge of gene control deepened, it became increasingly clear that expression relied on many more levels of regulation. In the process of dissecting mechanisms of gene expression, specific small-molecule inhibitors played an important role and became valuable tools of investigation. Small molecules offer significant advantages over genetic tools, as they allow inhibiting a process at any desired time point, whereas mutating or altering the gene of an important regulator would likely result in a dead organism. With the advent of modern sequencing technology, it has become possible to monitor global cellular effects of small-molecule treatment and thereby overcome the limitations of classical biochemistry, which usually looks at a biological system in isolation. This review focuses on several molecules, especially natural products, that have played an important role in dissecting gene expression and have opened up new fields of investigation as well as clinical venues for disease treatment.

PNPT1 Release from Mitochondria during Apoptosis Triggers Decay of Poly(A) RNAs.

Widespread mRNA decay, an unappreciated feature of apoptosis, enhances cell death and depends on mitochondrial outer membrane permeabilization (MOMP), TUTases, and DIS3L2. Which RNAs are decayed and the decay-initiating event are unknown. Here, we show extensive decay of mRNAs and poly(A) noncoding (nc)RNAs at the 3' end, triggered by the mitochondrial intermembrane space 3'-to-5' exoribonuclease PNPT1, released during MOMP. PNPT1 knockdown inhibits apoptotic RNA decay and reduces apoptosis, while ectopic expression of PNPT1, but not an RNase-deficient mutant, increases RNA decay and cell death. The 3' end of PNPT1 substrates thread through a narrow channel. Many non-poly(A) ncRNAs contain 3'-secondary structures or bind proteins that may block PNPT1 activity. Indeed, mutations that disrupt the 3'-stem-loop of a decay-resistant ncRNA render the transcript susceptible, while adding a 3'-stem-loop to an mRNA prevents its decay. Thus, PNPT1 release from mitochondria during MOMP initiates apoptotic decay of RNAs lacking 3'-structures.

Iron Drives T Helper Cell Pathogenicity by Promoting RNA-Binding Protein PCBP1-Mediated Proinflammatory Cytokine Production.

Iron deposition is frequently observed in human autoinflammatory diseases, but its functional significance is largely unknown. Here we showed that iron promoted proinflammatory cytokine expression in T cells, including GM-CSF and IL-2, via regulating the stability of an RNA-binding protein PCBP1. Iron depletion or Pcbp1 deficiency in T cells inhibited GM-CSF production by attenuating Csf2 3' untranslated region (UTR) activity and messenger RNA stability. Pcbp1 deficiency or iron uptake blockade in autoreactive T cells abolished their capacity to induce experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. Mechanistically, intracellular iron protected PCBP1 protein from caspase-mediated proteolysis, and PCBP1 promoted messenger RNA stability of Csf2 and Il2 by recognizing UC-rich elements in the 3' UTRs. Our study suggests that iron accumulation can precipitate autoimmune diseases by promoting proinflammatory cytokine production. RNA-binding protein-mediated iron sensing may represent a simple yet effective means to adjust the inflammatory response to tissue homeostatic alterations.

PD-L1 (B7-H1) Competes with the RNA Exosome to Regulate the DNA Damage Response and Can Be Targeted to Sensitize to Radiation or Chemotherapy.

Programmed death ligand 1 (PD-L1, also called B7-H1) is an immune checkpoint protein that inhibits immune function through its binding of the programmed cell death protein 1 (PD-1) receptor. Clinically approved antibodies block extracellular PD-1 and PD-L1 binding, yet the role of intracellular PD-L1 in cancer remains poorly understood. Here, we discovered that intracellular PD-L1 acts as an RNA binding protein that regulates the mRNA stability of NBS1, BRCA1, and other DNA damage-related genes. Through competition with the RNA exosome, intracellular PD-L1 protects targeted RNAs from degradation, thereby increasing cellular resistance to DNA damage. RNA immunoprecipitation and RNA-seq experiments demonstrated that PD-L1 regulates RNA stability genome-wide. Furthermore, we developed a PD-L1 antibody, H1A, which abrogates the interaction of PD-L1 with CMTM6, thereby promoting PD-L1 degradation. Intracellular PD-L1 may be a potential therapeutic target to enhance the efficacy of radiotherapy and chemotherapy in cancer through the inhibition of DNA damage response and repair.

Fluoropyrimidines trigger decay of hypomodified tRNA in yeast.

