Translational repression of pre-formed cytokine-encoding mRNA prevents chronic activation of memory T cells.

Memory T cells are critical for the immune response to recurring infections. Their instantaneous reactivity to pathogens is empowered by the persistent expression of cytokine-encoding mRNAs. How the translation of proteins from pre-formed cytokine-encoding mRNAs is prevented in the absence of infection has remained unclear. Here we found that protein production in memory T cells was blocked via a 3' untranslated region (3' UTR)-mediated process. Germline deletion of AU-rich elements (AREs) in the Ifng-3' UTR led to chronic cytokine production in memory T cells. This aberrant protein production did not result from increased expression and/or half-life of the mRNA. Instead, AREs blocked the recruitment of cytokine-encoding mRNA to ribosomes; this block depended on the ARE-binding protein ZFP36L2. Thus, AREs mediate repression of translation in mouse and human memory T cells by preventing undesirable protein production from pre-formed cytokine-encoding mRNAs in the absence of infection.

The favorable IFNL3 genotype escapes mRNA decay mediated by AU-rich elements and hepatitis C virus-induced microRNAs.

IFNL3, which encodes interferon-λ3 (IFN-λ3), has received considerable attention in the hepatitis C virus (HCV) field, as many independent genome-wide association studies have identified a strong association between polymorphisms near IFNL3 and clearance of HCV. However, the mechanism underlying this association has remained elusive. In this study, we report the identification of a functional polymorphism (rs4803217) in the 3' untranslated region (UTR) of IFNL3 mRNA that dictated transcript stability. We found that this polymorphism influenced AU-rich element (ARE)-mediated decay (AMD) of IFNL3 mRNA, as well as the binding of HCV-induced microRNAs during infection. Together these pathways mediated robust repression of the unfavorable IFNL3 polymorphism. Our data reveal a previously unknown mechanism by which HCV attenuates the antiviral response and indicate new potential therapeutic targets for HCV treatment.

Systematic Characterization of Stress-Induced RNA Granulation.

Upon stress, cytoplasmic mRNA is sequestered to insoluble ribonucleoprotein (RNP) granules, such as the stress granule (SG). Partially due to the belief that translationally suppressed mRNAs are recruited to SGs in bulk, stress-induced dynamic redistribution of mRNA has not been thoroughly characterized. Here, we report that endoplasmic reticulum (ER) stress targets only a small subset of translationally suppressed mRNAs into the insoluble RNP granule fraction (RG). This subset, characterized by extended length and adenylate-uridylate (AU)-rich motifs, is highly enriched with genes critical for cell survival and proliferation. This pattern of RG targeting was conserved for two other stress types, heat shock and arsenite toxicity, which induce distinct responses in the total cytoplasmic transcriptome. Nevertheless, stress-specific RG-targeting motifs, such as guanylate-cytidylate (GC)-rich motifs in heat shock, were also identified. Previously underappreciated, transcriptome profiling in the RG may contribute to understanding human diseases associated with RNP dysfunction, such as cancer and neurodegeneration.

Hydroxychloroquine attenuates double-stranded RNA-stimulated hyper-phosphorylation of tristetraprolin/ZFP36 and AU-rich mRNA stabilization.

The human innate immune system recognizes dsRNA as a pathogen-associated molecular pattern that induces a potent inflammatory response. The primary source of pathogenic dsRNA is cells infected with replicating viruses, but can also be released from uninfected necrotic cells. Here, we show that the dsRNA poly(I:C) challenge in human macrophages activates the p38 MAPK-MK2 signalling pathway and subsequently the phosphorylation of tristetraprolin (TTP/ZFP36). The latter is an mRNA decay-promoting protein that controls the stability of AU-rich mRNAs (AREs) that code for many inflammatory mediators. Hydroxychloroquine (HCQ), a common anti-malaria drug, is used to treat inflammatory and autoimmune disorders and, controversially, during acute COVID-19 disease. We found that HCQ reduced the dsRNA-dependent phosphorylation of p38 MAPK and its downstream kinase MK2. Subsequently, HCQ reduced the abundance and protein stability of the inactive (phosphorylated) form of TTP. HCQ reduced the levels and the mRNA stability of poly (I:C)-induced cytokines and inflammatory mRNAs like TNF, IL-6, COX-2, and IL-8 in THP-1 and primary blood monocytes. Our results demonstrate a new mechanism of the anti-inflammatory role of HCQ at post-transcriptional level (TTP phosphorylation) in a model of dsRNA activation, which usually occurs in viral infections or RNA release from necrotic tissue.

