Search

Supplementary Figure 1 from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA ligases. Three compounds that differed in their specificity for the three human DNA ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA ligases I and III, and L189 inhibited DNA ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents. [Cancer Res 2008;68(9):3169–77]

Supplementary Figure 5 from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

Supplementary Figure 2 from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

Supplementary Figure 4 from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

Supplementary Methods and Materials from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

Supplementary Figure 3 from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

The mammalian transcriptome comprises a vast family of long noncoding (lnc)RNAs implicated in physiologic processes such as myogenesis, through which muscle forms during embryonic development and regenerates in the adult. However, the specific molecular mechanisms by which lncRNAs regulate human myogenesis are poorly understood. Here, we identified a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis. Overexpression of lncFAM promoted differentiation of human myoblasts into myotubes, while silencing lncFAM suppressed this process. As lncFAM resides in the nucleus, chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) analysis was employed to identify the molecular mechanisms whereby it might promote myogenesis. Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYBPC2. These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL, elevates MYBPC2 expression transcriptionally to promote myogenesis.

We have identified chemical probes that simultaneously inhibit cancer cell progression and an immune checkpoint. Using the computational Site Identification by Ligand Competitive Saturation (SILCS) technology, structural biology and cell-based assays, we identify small molecules that directly and selectively bind to the RNA Recognition Motif (RRM) of hnRNP A18, a regulator of protein translation in cancer cells. hnRNP A18 recognizes a specific RNA signature motif in the 3′UTR of transcripts associated with cancer cell progression (Trx, VEGF, RPA) and, as shown here, a tumor immune checkpoint (CTLA-4). Post-transcriptional regulation of immune checkpoints is a potential therapeutic strategy that remains to be exploited. The probes target hnRNP A18 RRM in vitro and in cells as evaluated by cellular target engagement. As single agents, the probes specifically disrupt hnRNP A18–RNA interactions, downregulate Trx and CTLA-4 protein levels and inhibit proliferation of several cancer cell lines without affecting the viability of normal epithelial cells. These first-in-class chemical probes will greatly facilitate the elucidation of the underexplored biological function of RNA Binding Proteins (RBPs) in cancer cells, including their effects on proliferation and immune checkpoint activation.

Pyrrolo[3,2-d]pyrimidines have been studied for many years as potential lead compounds for the development of antiproliferative agents. Much of the focus has been on modifications to the pyrimidine ring, with enzymatic recognition often modulated by C2 and C4 substituents. In contrast, this work focuses on the N5 of the pyrrole ring by means of a series of novel N5-substituted pyrrolo[3,2-d]pyrimidines. The compounds were screened against the NCI-60 Human Tumor Cell Line panel, and the results were analyzed using the COMPARE algorithm to elucidate potential mechanisms of action. COMPARE analysis returned strong correlation to known DNA alkylators and groove binders, corroborating the hypothesis that these pyrrolo[3,2-d]pyrimidines act as DNA or RNA alkylators. In addition, N5 substitution reduced the EC50 against CCRF-CEM leukemia cells by up to 7-fold, indicating that this position is of interest in the development of antiproliferative lead compounds based on the pyrrolo[3,2-d]pyrimidine scaffold.

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a key protein involved in pre-mRNA processing. CPSF30 contains five Cys3His domains (annotated as "zinc-finger" domains). Using inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy, and UV-visible spectroscopy, we report that CPSF30 is isolated with iron, in addition to zinc. Iron is present in CPSF30 as a 2Fe-2S cluster and uses one of the Cys3His domains; 2Fe-2S clusters with a Cys3His ligand set are rare and notably have also been identified in MitoNEET, a protein that was also annotated as a zinc finger. These findings support a role for iron in some zinc-finger proteins. Using electrophoretic mobility shift assays and fluorescence anisotropy, we report that CPSF30 selectively recognizes the AU-rich hexamer (AAUAAA) sequence present in pre-mRNA, providing the first molecular-based evidence to our knowledge for CPSF30/RNA binding. Removal of zinc, or both zinc and iron, abrogates binding, whereas removal of just iron significantly lessens binding. From these data we propose a model for RNA recognition that involves a metal-dependent cooperative binding mechanism.

Skeletal muscle contains long multinucleated and contractile structures known as muscle fibers, which arise from the fusion of myoblasts into multinucleated myotubes during myogenesis. The myogenic regulatory factor (MRF) MYF5 is the earliest to be expressed during myogenesis and functions as a transcription factor in muscle progenitor cells (satellite cells) and myocytes. In mouse C2C12 myocytes, MYF5 is implicated in the initial steps of myoblast differentiation into myotubes. Here, using ribonucleoprotein immunoprecipitation (RIP) analysis, we discovered a novel function for MYF5 as an RNA-binding protein which associated with a subset of myoblast mRNAs. One prominent MYF5 target was Ccnd1 mRNA, which encodes the key cell cycle regulator CCND1 (Cyclin D1). Biotin-RNA pulldown, UV-crosslinking and gel shift experiments indicated that MYF5 was capable of binding the 3' untranslated region (UTR) and the coding region (CR) of Ccnd1 mRNA. Silencing MYF5 expression in proliferating myoblasts revealed that MYF5 promoted CCND1 translation and modestly increased transcription of Ccnd1 mRNA. Accordingly, overexpressing MYF5 in C2C12 cells upregulated CCND1 expression while silencing MYF5 reduced myoblast proliferation as well as differentiation of myoblasts into myotubes. Moreover, MYF5 silencing reduced myogenesis, while ectopically restoring CCND1 abundance partially rescued the decrease in myogenesis seen after MYF5 silencing. We propose that MYF5 enhances early myogenesis in part by coordinately elevating Ccnd1 transcription and Ccnd1 mRNA translation.