Therapeutic fluoropyrimidines 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC) are in long use for treatment of human cancers and severe invasive fungal infections, respectively. 5-Fluorouridine triphosphate represents a bioactive metabolite of both drugs and is incorporated into target cells' RNA. Here we use the model fungus Saccharomyces cerevisiae to define fluorinated tRNA as a key mediator of 5-FU and 5-FC cytotoxicity when specific tRNA methylations are absent. tRNA methylation deficiency caused by loss of Trm4 and Trm8 was previously shown to trigger an RNA quality control mechanism resulting in partial destabilization of hypomodified tRNAValAAC. We demonstrate that, following incorporation into tRNA, fluoropyrimidines strongly enhance degradation of yeast tRNAValAAC lacking Trm4 and Trm8 dependent methylations. At elevated temperature, such effect occurs already in absence of Trm8 alone. Genetic approaches and quantification of tRNA modification levels reveal that enhanced fluoropyrimidine cytotoxicity results from additional, drug induced uridine modification loss and activation of tRNAValAAC decay involving the exonuclease Xrn1. These results suggest that inhibition of tRNA methylation may be exploited to boost therapeutic efficiency of 5-FU and 5-FC.

Collection and transportation of SARS-CoV-2 and influenza A virus diagnostic samples: Optimizing the usage of guanidine-based chaotropic salts for enhanced biosafety and viral genome preservation.

Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.

IGF2BP3 stabilizes SESN1 mRNA to mitigate oxidized low-density lipoprotein-induced oxidative stress and endothelial dysfunction in human umbilical vein endothelial cells by activating Nrf2 signaling.

Atherosclerosis (AS) represents a prevalent initiating factor for cardiovascular events. Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) is an oncofetal RNA-binding protein that participates in cardiovascular diseases. This work aimed to elaborate the effects of IGF2BP3 on AS and the probable mechanism by using an oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) model. Results indicated that IGF2BP3 expression was declined in the blood of AS patients and ox-LDL-induced HUVECs. IGF2BP3 elevation alleviated ox-LDL-provoked viability loss, apoptosis, oxidative DNA damage and endothelial dysfunction in HUVECs. Moreover, IGF2BP3 bound SESN1 and stabilized SESN1 mRNA. Furthermore, SESN1 interference reversed the impacts of IGF2BP3 overexpression on the apoptosis, oxidative DNA damage and endothelial dysfunction of ox-LDL-challenged HUVECs. Additionally, the activation of Nrf2 signaling mediated by IGF2BP3 up-regulation in ox-LDL-treated HUVECs was blocked by SESN1 absence. Collectively, SESN1 stabilized by IGF2BP3 might protect against AS by activating Nrf2 signaling.

Targeted i6A-RNA degradation through sequential Fluorination-Azidation and Click reaction with imidazole-based probes.

We report the use of trimethylsilyl azide and Selectfluor to implement a standard protocol targeted at the prenylated nucleic acid known as i6A-RNA. After optimizing the conditions, we applied this method to regulate a wide range of i6A-RNA species using synthetic imidazole-based probes (I-IV). We observed that prenylated nucleic acid plays a crucial role in the cell hemostasis in A549 cell lines.

Deoxynivalenol induces m6A-mediated upregulation of p21 and growth arrest of mouse hippocampal neuron cells in vitro.

Hippocampal neurons maintain the ability of proliferation throughout life to support neurogenesis. Deoxynivalenol (DON) is a mycotoxin that exhibits brain toxicity, yet whether and how DON affects hippocampal neurogenesis remains unknown. Here, we use mouse hippocampal neuron cells (HT-22) as a model to illustrate the effects of DON on neuron proliferation and to explore underlying mechanisms. DON exposure significantly inhibits the proliferation of HT-22 cells, which is associated with an up-regulation of cell cycle inhibitor p21 at both mRNA and protein levels. Global and site-specific m6A methylation levels on the 3'UTR of p21 mRNA are significantly increased in response to DON treatment, whereas inhibition of m6A hypermethylation significantly alleviates DON-induced cell cycle arrest. Further mechanistic studies indicate that the m6A readers YTHDF1 and IGF2BP1 are responsible for m6A-mediated increase in p21 mRNA stability. Meanwhile, 3'UTR of E3 ubiquitin ligase TRIM21 mRNA is also m6A hypermethylated, and another m6A reader YTHDF2 binds to the m6A sites, leading to decreased TRIM21 mRNA stability. Consequently, TRIM21 suppression impairs ubiquitin-mediated p21 protein degradation. Taken together, m6A-mediated upregulation of p21, at both post-transcriptional and post-translational levels, contributes to DON-induced inhibition of hippocampal neuron proliferation. These results may provide new insights for epigenetic therapy of neurodegenerative diseases.