Multiple roles for AU-rich RNA binding proteins in the development of haematologic malignancies and their resistance to chemotherapy.

Post-transcriptional regulation by RNA binding proteins can determine gene expression levels and drive changes in cancer cell proteomes. Identifying mechanisms of protein-RNA binding, including preferred sequence motifs bound in vivo, provides insights into protein-RNA networks and how they impact mRNA structure, function, and stability. In this review, we will focus on proteins that bind to AU-rich elements (AREs) in nascent or mature mRNA where they play roles in response to stresses encountered by cancer cells. ARE-binding proteins (ARE-BPs) specifically impact alternative splicing, stability, decay and translation, and formation of RNA-rich biomolecular condensates like cytoplasmic stress granules (SGs). For example, recent findings highlight the role of ARE-BPs - like TIAR and HUR - in chemotherapy resistance and in translational regulation of mRNAs encoding pro-inflammatory cytokines. We will discuss emerging evidence that different modes of ARE-BP activity impact leukaemia and lymphoma development, progression, adaptation to microenvironment and chemotherapy resistance.

Structural basis for the recognition of transiently structured AU-rich elements by Roquin.

Adenylate/uridylate-rich elements (AREs) are the most common cis-regulatory elements in the 3'-untranslated region (UTR) of mRNAs, where they fine-tune turnover by mediating mRNA decay. They increase plasticity and efficacy of mRNA regulation and are recognized by several ARE-specific RNA-binding proteins (RBPs). Typically, AREs are short linear motifs with a high content of complementary A and U nucleotides and often occur in multiple copies. Although thermodynamically rather unstable, the high AU-content might enable transient secondary structure formation and modify mRNA regulation by RBPs. We have recently suggested that the immunoregulatory RBP Roquin recognizes folded AREs as constitutive decay elements (CDEs), resulting in shape-specific ARE-mediated mRNA degradation. However, the structural evidence for a CDE-like recognition of AREs by Roquin is still lacking. We here present structures of CDE-like folded AREs, both in their free and protein-bound form. Moreover, the AREs in the UCP3 3'-UTR are additionally bound by the canonical ARE-binding protein AUF1 in their linear form, adopting an alternative binding-interface compared to the recognition of their CDE structure by Roquin. Strikingly, our findings thus suggest that AREs can be recognized in multiple ways, allowing control over mRNA regulation by adapting distinct conformational states, thus providing differential accessibility to regulatory RBPs.

Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM.

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.

Muscle development and regeneration controlled by AUF1-mediated stage-specific degradation of fate-determining checkpoint mRNAs.

AUF1 promotes rapid decay of mRNAs containing 3' untranslated region (3'UTR) AU-rich elements (AREs). AUF1 depletion in mice accelerates muscle loss and causes limb girdle muscular dystrophy. Here, we demonstrate that the selective, targeted degradation by AUF1 of key muscle stem cell fate-determining checkpoint mRNAs regulates each stage of muscle development and regeneration by reprogramming each myogenic stage. Skeletal muscle stem (satellite) cell explants show that Auf1 transcription is activated with satellite cell activation by stem cell regulatory factor CTCF. AUF1 then targets checkpoint ARE-mRNAs for degradation, progressively reprogramming the transcriptome through each stage of myogenesis. Transition steps in myogenesis, from stem cell proliferation to differentiation to muscle fiber development, are each controlled by fate-determining checkpoint mRNAs, which, surprisingly, were found to be controlled in their expression by AUF1-targeted mRNA decay. Checkpoint mRNAs targeted by AUF1 include Twist1, decay of which promotes myoblast development; CyclinD1, decay of which blocks myoblast proliferation and initiates differentiation; and RGS5, decay of which activates Sonic Hedgehog (SHH) pathway-mediated differentiation of mature myotubes. AUF1 therefore orchestrates muscle stem cell proliferation, self-renewal, myoblast differentiation, and ultimately formation of muscle fibers through targeted, staged mRNA decay.

Novel AU-rich proximal UTR sequences (APS) enhance CXCL8 synthesis upon the induction of rpS6 phosphorylation.