The main characteristic of cancers, including breast cancer, is the ability of cancer cells to proliferate uncontrollably. However, the underlying mechanisms of cancer cell proliferation, especially those regulated by the RNA binding protein tristetraprolin (TTP), are not completely understood. In this study, we found that TTP inhibits cell proliferation in vitro and suppresses tumor growth in vivo through inducing cell cycle arrest at the S phase. Our studies demonstrate that TTP inhibits c-Jun expression through the C-terminal Zn finger and therefore increases Wee1 expression, a regulatory molecule which controls cell cycle transition from the S to the G2 phase. In contrast to the well-known function of TTP in regulating mRNA stability, TTP inhibits c-Jun expression at the level of transcription by selectively blocking NF-κB p65 nuclear translocation. Reconstitution of NF-κB p65 completely abolishes the inhibition of c-Jun transcription by TTP. Moreover, reconstitution of c-Jun in TTP-expressing breast tumor cells diminishes Wee1 overexpression and promotes cell proliferation. Our results indicate that TTP suppresses c-Jun expression that results in Wee1 induction which causes cell cycle arrest at the S phase and inhibition of cell proliferation. Our study provides a new pathway for TTP function as a tumor suppressor which could be targeted in tumor treatment.

Eukaryotic gene expression is tightly regulated post-transcriptionally by RNA-binding proteins (RBPs) and microRNAs. The RBP AU-rich-binding factor 1 (AUF1) isoform p37 was found to have high affinity for the microRNA let-7b in vitro (Kd = ∼6 nM) in cells. Ribonucleoprotein immunoprecipitation, in vitro association, and single-molecule-binding analyses revealed that AUF1 promoted let-7b loading onto Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex (RISC). In turn, AGO2–let-7 triggered target mRNA decay. Our findings uncover a novel mechanism by which AUF1 binding and transfer of microRNA let-7 to AGO2 facilitates let-7-elicited gene silencing.

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9–Rad1–Hus1 (9–1–1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9–1–1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9–1–1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134–155) is a key determinant of MYH binding. Both the N-(residues 1–146) and C-terminal (residues 147–280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1K136A mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad91−266 (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 91−266–1–1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 91−266–1–1 to 5′-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9–1–1 subunits to MYH-directed BER based on subunit asymmetry in protein–protein interactions and DNA binding events.

Summary Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins bind to AU-rich regions in target mRNAs, leading to their deadenylation and decay. Family members in Saccharomyces cerevisiae influence iron metabolism, whereas the single protein expressed in Schizosaccharomyces pombe, Zfs1, regulates cell–cell interactions. In the human pathogen Candida albicans, deep sequencing of mutants lacking the orthologous protein, Zfs1, revealed significant increases (> 1.5-fold) in 156 transcripts. Of these, 113 (72%) contained at least one predicted TTP family member binding site in their 3′UTR, compared with only 3 of 56 (5%) down-regulated transcripts. The zfs1Δ/Δ mutant was resistant to 3-amino-1,2,4-triazole, perhaps because of increased expression of the potential target transcript encoded by HIS3. Sequences of the proteins encoded by the putative Zfs1 targets were highly conserved among other species within the fungal CTG clade, while the predicted Zfs1 binding sites in these mRNAs often ‘disappeared’ with increasing evolutionary distance from the parental species. C. albicans Zfs1 bound to the ideal mammalian TTP binding site with high affinity, and Zfs1 was associated with target transcripts after co-immunoprecipitation. Thus, the biochemical activities of these proteins in fungi are highly conserved, but Zfs1-like proteins may target different transcripts in each species.

Hsp70 is a protein chaperone that prevents protein aggregation and aids protein folding by binding to hydrophobic peptide domains through a reversible mechanism directed by an ATPase cycle. However, Hsp70 also binds U-rich RNA including some AU-rich elements (AREs) that regulate the decay kinetics of select mRNAs and has recently been shown to bind and stabilize some ARE-containing transcripts in cells. Previous studies indicated that both the ATP- and peptide-binding domains of Hsp70 contributed to the stability of Hsp70–RNA complexes and that ATP might inhibit RNA recruitment. This suggested the possibility that RNA binding by Hsp70 might mimic features of its peptide-directed chaperone activities. Here, using purified, cofactor-free preparations of recombinant human Hsp70 and quantitative biochemical approaches, we found that high-affinity RNA binding requires at least 30 nucleotides of RNA sequence but is independent of Hsp70's nucleotide-bound status, ATPase activity, or peptide-binding roles. Furthermore, although both the ATP- and peptide-binding domains of Hsp70 could form complexes with an ARE sequence from VEGFA mRNA in vitro, only the peptide-binding domain could recover cellular VEGFA mRNA in ribonucleoprotein immunoprecipitations. Finally, Hsp70-directed stabilization of VEGFA mRNA in cells was mediated exclusively by the protein's peptide-binding domain. Together, these findings indicate that the RNA-binding and mRNA-stabilizing functions of Hsp70 are independent of its protein chaperone cycle but also provide potential mechanical explanations for several well-established and recently discovered cytoprotective and RNA-based Hsp70 functions.