K63-Ubiquitylation and TRAF6 Pathways Regulate Mammalian P-Body Formation and mRNA Decapping.

Signals and posttranslational modifications regulating the decapping step in mRNA degradation pathways are poorly defined. In this study we reveal the importance of K63-linked ubiquitylation for the assembly of decapping factors, P-body formation, and constitutive decay of instable mRNAs encoding mediators of inflammation by various experimental approaches. K63-branched ubiquitin chains also regulate IL-1-inducible phosphorylation of the P-body component DCP1a. The E3 ligase TRAF6 binds to DCP1a and indirectly regulates DCP1a phosphorylation, expression of decapping factors, and gene-specific mRNA decay. Mutation of six C-terminal lysines of DCP1a suppresses decapping activity and impairs the interaction with the mRNA decay factors DCP2, EDC4, and XRN1, but not EDC3, thus remodeling P-body architecture. The usage of ubiquitin chains for the proper assembly and function of the decay-competent mammalian decapping complex suggests an additional layer of control to allow a coordinated function of decapping activities and mRNA metabolism in higher eukaryotes.

Engineered Covalent Inactivation of TFIIH-Kinase Reveals an Elongation Checkpoint and Results in Widespread mRNA Stabilization.

During transcription initiation, the TFIIH-kinase Kin28/Cdk7 marks RNA polymerase II (Pol II) by phosphorylating the C-terminal domain (CTD) of its largest subunit. Here we describe a structure-guided chemical approach to covalently and specifically inactivate Kin28 kinase activity in vivo. This method of irreversible inactivation recapitulates both the lethal phenotype and the key molecular signatures that result from genetically disrupting Kin28 function in vivo. Inactivating Kin28 impacts promoter release to differing degrees and reveals a "checkpoint" during the transition to productive elongation. While promoter-proximal pausing is not observed in budding yeast, inhibition of Kin28 attenuates elongation-licensing signals, resulting in Pol II accumulation at the +2 nucleosome and reduced transition to productive elongation. Furthermore, upon inhibition, global stabilization of mRNA masks different degrees of reduction in nascent transcription. This study resolves long-standing controversies on the role of Kin28 in transcription and provides a rational approach to irreversibly inhibit other kinases in vivo.

Noncanonical immune response to the inhibition of DNA methylation by Staufen1 via stabilization of endogenous retrovirus RNAs.

DNA-methyltransferase inhibitors (DNMTis), such as azacitidine and decitabine, are used clinically to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Decitabine activates the transcription of endogenous retroviruses (ERVs), which can induce immune response by acting as cellular double-stranded RNAs (dsRNAs). Yet, the posttranscriptional regulation of ERV dsRNAs remains uninvestigated. Here, we find that the viral mimicry and subsequent cell death in response to decitabine require the dsRNA-binding protein Staufen1 (Stau1). We show that Stau1 directly binds to ERV RNAs and stabilizes them in a genome-wide manner. Furthermore, Stau1-mediated stabilization requires a long noncoding RNA TINCR, which enhances the interaction between Stau1 and ERV RNAs. Analysis of a clinical patient cohort reveals that MDS and AML patients with lower Stau1 and TINCR expressions exhibit inferior treatment outcomes to DNMTi therapy. Overall, our study reveals the posttranscriptional regulatory mechanism of ERVs and identifies the Stau1-TINCR complex as a potential target for predicting the efficacy of DNMTis and other drugs that rely on dsRNAs.

The inducible kinase IKKi is required for IL-17-dependent signaling associated with neutrophilia and pulmonary inflammation.

Interleukin 17 (IL-17) is critical in the pathogenesis of inflammatory and autoimmune diseases. Here we report that Act1, the key adaptor for the IL-17 receptor (IL-7R), formed a complex with the inducible kinase IKKi after stimulation with IL-17. Through the use of IKKi-deficient mice, we found that IKKi was required for IL-17-induced expression of genes encoding inflammatory molecules in primary airway epithelial cells, neutrophilia and pulmonary inflammation. IKKi deficiency abolished IL-17-induced formation of the complex of Act1 and the adaptors TRAF2 and TRAF5, activation of mitogen-activated protein kinases (MAPKs) and mRNA stability, whereas the Act1-TRAF6-transcription factor NF-κB axis was retained. IKKi was required for IL-17-induced phosphorylation of Act1 on Ser311, adjacent to a putative TRAF-binding motif. Substitution of the serine at position 311 with alanine impaired the IL-17-mediated Act1-TRAF2-TRAF5 interaction and gene expression. Thus, IKKi is a kinase newly identified as modulating IL-17 signaling through its effect on Act1 phosphorylation and consequent function.