The role of ribosomal protein S6 (rpS6) phosphorylation in mRNA translation remains poorly understood. Here, we reveal a potential role in modulating the translation rate of chemokine (C-X-C motif) ligand 8 (CXCL8 or Interleukin 8, IL8). We observed that more CXCL8 protein was being secreted from less CXCL8 mRNA in primary macrophages and macrophage-like HL-60 cells relative to other cell types. This correlated with an increase in CXCL8 polyribosome association, suggesting an increase in the rate of CXCL8 translation in macrophages. The cell type-specific expression levels were replicated by a CXCL8- UTR-reporter (Nanoluc reporter flanked by the 5' and 3' UTR of CXCL8). Mutations of the CXCL8-UTR-reporter revealed that cell type-specific expression required: 1) a 3' UTR of at least three hundred bases; and 2) an AU base content that exceeds fifty percent in the first hundred bases of the 3' UTR immediately after the stop codon, which we dub AU-rich proximal UTR sequences (APS). The 5' UTR of CXCL8 enhanced expression at the protein level and conferred cell type-specific expression when paired with a 3' UTR. A search for other APS-positive mRNAs uncovered TNF alpha induced protein 6 (TNFAIP6), another mRNA that was translationally upregulated in macrophages. The elevated translation of APS-positive mRNAs in macrophages coincided with elevated rpS6 S235/236 phosphorylation. Both were attenuated by the ERK1/2 signaling inhibitors, U0126 and AZD6244. In A549 cells, rpS6 S235/236 phosphorylation was induced by TAK1, Akt or PKA signaling. This enhanced the translation of the CXCL8-UTR-reporters. Thus, we propose that the induction of rpS6 S235/236 phosphorylation enhances the translation of mRNAs that contain APS motifs, such as CXCL8 and TNFAIP6. This may contribute to the role of macrophages as the primary producer of CXCL8, a cytokine that is essential for immune cell recruitment and activation.

Post-transcriptional control of T-cell cytokine production: Implications for cancer therapy.

As part of the adaptive immune system, T cells are vital for the eradication of infected and malignantly transformed cells. To perform their protective function, T cells produce effector molecules that are either directly cytotoxic, such as granzymes, perforin, interferon-γ and tumour necrosis factor α, or attract and stimulate (immune) cells, such as interleukin-2. As these molecules can also induce immunopathology, tight control of their production is required. Indeed, inflammatory cytokine production is regulated on multiple levels. Firstly, locus accessibility and transcription factor availability and activity determine the amount of mRNA produced. Secondly, post-transcriptional mechanisms, influencing mRNA splicing/codon usage, stability, decay, localization and translation rate subsequently determine the amount of protein that is produced. In the immune suppressive environments of tumours, T cells gradually lose the capacity to produce effector molecules, resulting in tumour immune escape. Recently, the role of post-transcriptional regulation in fine-tuning T-cell effector function has become more appreciated. Furthermore, several groups have shown that exhausted or dysfunctional T cells from cancer patients or murine models possess mRNA for inflammatory mediators, but fail to produce effector molecules, hinting that post-transcriptional events also play a role in hampering tumour-infiltrating lymphocyte effector function. Here, the post-transcriptional regulatory events governing T-cell cytokine production are reviewed, with a specific focus on the importance of post-transcriptional regulation in anti-tumour responses. Furthermore, potential approaches to circumvent tumour-mediated dampening of T-cell effector function through the (dis)engagement of post-transcriptional events are explored, such as CRISPR/Cas9-mediated genome editing or chimeric antigen receptors.

Human T cells employ conserved AU-rich elements to fine-tune IFN-γ production.

Long-lasting CD8+ T cell responses are critical in combatting infections and tumors. The pro-inflammatory cytokine IFN-γ is a key effector molecule herein. We recently showed that in murine T cells the production of IFN-γ is tightly regulated through adenylate uridylate-rich elements (AREs) that are located in the 3' untranslated region (UTR) of the Ifng mRNA molecule. Loss of AREs resulted in prolonged cytokine production in activated T cells and boosted anti-tumoral T cell responses. Here, we investigated whether these findings can be translated to primary human T cells. Utilizing CRISPR-Cas9 technology, we deleted the ARE region from the IFNG 3' UTR in peripheral blood-derived human T cells. Loss of AREs stabilized the IFNG mRNA in T cells and supported a higher proportion of IFN-γ protein-producing T cells. Importantly, combining MART-1 T cell receptor engineering with ARE-Del gene editing showed that this was also true for antigen-specific activation of T cells. MART-1-specific ARE-Del T cells showed higher percentages of IFN-γ producing T cells in response to MART-1 expressing tumor cells. Combined, our study reveals that ARE-mediated posttranscriptional regulation is conserved between murine and human T cells. Furthermore, generating antigen-specific ARE-Del T cells is feasible, a feature that could potentially be used for therapeutical purposes.