Halogenated thieno[3,2-d]pyrimidines exhibit antiproliferative activity against a variety of cancer cell models, such as the mouse lymphocytic leukemia cell line L1210 in which they induce apoptosis independent of cell cycle arrest. Here we assessed these activities on MDA-MB-231 cells, a well-established model of aggressive, metastatic breast cancer. While 2,4-dichloro[3,2-d]pyrimidine was less toxic to MDA-MB-231 cells than previously observed in the L1210 model, flow cytometry analysis showed that MDA-MB-231 cell death involved arrest at the G2/M stage of the cell cycle. Conversely, the introduction of bromine at C7 of the 2,4-dichloro[3,2-d]pyrimidine eliminated cell type-dependent differences in cytotoxicity or cell cycle status. Together, these data indicate that a substituent at C7 can profoundly modify the cytotoxic mechanism of halogenated thieno[3,2-d]pyrimidines in a cell type-specific manner.

AU-rich element RNA-binding protein 1 (AUF1) regulates the stability and/or translational efficiency of diverse mRNA targets, including many encoding products controlling the cell cycle, apoptosis, and inflammation by associating with AU-rich elements residing in their 3′-untranslated regions. Previous biochemical studies showed that optimal AUF1 binding requires 33–34 nucleotides with a strong preference for U-rich RNA despite observations that few AUF1-associated cellular mRNAs contain such extended U-rich domains. Using the smallest AUF1 isoform (p37AUF1) as a model, we employed fluorescence anisotropy-based approaches to define thermodynamic parameters describing AUF1 ribonucleoprotein (RNP) complex formation across a panel of RNA substrates. These data demonstrated that 15 nucleotides of AU-rich sequence were sufficient to nucleate high affinity p37AUF1 RNP complexes within a larger RNA context. In particular, p37AUF1 binding to short AU-rich RNA targets was significantly stabilized by interactions with a 3′-purine residue and largely base-independent but non-ionic contacts 5′ of the AU-rich site. RNP stabilization by the upstream RNA domain was associated with an enhanced negative change in heat capacity consistent with conformational changes in protein and/or RNA components, and fluorescence resonance energy transfer-based assays demonstrated that these contacts were required for p37AUF1 to remodel local RNA structure. Finally, reporter mRNAs containing minimal high affinity p37AUF1 target sequences associated with AUF1 and were destabilized in a p37AUF1-dependent manner in cells. These findings provide a mechanistic explanation for the diverse population of AUF1 target mRNAs but also suggest how AUF1 binding could regulate protein and/or microRNA binding events at adjacent sites. Background: AUF1 post-transcriptionally regulates mRNA targets. Results: Short AU-rich sequences nucleate AUF1 ribonucleoproteins but require non-ionic contacts with flanking RNA to stabilize complexes and manipulate local RNA structure. Conclusion: AUF1 uses several molecular determinants to bind and remodel RNA targets. Significance: This model explains AUF1 interactome diversity and predicts allosteric effects on protein and microRNA trans-factor binding to proximal sites.

The RNA-binding protein AUF1 binds AU-rich elements in 3'-untranslated regions to regulate mRNA degradation and/or translation. Many of these mRNAs are predicted microRNA targets as well. An emerging theme in post-transcriptional control of gene expression is that RNA-binding proteins and microRNAs co-regulate mRNAs. Recent experiments and bioinformatic analyses suggest this type of co-regulation may be widespread across the transcriptome. Here, we identified mRNA targets of AUF1 from a complex pool of cellular mRNAs and examined a subset of these mRNAs to explore the links between RNA binding and mRNA degradation for both AUF1 and Argonaute 2 (AGO2), which is an essential effector of microRNA-induced gene silencing. Depending on the specific mRNA examined, AUF1 and AGO2 binding is proportional/cooperative, reciprocal/competitive or independent. For most mRNAs in which AUF1 affects their decay rates, mRNA degradation requires AGO2. Thus, AUF1 and AGO2 present mRNA-specific allosteric binding relationships for co-regulation of mRNA degradation.

The AU-rich elements (AREs) encoded within many mRNA 3' untranslated regions (3'UTRs) are targets for factors that control transcript longevity and translational efficiency. Hsp70, best known as a protein chaperone with well-defined peptide-refolding properties, is known to interact with ARE-like RNA substrates in vitro. Here, we show that cofactor-free preparations of Hsp70 form direct, high-affinity complexes with ARE substrates based on specific recognition of U-rich sequences by both the ATP- and peptide-binding domains. Suppressing Hsp70 in HeLa cells destabilized an ARE reporter mRNA, indicating a novel ARE-directed mRNA-stabilizing role for this protein. Hsp70 also bound and stabilized endogenous ARE-containing mRNAs encoding vascular endothelial growth factor (VEGF) and Cox-2, which involved a mechanism that was unaffected by an inhibitor of its protein chaperone function. Hsp70 recognition and stabilization of VEGF mRNA was mediated by an ARE-like sequence in the proximal 3'UTR. Finally, stabilization of VEGF mRNA coincided with the accumulation of Hsp70 protein in HL60 promyelocytic leukemia cells recovering from acute thermal stress. We propose that the binding and stabilization of selected ARE-containing mRNAs may contribute to the cytoprotective effects of Hsp70 following cellular stress but may also provide a novel mechanism linking constitutively elevated Hsp70 expression to the development of aggressive neoplastic phenotypes.

Vascular calcification is a major risk factor of cardiovascular mortality, particularly for patients with end-stage renal disease and diabetes. Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood. To clarify this, we studied how nuclear factor-kappa B (NF-κB) induction, a mediator of inflammation, might promote vascular calcification. Activation of NF-κB by tumor necrosis factor (TNF) promoted inorganic phosphate-induced calcification in human aortic smooth muscle cells. Pyrophosphate (an inhibitor of calcification) efflux to the extracellular matrix was suppressed along with the decreased expression of ankylosis protein homolog (ANKH), a transmembrane protein that controls pyrophosphate efflux of cells. The restoration of ANKH expression in these cells overcame the decreased pyrophosphate efflux and calcification. Tristetraprolin, a downstream product of NF-κB activation, may mediate destablization of ANKH mRNA since its knockdown by shRNA increased ANKH expression and decreased calcification. Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels. In contrast, the inhibition of NF-κB maintained ANKH expression and attenuated vascular calcification both in vivo and in vitro. Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression. Thus, TNF-activated NF-κB promotes inflammation-accelerated vascular calcification by inhibiting ankylosis protein homolog expression and consequent pyrophosphate secretion.