Treatment with IL-17 prolongs the half-life of chemokine CXCL1 mRNA via the adaptor TRAF5 and the splicing-regulatory factor SF2 (ASF).

Interleukin 17 (IL-17) promotes the expression of chemokines and cytokines via the induction of gene transcription and post-transcriptional stabilization of mRNA. We show here that IL-17 enhanced the stability of chemokine CXCL1 mRNA and other mRNAs through a pathway that involved the adaptor Act1, the adaptors TRAF2 or TRAF5 and the splicing factor SF2 (also known as alternative splicing factor (ASF)). TRAF2 and TRAF5 were necessary for IL-17 to signal the stabilization of CXCL1 mRNA. Furthermore, IL-17 promoted the formation of complexes of TRAF5-TRAF2, Act1 and SF2 (ASF). Overexpression of SF2 (ASF) shortened the half-life of CXCL1 mRNA, whereas depletion of SF2 (ASF) prolonged it. SF2 (ASF) bound chemokine mRNA in unstimulated cells, whereas the SF2 (ASF)-mRNA interaction was much lower after stimulation with IL-17. Our findings define an IL-17-induced signaling pathway that links to the stabilization of selected mRNA species through Act1, TRAF2-TRAF5 and the RNA-binding protein SF2 (ASF).

Global RNA profiles show target selectivity and physiological effects of peptide-delivered antisense antibiotics.

Antisense peptide nucleic acids (PNAs) inhibiting mRNAs of essential genes provide a straight-forward way to repurpose our knowledge of bacterial regulatory RNAs for development of programmable species-specific antibiotics. While there is ample proof of PNA efficacy, their target selectivity and impact on bacterial physiology are poorly understood. Moreover, while antibacterial PNAs are typically designed to block mRNA translation, effects on target mRNA levels are not well-investigated. Here, we pioneer the use of global RNA-seq analysis to decipher PNA activity in a transcriptome-wide manner. We find that PNA-based antisense oligomer conjugates robustly decrease mRNA levels of the widely-used target gene, acpP, in Salmonella enterica, with limited off-target effects. Systematic analysis of several different PNA-carrier peptides attached not only shows different bactericidal efficiency, but also activation of stress pathways. In particular, KFF-, RXR- and Tat-PNA conjugates especially induce the PhoP/Q response, whereas the latter two additionally trigger several distinct pathways. We show that constitutive activation of the PhoP/Q response can lead to Tat-PNA resistance, illustrating the utility of RNA-seq for understanding PNA antibacterial activity. In sum, our study establishes an experimental framework for the design and assessment of PNA antimicrobials in the long-term quest to use these for precision editing of microbiota.

Contributions and competition of Mg2+ and K+ in folding and stabilization of the Twister ribozyme.

Native folded and compact intermediate states of RNA typically involve tertiary structures in the presence of divalent ions such as Mg2+ in a background of monovalent ions. In a recent study, we have shown how the presence of Mg2+ impacts the transition from partially unfolded to folded states through a "push-pull" mechanism where the ion both favors and disfavors the sampling of specific phosphate-phosphate interactions. To further understand the ion atmosphere of RNA in folded and partially folded states results from atomistic umbrella sampling and oscillating chemical potential grand canonical Monte Carlo/molecular dynamics (GCMC/MD) simulations are used to obtain atomic-level details of the distributions of Mg2+ and K+ ions around Twister RNA. Results show the presence of 100 mM Mg2+ to lead to increased charge neutralization over that predicted by counterion condensation theory. Upon going from partially unfolded to folded states, overall charge neutralization increases at all studied ion concentrations that, while associated with an increase in the number of direct ion-phosphate interactions, is fully accounted for by the monovalent K+ ions. Furthermore, K+ preferentially interacts with purine N7 atoms of helical regions in partially unfolded states, thereby potentially stabilizing the helical regions. Thus, both secondary helical structures and formation of tertiary structures leads to increased counterion condensation, thereby stabilizing those structural features of Twister. Notably, it is shown that K+ can act as a surrogate for Mg2+ by participating in specific interactions with nonsequential phosphate pairs that occur in the folded state, explaining the ability of Twister to self-cleave at submillimolar Mg2+ concentrations.

Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast.

Assessing variations in mRNA stability typically involves inhibiting transcription either globally or in a gene-specific manner. Alternatively, mRNA pulse-labeling strategies offer a means to calculate mRNA stability without inhibiting transcription. However, key stress-responsive cell signaling pathways, which affect mRNA stability, may themselves be perturbed by the approaches used to measure mRNA stability, leading to artifactual results. Here, we have focused on common strategies to measure mRNA half-lives in yeast and determined that commonly used transcription inhibitors thiolutin and 1,10 phenanthroline inhibit TORC1 signaling, PKC signaling, and partially activate HOG signaling. Additionally, 4-thiouracil (4tU), a uracil analog used in mRNA pulse-labeling approaches, modestly induces P-bodies, mRNA-protein granules implicated in storage and decay of nontranslating mRNA. Thiolutin also induces P-bodies, whereas phenanthroline has no effect. Doxycycline, which controls "Tet On/Tet Off" regulatable promoters, shows no impact on the above signaling pathways or P-bodies. In summary, our data argues that broad-acting transcriptional inhibitors are problematic for determining mRNA half-life, particularly if studying the impacts of the TORC1, HOG, or PKC pathway on mRNA stability. Regulatable promoter systems are a preferred approach for individual mRNA half-life studies, with 4tU labeling representing a good approach to global mRNA half-life analysis, despite modestly inducing P-bodies.

Drugging the "undruggable" microRNAs.

As a naturally occurring class of gene regulators, microRNAs (miRNAs) have attracted much attention as promising targets for therapeutic development. However, RNAs including miRNAs have long been considered undruggable, and most efforts have been devoted to using synthetic oligonucleotides to regulate miRNAs. Encouragingly, recent findings have revealed that miRNAs can also be drugged with small molecules that directly target miRNAs. In this review paper, we give a summary of recently emerged small-molecule inhibitors (SMIs) and small-molecule degraders (SMDs) for miRNAs. SMIs are small molecules that directly bind to miRNAs to inhibit their biogenesis, and SMDs are bifunctional small molecules that upon binding to miRNAs induce miRNA degradation. Strategies for discovering SMIs and developing SMDs were summarized. Applications of SMIs and SMDs in miRNA inhibition and cancer therapy were also introduced. Overall, SMIs and SMDs introduced here have high potency and specificity in miRNA inhibition. We envision that these small molecules will pave the way for developing novel therapeutics toward miRNAs that were previously considered undruggable.

Targeted inhibition of eIF4A suppresses B-cell receptor-induced translation and expression of MYC and MCL1 in chronic lymphocytic leukemia cells.

Signaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

A primate-specific RNA-binding protein (RBMXL3) is involved in glucocorticoid regulation of human pulmonary surfactant protein B (SP-B) mRNA stability.

The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate levels of surfactant protein B (SP-B). Dexamethasone (DEX) increases human SP-B expression, in part, through increased SP-B mRNA stability. A 30-nt-long hairpin element (RBE) in the 3'-untranslated region of human SP-B mRNA mediates both DEX-induced and intrinsic mRNA stabilities, but the mechanism is unknown. Proteomic analysis of RBE-interacting proteins identified a primate-specific protein, RNA-binding motif X-linked-like-3 (RBMXL3). siRNA directed against RBMXL3 reduces DEX-induced SP-B mRNA expression in human bronchoalveolar cells. Human SP-B mRNA stability, measured by our dual cistronic plasmid assay, is unaffected by DEX in mouse lung epithelial cells lacking RBMXL3, but DEX increases human SP-B mRNA stability when RBMXL3 is expressed and requires the RBE. In the absence of DEX, RBE interacts with cellular proteins, reducing intrinsic SP-B mRNA stability in human and mouse lung epithelial cells. RBMXL3 specifically binds the RBE in vitro, whereas RNA immunoprecipitation and affinity chromatography analyses indicate that binding is enhanced in the presence of DEX. These results describe a model where intrinsic stability of human SP-B mRNA is reduced through binding of cellular mRNA decay factors to RBE, which is then relieved through DEX-enhanced binding of primate-specific RBMXL3.