Synergistic regulation of Rgs4 mRNA by HuR and miR-26/RISC in neurons.

The negative regulator of G-protein signalling 4 (Rgs4) is linked to several neurologic diseases, e.g. schizophrenia, addiction, seizure and pain perception. Consequently, Rgs4 expression is tightly regulated, resulting in high mRNA and protein turnover. The post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. Here, we show that in neurons the RBP HuR reduces endogenous Rgs4 expression by destabilizing Rgs4 mRNA. Interestingly, in smooth muscle cells, Rgs4 is stabilized by HuR, indicating tissue-dependent differences in HuR function. Using in vitro RNA-based pulldown experiments, we identify the functional AU-rich element (ARE) within the Rgs4 3'-UTR that is recognized and bound by HuR. Bioinformatic analysis uncovered that this ARE lies within a highly conserved area next to a miR-26 binding site. We find that the neuronal-enriched miR-26 negatively influences Rgs4 expression in neurons. Further, HuR and miR-26 act synergistically in fluorescent reporter assays. Together, our data suggest a regulatory mechanism, in which an RBP selectively destabilizes a target mRNA in cooperation with a miRNA and the RISC machinery.

Insight into novel RNA-binding activities via large-scale analysis of lncRNA-bound proteome and IDH1-bound transcriptome.

RNA-binding proteins (RBPs) play pivotal roles in directing RNA fate and function. Yet the current annotation of RBPs is largely limited to proteins carrying known RNA-binding domains. To systematically reveal dynamic RNA-protein interactions, we surveyed the human proteome by a protein array-based approach and identified 671 proteins with RNA-binding activity. Among these proteins, 525 lack annotated RNA-binding domains and are enriched in transcriptional and epigenetic regulators, metabolic enzymes, and small GTPases. Using an improved CLIP (crosslinking and immunoprecipitation) method, we performed genome-wide target profiling of isocitrate dehydrogenase 1 (IDH1), a novel RBP. IDH1 binds to thousands of RNA transcripts with enriched functions in transcription and chromatin regulation, cell cycle and RNA processing. Purified IDH1, but not an oncogenic mutant, binds directly to GA- or AU-rich RNA that are also enriched in IDH1 CLIP targets. Our study provides useful resources of unconventional RBPs and IDH1-bound transcriptome, and convincingly illustrates, for the first time, the in vivo and in vitro RNA targets and binding preferences of IDH1, revealing an unanticipated complexity of RNA regulation in diverse cellular processes.

Yeast Cth2 protein represses the translation of ARE-containing mRNAs in response to iron deficiency.

In response to iron deficiency, the budding yeast Saccharomyces cerevisiae undergoes a metabolic remodeling in order to optimize iron utilization. The tandem zinc finger (TZF)-containing protein Cth2 plays a critical role in this adaptation by binding and promoting the degradation of multiple mRNAs that contain AU-rich elements (AREs). Here, we demonstrate that Cth2 also functions as a translational repressor of its target mRNAs. By complementary approaches, we demonstrate that Cth2 protein inhibits the translation of SDH4, which encodes a subunit of succinate dehydrogenase, and CTH2 mRNAs in response to iron depletion. Both the AREs within SDH4 and CTH2 transcripts, and the Cth2 TZF are essential for translational repression. We show that the role played by Cth2 as a negative translational regulator extends to other mRNA targets such as WTM1, CCP1 and HEM15. A structure-function analysis of Cth2 protein suggests that the Cth2 amino-terminal domain (NTD) is important for both mRNA turnover and translation inhibition, while its carboxy-terminal domain (CTD) only participates in the regulation of translation, but is dispensable for mRNA degradation. Finally, we demonstrate that the Cth2 CTD is physiologically relevant for adaptation to iron deficiency.

Deficiency of Tristetraprolin Triggers Hyperthermia through Enhancing Hypothalamic Inflammation.