PKCδ increases keratinocyte differentiation and suppresses keratinocyte proliferation and survival. However, the mechanism of proliferation suppression is not well understood. The present studies show that PKCδ overexpression increases p21(Cip1) mRNA and protein level and promoter activity and that treatment with dominant-negative PKCδ, PKCδ-siRNA, or rottlerin inhibits promoter activation. Analysis of the p21(Cip1) promoter upstream regulatory region reveals three DNA segments that mediate PKCδ-dependent promoter activation. The PKCδ response element most proximal to the transcription start site encodes six GC-rich DNA elements. Mutation of these sites results in a loss of PKCδ-dependent promoter activation. Gel mobility supershift and chromatin immunoprecipitation reveal that these DNA elements bind the Kruppel-like transcription factor KLF4. PKCδ increases KLF4 mRNA and protein level and KLF4 binding to the GC-rich elements in the p21(Cip1) proximal promoter. In addition, KLF4-siRNA inhibits PKCδ-dependent p21(Cip1) promoter activity. PKCδ increases KLF4 expression leading to enhanced KLF4 interaction with the GC-rich elements in the p21(Cip1) promoter to activate transcription.

AU-rich element-binding proteins (ARE-BP) regulate the stability and/or translational efficiency of mRNAs containing cognate binding sites. Many targeted transcripts encode factors that control processes such as cell division, apoptosis, and angiogenesis, suggesting that dysregulated ARE-BP expression could dramatically influence oncogenic phenotypes. Using several approaches, we evaluated the expression of four well-characterized ARE-BPs across a variety of human neoplastic syndromes. AUF1, TIA-1, and HuR mRNAs were not systematically dysregulated in cancers; however, tristetraprolin mRNA levels were significantly decreased across many tumor types, including advanced cancers of the breast and prostate. Restoring tristetraprolin expression in an aggressive tumor cell line suppressed three key tumorgenic phenotypes: cell proliferation, resistance to proapoptotic stimuli, and expression of vascular endothelial growth factor mRNA. However, the cellular consequences of tristetraprolin expression varied across different cell models. Analyses of gene array data sets revealed that suppression of tristetraprolin expression is a negative prognostic indicator in breast cancer, because patients with low tumor tristetraprolin mRNA levels were more likely to present increased pathologic tumor grade, vascular endothelial growth factor expression, and mortality from recurrent disease. Collectively, these data establish that tristetraprolin expression is frequently suppressed in human cancers, which in turn can alter tumorigenic phenotypes that influence patient outcomes. [Cancer Res 2009;69(12):5168–76]

LDL is the most abundant cholesterol transport vehicle in plasma and a major prognostic indicator of atherosclerosis. Hepatic LDL receptors limit circulating LDL levels, since cholesterol internalized by the liver can be excreted. As such, mechanisms regulating LDL receptor expression in liver cells are appealing targets for cholesterol-lowering therapeutic strategies. Activation of HepG2 cells with phorbol esters enhances LDL receptor mRNA levels through transcriptional and posttranscriptional mechanisms. Here, we show that 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced stabilization of receptor mRNA requires the activity of protein kinase C and is accompanied by activation of the major mitogen activated protein kinase pathways. Inhibitor studies demonstrated that receptor mRNA stabilization is independent of the extracellular signal-regulated kinase or p38(MAPK), but requires activation of the c-Jun N-terminal kinase (JNK). An essential role for JNK in stabilizing receptor mRNA was further confirmed through small interfering RNA (siRNA) experiments and by activating JNK through two protein kinase C-independent mechanisms. Finally, prolonged JNK activation increased steady-state levels of receptor mRNA and protein, and significantly enhanced cellular LDL-binding activity. These data suggest that JNK may play an important role in posttranscriptional control of LDL receptor expression, thus constituting a novel mechanism to enhance plasma LDL clearance by liver cells.

The fluorescent adenine analogue 2-aminopurine (2AP) has been used extensively to monitor conformational changes and macromolecular binding events involving nucleic acids because its fluorescence properties are highly sensitive to changes in chemical environment. Furthermore, site-specific incorporation of 2AP permits local DNA and RNA conformational events to be discriminated from the global structural changes monitored by UV/Vis spectroscopy and circular dichroism. However, while the steady-state fluorescence properties of 2AP have been well defined in diverse settings, interpretation of 2AP fluorescence lifetime parameters has been hampered by the heterogeneous nature of multi-exponential 2AP intensity decays observed across populations of microenvironments. To resolve this problem, we have tested the utility of multi-exponential versus continuous Lorentzian lifetime distribution models to describe fluorescence intensity decays from 2AP in diverse chemical backgrounds and within the context of RNA. Heterogeneity was introduced into 2AP intensity decays by mixing solvents of differing polarities, or by adding quenchers under high viscosity to evaluate the transient effect. Heterogeneity of 2AP fluorescence within the context of a synthetic RNA hairpin was introduced by structural remodeling using a magnesium salt. In each case, except folded RNA which required a bimodal distribution, 2AP lifetime properties were well described by single Lorentzian distribution functions, abrogating the need to introduce additional discrete lifetime subpopulations. Rather, heterogeneity in fluorescence decay processes was accommodated by the breadth of each distribution. This approach also permitted solvent relaxation effects on 2AP emission to be assessed by comparing lifetime distributions at multiple wavelengths. Together, these studies provide a new perspective for the interpretation of 2AP fluorescence lifetime properties, which will further the utility of this fluorophore in analyses of the complex and heterogeneous structural microenvironments associated with nucleic acids.

Mtr4p belongs to the Ski2p family of DEVH-box containing proteins and is required for processing and degradation of a variety of RNA substrates in the nucleus. In particular, Mtr4p is required for creating the 5.8 S ribosomal RNA from its 7 S precursor, proper 3′-end processing of the U4 small nuclear RNA and some small nucleolar RNAs, and degradation of aberrant mRNAs and tRNAs. In these studies we have shown that Mtr4p has RNA-dependent ATPase (or dATPase) activity that is stimulated effectively by likely substrates (e.g. tRNA) but surprisingly weakly by poly(A). Using an RNA strand-displacement assay, we have demonstrated that Mtr4p can, in the presence of ATP or dATP, unwind the duplex region of a partial duplex RNA substrate in the 3′→5′ direction. We have examined the ability of Mtr4p to bind model RNA substrates in the presence of nucleotides that mimic the stages (i.e. ATP-bound, ADP-bound, and nucleotide-free) of the unwinding reaction. Our results demonstrate that the presence of a non-hydrolyzable ATP analog allows Mtr4p to discriminate between partial duplex RNA substrates, binding a 3′-tailed substrate with 5-fold higher affinity than a 5′-tailed substrate. In addition, Mtr4p displays a marked preference for binding to poly(A) RNA relative to an oligoribonucleotide of the same length and a random sequence. This binding exhibits apparent cooperativity and different dynamic behavior from binding to the random single-stranded RNA. This unique binding mode might be employed primarily for degradation.

The fluorescent base analogue 2-aminopurine (2-AP) is commonly used to study specific conformational and protein binding events involving nucleic acids. Here, combinations of steady-state and time-resolved fluorescence spectroscopy of 2-AP were employed to monitor conformational transitions within a model hairpin RNA from diverse structural perspectives. RNA substrates adopting stable, unambiguous secondary structures were labeled with 2-AP at an unpaired base, within the loop, or inside the base-paired stem. Steady-state fluorescence was monitored as the RNA hairpins made the transitions between folded and unfolded conformations using thermal denaturation, urea titration, and cation-mediated folding. Unstructured control RNA substrates permitted the effects of higher-order RNA structures on 2-AP fluorescence to be distinguished from stimulus-dependent changes in intrinsic 2-AP photophysics and/or interactions with adjacent residues. Thermodynamic parameters describing local conformational changes were thus resolved from multiple perspectives within the model RNA hairpin. These data provided energetic bases for construction of folding mechanisms, which varied among different folding-unfolding stimuli. Time-resolved fluorescence studies further revealed that 2-AP exhibits characteristic signatures of component fluorescence lifetimes and respective fractional contributions in different RNA structural contexts. Together, these studies demonstrate localized conformational events contributing to RNA folding and unfolding that could not be observed by approaches monitoring only global structural transitions.

Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pump function is common, development of effective therapies to stimulate contraction has proven difficult. This is thought to be attributable to their frequent reliance on cAMP stimulation to increase activator Ca 2+ . A potential alternative is nitroxyl (HNO), the 1-electron reduction product of nitric oxide (NO) that improves contraction and relaxation in normal and failing hearts in vivo. The mechanism for myocyte effects remains unknown. Here, we show that this activity results from a direct interaction of HNO with the sarcoplasmic reticulum Ca 2+ pump and the ryanodine receptor 2, leading to increased Ca 2+ uptake and release from the sarcoplasmic reticulum. HNO increases the open probability of isolated ryanodine-sensitive Ca 2+ -release channels and accelerates Ca 2+ reuptake into isolated sarcoplasmic reticulum by stimulating ATP-dependent Ca 2+ transport. Contraction improves with no net rise in diastolic calcium. These changes are not induced by NO, are fully reversible by addition of reducing agents (redox sensitive), and independent of both cAMP/protein kinase A and cGMP/protein kinase G signaling. Rather, the data support HNO/thiolate interactions that enhance the activity of intracellular Ca 2+ cycling proteins. These findings suggest HNO donors are attractive candidates for the pharmacological treatment of heart failure.

In vitro evaluation of the halogenated pyrrolo[3,2-d]pyrimidines identified antiproliferative activities in compounds 1 and 2 against four different cancer cell lines. Upon screening of a series of pyrrolo[3,2-d]pyrimidines, the 2,4-Cl compound 1 was found to exhibit antiproliferative activity at low micromolar concentrations. Introduction of iodine at C7 resulted in significant enhancement of potency by reducing the IC50 into sub-micromolar levels, thereby suggesting the importance of a halogen at C7. This finding was further supported by an increased antiproliferative effect for 4 as compared to 3. Cell-cycle and apoptosis studies conducted on the two potent compounds 1 and 2 showed differences in their cytotoxic mechanisms in triple negative breast cancer MDA-MB-231 cells, wherein compound 1 induced cells to accumulate at the G2/M stage with little evidence of apoptotic death. In contrast, compound 2 robustly induced apoptosis with concomitant G2/M cell cycle arrest in this cell model. ispartof: Bioorganic & Medicinal Chemistry vol:23 issue:15 pages:4354-4363 ispartof: location:England status: published

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3'-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD(+) binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces.

Association of tristetraprolin (TTP) with mRNAs containing selected AU-rich mRNA-destabilizing elements (AREs) initiates rapid cytoplasmic degradation of these transcripts. The RNA-binding activity of TTP is mediated by an internal tandem zinc finger domain that preferentially recognizes U-rich RNA ligands containing adjacent UUAU half-sites, and is accompanied by conformational changes within the peptide. Here, we have used analogues of the TTP RNA-binding domain containing specific tryptophan substitutions to probe the Zn2+- and RNA substrate-dependence of conformational events within individual zinc fingers. Fluorescence methods demonstrate that the N-terminal, but not C-terminal zinc finger domain adopts a stably folded conformation in the presence of Zn2+. Denaturant titrations suggest that both the N- and C-terminal zinc fingers exhibit limited structural heterogeneity in the absence of RNA substrates, although this is more pronounced for the C-terminal finger. Binding to a cognate ARE substrate induced significant conformational changes within each zinc finger, which also included increased resistance to chemical denaturation. Studies with mutant ARE ligands revealed that a single UUAU half-site was sufficient to induce structural modulation of the N-terminal finger. However, RNA-dependent folding of the C-terminal zinc finger was only observed in the presence of tandem UUAU half-sites, suggesting that the conformation of this domain is linked not only to RNA substrate recognition, but also to the ligand occupancy and/or conformational status of the N-terminal finger. Coupled with previous structural and thermodynamic analyses, these data provide a mechanistic framework for discrimination of RNA substrates involving ligand-dependent conformational adaptation of both zinc fingers within the TTP RNA-binding domain.

In mammals, rapid mRNA turnover directed by AU-rich elements (AREs) is mediated by selective association of cellular ARE-binding proteins. These trans-acting factors display overlapping RNA substrate specificities and may act to either stabilize or destabilize targeted transcripts; however, the mechanistic features of AREs that promote preferential binding of one trans-factor over another are not well understood. Here, we describe a hairpin-like structure adopted by the ARE from tumor necrosis factor alpha (TNFalpha) mRNA that modulates its affinity for selected ARE-binding proteins. In particular, association of the mRNA-destabilizing factor p37(AUF1) was strongly inhibited by adoption of the higher order ARE structure, whereas binding of the inducible heat shock protein Hsp70 was less severely compromised. By contrast, association of the mRNA-stabilizing protein HuR was only minimally affected by changes in ARE folding. Consistent with the inverse relationship between p37(AUF1) binding affinity and the stability of ARE folding, mutations that stabilized the ARE hairpin also inhibited its ability to direct rapid mRNA turnover in transfected cells. Finally, phylogenetic analyses and structural modeling indicate that TNFalpha mRNA sequences flanking the ARE are highly conserved and may stabilize the hairpin fold in vivo. Taken together, these data suggest that local higher order structures involving AREs may function as potent regulators of mRNA turnover in mammalian cells by modulating trans-factor binding selectivity.

Escherichia coli MutY is an adenine and weak guanine DNA glycosylase involved in reducing the mutagenic effects of 7,8-dihydro-8-oxoguanine (GO). MutY contains three structural domains: an iron-sulfur module, a six-helix barrel module with the helix-hairpin-helix motif, and a C-terminal domain. Here, we demonstrate that the mutant MutY(Δ26–134), which lacks the six-helix barrel domain, cannot complement the mutator phenotype of a mutY mutant in vivo. However, the mutant can still bind DNA and has weak catalytic activity at high enzyme concentrations. The mutant is a dimer in solution and assembled into two and multiple (up to five) complexes with 20- and 44-bp DNA fragments, respectively, in a concentration-dependent manner. Higher order complexes with DNA substrates containing A/GO mismatches were formed at lower protein concentrations than with the A/G mismatch and homoduplex DNA. Measurement of equilibrium binding using fluorescence anisotropy showed that the mutant protein retains some specificity for A/GO-containing DNA substrates and that the binding event is highly cooperative. This is consistent with the MutY structure determined, which indicates that GO specificity is contributed by both the six-helix barrel and C-terminal domains. The nonspecific binding of MutY(Δ26–134) to DNA suggests a model in which the specific binding of mismatched DNA by MutY involves sequential interactions, in which one MutY molecule scans the DNA and enhances binding of another MutY molecule to the A/GO mismatch.

Tristetraprolin (TTP) binds AU-rich elements (AREs) encoded within selected labile mRNAs and targets these transcripts for rapid cytoplasmic decay. RNA binding by TTP is mediated by an ∼70-amino acid domain containing two tandemly arrayed CCCH zinc fingers. Here we show that a 73-amino acid peptide spanning the TTP zinc finger domain, denoted TTP73, forms a dynamic, equimolar RNA·peptide complex with a 13-nucleotide fragment of the ARE from tumor necrosis factor α mRNA, which includes small but significant contributions from ionic interactions. Association of TTP73 with high affinity RNA substrates is accompanied by a large negative change in heat capacity without substantial modification of RNA structure, consistent with conformational changes in the peptide moiety during RNA binding. Analyses using mutant ARE substrates indicate that two adenylate residues located 3–6 bases apart within a uridylate-rich sequence are sufficient for high affinity recognition by TTP73 (Kd

Members of the mammalian tristetraprolin family of CCCH tandem zinc finger proteins can bind to certain AU-rich elements (AREs) in mRNAs, leading to their deadenylation and destabilization. Mammals express three or four members of this family, but Drosophila melanogaster and other insects appear to contain a single gene, Tis11. We found that recombinant Drosophila Tis11 protein could bind to ARE-containing RNA oligonucleotides with low nanomolar affinity. Remarkably, co-expression in mammalian cells with "target" RNAs demonstrated that Tis11 could promote destabilization of ARE-containing mRNAs and that this was partially dependent on a conserved C-terminal sequence resembling the mammalian NOT1 binding domain. Drosophila Tis11 promoted both deadenylation and decay of a target transcript in this heterologous cell system. We used chromosome deletion/duplication and P element insertion to produce two types of Tis11 deficiency in adult flies, both of which were viable and fertile. To address the hypothesis that Tis11 deficiency would lead to the abnormal accumulation of potential target transcripts, we analyzed gene expression in adult flies by deep mRNA sequencing. We identified 69 transcripts from 56 genes that were significantly up-regulated more than 1.5-fold in both types of Tis11-deficient flies. Ten of the up-regulated transcripts encoded probable proteases, but many other functional classes of proteins were represented. Many of the up-regulated transcripts contained potential binding sites for tristetraprolin family member proteins that were conserved in other Drosophila species. Tis11 is thus an ARE-binding, mRNA-destabilizing protein that may play a role in post-transcriptional gene expression in Drosophila and other insects.

The cellular response to mitogens is tightly regulated via transcriptional and post-transcriptional mechanisms to rapidly induce genes that promote proliferation and efficiently attenuate their expression to prevent malignant growth. RNase L is an endoribonuclease that mediates diverse antiproliferative activities, and tristetraprolin (TTP) is a mitogen-induced RNA-binding protein that directs the decay of proliferation-stimulatory mRNAs. In light of their roles as endogenous proliferative constraints, we examined the mechanisms and functional interactions of RNase L and TTP to attenuate a mitogenic response. Mitogen stimulation of RNase L-deficient cells significantly increased TTP transcription and the induction of other mitogen-induced mRNAs. This regulation corresponded with elevated expression of serum-response factor (SRF), a master regulator of mitogen-induced transcription. RNase L destabilized the SRF transcript and formed a complex with SRF mRNA in cells providing a mechanism by which RNase L down-regulates SRF-induced genes. TTP and RNase L proteins interacted in cells suggesting that RNase L is directed to cleave TTP-bound RNAs as a mechanism of substrate specificity. Consistent with their concerted function in RNA turnover, the absence of either RNase L or TTP stabilized SRF mRNA, and a subset of established TTP targets was also regulated by RNase L. RNase L deficiency enhanced mitogen-induced proliferation demonstrating its functional role in limiting the mitogenic response. Our findings support a model of feedback regulation in which RNase L and TTP target SRF mRNA and SRF-induced transcripts. Accordingly, meta-analysis revealed an enrichment of RNase L and TTP targets among SRF-regulated genes suggesting that the RNase L/TTP axis represents a viable target to inhibit SRF-driven proliferation in neoplastic diseases.

Post-transcriptional gene regulation is robustly regulated by RNA-binding proteins (RBPs). Here we describe the collection of RNAs regulated by AUF1 (AU-binding factor 1), an RBP linked to cancer, inflammation and aging. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis reveals that AUF1 primarily recognizes U-/GU-rich sequences in mRNAs and noncoding RNAs and influences target transcript fate in three main directions. First, AUF1 lowers the steady-state levels of numerous target RNAs, including long noncoding RNA NEAT1, in turn affecting the organization of nuclear paraspeckles. Second, AUF1 does not change the abundance of many target RNAs, but ribosome profiling reveals that AUF1 promotes the translation of numerous mRNAs in this group. Third, AUF1 unexpectedly enhances the steady-state levels of several target mRNAs encoding DNA-maintenance proteins. Through its actions on target RNAs, AUF1 preserves genomic integrity, in agreement with the AUF1-elicited prevention of premature cellular senescence.

Rapid mRNA degradation directed by A + U-rich elements (AREs) is mediated by the interaction of specific RNA-binding proteins to these sequences. The protein chaperone Hsp70 has been identified in a cellular complex containing the ARE-binding protein AUF1 and has also been detected in direct contact with A + U-rich RNA substrates, indicating that Hsp70 may be involved in the regulation of ARE-directed mRNA turnover. By using gel mobility shift and fluorescence anisotropy assays, we have determined that Hsp70 directly and specifically associates with U-rich RNA substrates in solution. With the ARE from tumor necrosis factor alpha (TNFalpha) mRNA, Hsp70 forms a dynamic complex consistent with a 1:1 association of protein:RNA but demonstrates cooperative binding behavior on polyuridylate substrates. Unlike AUF1, the RNA binding activity of Hsp70 is not regulated by ion-dependent folding of the TNFalpha ARE, suggesting that AUF1 and Hsp70 recognize distinct binding determinants on this RNA substrate. Binding of Hsp70 to the TNFalpha ARE is driven entirely by enthalpy at physiological temperatures, indicating that burial of hydrophobic surfaces is likely the principal mechanism stabilizing the Hsp70.RNA complex. Potential roles for the interaction of Hsp70 with ARE-containing mRNAs in the regulation of mRNA turnover and/or translational efficiency are discussed.

In mammals, A+U-rich elements (AREs) are potentcis-acting determinants of rapid cytoplasmic mRNA turnover. Recognition of these sequences by AUF1 is associated with acceleration of mRNA decay, likely involving recruitment or assembly of multi-subunit trans-acting complexes. Previously, we demonstrated that AUF1 deletion mutants formed tetramers on U-rich RNA substrates by sequential addition of protein dimers (Wilson, G. M., Sun, Y., Lu, H., and Brewer, G. (1999)J. Biol. Chem. 274, 33374–33381). Here, we show that binding of the full-length p37 isoform of AUF1 to these RNAs proceedsvia a similar mechanism, allowing delineation of equilibrium binding constants for both stages of tetramer assembly. However, association of AUF1 with the ARE from tumor necrosis factor (TNFα) mRNA was significantly inhibited by magnesium ions. Further fluorescence and hydrodynamic experiments indicated that Mg2+ induced or stabilized a conformational change in the TNFα ARE. Based on the solution of parameters describing both the protein-RNA and Mg2+-RNA equilibria, we present a dynamic, global equilibrium binding model describing the relationship between Mg2+ and AUF1 binding to the TNFα ARE. These studies provide the first evidence that some AREs may adopt higher order RNA structures that regulate their interaction withtrans-acting factors and indicate that mRNA structural remodeling has the potential to modulate the turnover rates of some ARE-containing mRNAs.

A cell-free mRNA decay assay has been adapted to permit the kinetics of 3' --> 5' exoribonuclease activities to be monitored in real time. RNA probes containing 5' caps and 3' poly(A) tails generated by transcription in vitro are 3' labeled using fluorescein-N6-ATP and poly(A) polymerase. Release of fluorescein-conjugated adenosine residues from the 3' end of the RNA substrate is monitored by a time-dependent decrease in fluorescence anisotropy in the presence of cytosolic proteins. To demonstrate the utility of the assay, an RNA probe was constructed containing a fragment of the c-myc 3' untranslated region and an 85-base poly(A) tail. Following 3' fluorescein labeling, the rate of 3'-terminal adenosine excision was monitored in the presence of an S100 cytosolic extract prepared from K562 erythroleukemia cells. Removal of the fluorescein-tagged A residues resolved to a first-order decay function, allowing the rate constant and enzyme-specific activity to be determined in this extract. Further applications and advantages of this technology are discussed.

Aims: Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca2+ uptake and myofilament Ca2+ sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) in a redox-dependent manner, improving Ca2+ handling in isolated myocytes/hearts. Results: Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln−/−) mice. Compared to WT, pln−/− myocytes displayed enhanced resting sarcomere shortening, peak Ca2+ transient, and blunted β-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca2+ transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln−/− cells/hearts. HNO enhanced SR Ca2+ uptake in WT but not pln−/− SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca2+-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available. Innovation: HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a. Conclusions: PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca2+ handling in failing hearts. Antioxid. Redox Signal. 19, 1185–1197.

KLF4 is a member of the Kruppel-like factor family of transcriptional regulators. KLF4 has been shown to be required for normal terminal differentiation of keratinocytes, but the molecular mechanism whereby KLF4 regulates genes associated with the differentiation process has not been studied. In the present study, we explore the impact of KLF4 on expression of involucrin, a gene that is specifically expressed in differentiated keratinocytes. KLF4 overexpression and knockdown studies show that involucrin mRNA and protein level correlates directly with KLF4 level. Moreover, studies of mutant KLF4 proteins indicate that transcriptionally inactive forms do not increase involucrin expression. PKCδ is a regulator of keratinocyte differentiation that increases expression of differentiation-associated target genes, including involucrin. Overexpression of KLF4 augments the PKCδ-dependent increase in involucrin expression, whereas KLF4 knockdown attenuates this response. The KLF4 induction of human involucrin (hINV) promoter activity is mediated via KLF4 binding to a GC-rich element located in the hINV promoter distal regulatory region, a region of the promoter required for in vivo involucrin expression. Mutation of the GC-rich element, an adjacent AP1 factor binding site, or both sites severely attenuates the response. Moreover, loss of KLF4 in an epidermal equivalent model of differentiation results in loss of hINV expression. These studies suggest that KLF4 is part of a multiprotein complex that interacts that the hINV promoter distal regulatory region to drive differentiation-dependent hINV gene expression in epidermis.

Although mammalian long non-coding (lnc)RNAs are best known for modulating transcription, their post-transcriptional influence on mRNA splicing, stability and translation is emerging. Here we report a post-translational function for the lncRNA HOTAIR as an inducer of ubiquitin-mediated proteolysis. HOTAIR associates with E3 ubiquitin ligases bearing RNA-binding domains, Dzip3 and Mex3b, as well as with their respective ubiquitination substrates, Ataxin-1 and Snurportin-1. In this manner, HOTAIR facilitates the ubiquitination of Ataxin-1 by Dzip3 and Snurportin-1 by Mex3b in cells and in vitro, and accelerates their degradation. HOTAIR levels are highly upregulated in senescent cells, causing rapid decay of targets Ataxin-1 and Snurportin-1, and preventing premature senescence. These results uncover a role for a lncRNA, HOTAIR, as a platform for protein ubiquitination.

AUF1 is a family of four proteins generated by alternative pre-mRNA splicing that form high affinity complexes with AU-rich, mRNA-destabilizing sequences located within the 3' untranslated regions of many labile mRNAs. While AUF1 binding is most frequently associated with accelerated mRNA decay, emerging examples have demonstrated roles as a mRNA stabilizer or even translational regulator for specific transcripts. In this review, we summarize recent advances in our understanding of mRNA recognition by AUF1 and the biochemical and functional consequences of these interactions. In addition, unique properties of individual AUF1 isoforms and the roles of these proteins in modulating expression of genes associated with inflammatory, neoplastic, and cardiac diseases are discussed. Finally, we describe mechanisms that regulate AUF1 expression in cells, and current knowledge of regulatory switches that modulate the cellular levels and/or activities of AUF1 isoforms through distinct protein post-translational modifications. This article is part of a Special Issue entitled: RNA Decay mechanisms.