Tristetraprolin (TTP), an RNA-binding protein, controls the stability of RNA by capturing AU-rich elements on their target genes. It has recently been identified that TTP serves as an anti-inflammatory protein by guiding the unstable mRNAs of pro-inflammatory proteins in multiple cells. However, it has not yet been investigated whether TTP affects the inflammatory responses in the hypothalamus. Since hypothalamic inflammation is tightly coupled to the disturbance of energy homeostasis, we designed the current study to investigate whether TTP regulates hypothalamic inflammation and thereby affects energy metabolism by utilizing TTP-deficient mice. We observed that deficiency of TTP led to enhanced hypothalamic inflammation via stimulation of a variety of pro-inflammatory genes. In addition, microglial activation occurred in the hypothalamus, which was accompanied by an enhanced inflammatory response. In line with these molecular and cellular observations, we finally confirmed that deficiency of TTP results in elevated core body temperature and energy expenditure. Taken together, our findings unmask novel roles of hypothalamic TTP on energy metabolism, which is linked to inflammatory responses in hypothalamic microglial cells.

AU-rich element-mediated mRNA decay via the butyrate response factor 1 controls cellular levels of polyadenylated replication-dependent histone mRNAs.

Replication-dependent histone (RDH) mRNAs have a nonpolyadenylated 3'-UTR that ends in a highly conserved stem-loop structure. Nonetheless, a subset of RDH mRNAs has a poly(A) tail under physiological conditions. The biological meaning of poly(A)-containing (+) RDH mRNAs and details of their biosynthesis remain elusive. Here, using HeLa cells and Western blotting, qRT-PCR, and biotinylated RNA pulldown assays, we show that poly(A)+ RDH mRNAs are post-transcriptionally regulated via adenylate- and uridylate-rich element-mediated mRNA decay (AMD). We observed that the rapid degradation of poly(A)+ RDH mRNA is driven by butyrate response factor 1 (BRF1; also known as ZFP36 ring finger protein-like 1) under normal conditions. Conversely, cellular stresses such as UV C irradiation promoted BRF1 degradation, increased the association of Hu antigen R (HuR; also known as ELAV-like RNA-binding protein 1) with the 3'-UTR of poly(A)+ RDH mRNAs, and eventually stabilized the poly(A)+ RDH mRNAs. Collectively, our results provide evidence that AMD surveils poly(A)+ RDH mRNAs via BRF1-mediated degradation under physiological conditions.

Tristetraprolin posttranscriptionally downregulates PFKFB3 in cancer cells.

The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases 3 (PFKFB3) catalyzes the first committed rate-limiting step of glycolysis and is upregulated in cancer cells. The mechanism of PFKFB3 expression upregulation in cancer cells has not been fully elucidated. The PFKFB3 3'-UTR is reported to contain AU-rich elements (AREs) that are important for regulating PFKFB3 mRNA stability. However, the mechanisms by which PFKFB3 mRNA stability is determined by its 3'-UTR are not well known. We demonstrated that tristetraprolin (TTP), an ARE-binding protein, has a critical function regulating PFKFB3 mRNA stability. Our results showed that PFKFB3 mRNA contains three AREs in the 3'-UTR. TTP bound to the 3rd ARE and enhanced the decay of PFKFB3 mRNA. Overexpression of TTP decreased PFKFB3 expression and ATP levels but increased GSH level in cancer cells. Overexpression of PFKFB3 cDNA without the 3'-UTR rescued ATP level and GSH level in TTP-overexpressing cells. Our results suggested that TTP post-transcriptionally downregulated PFKFB3 expression and that overexpression of TTP may contribute to suppression of glycolysis and energy production of cancer cells in part by downregulating PFKFB3 expression.

Enhanced oncolytic activity of E4orf6-deficient adenovirus by facilitating nuclear export of HuR.

An AU-rich element (ARE) is RNA element that enhances the rapid decay of mRNA. The RNA binding protein HuR stabilizes ARE-mRNA by exporting it to the cytoplasm. In most of cancer cells, HuR is exported to the cytoplasm and ARE-mRNA is stabilized. In addition, the viral gene product E4orf6 exports HuR to stabilize ARE-mRNA in adenovirus-infected cells and the stabilization is required for full virus replication. Previously we showed the oncolytic activity of E4orf6-deleted adenovirus dl355, which can replicate in cancer cells where ARE-mRNA is stabilized. In this study, we examined whether the further enhancement of HuR export can stimulate the replication and the oncolytic activity of dl355. We found that ethanol treatment promoted the cytoplasmic relocalization of HuR in cancer cells. In addition, the replication efficiency of dl355 increased in ethanol-treated cells, and in response, the cytolytic activity of the virus also increased in vitro and in vivo. Upregulation of a cleaved-PARP level in infected cells mediated by ethanol is suggesting that ethanol activated the apoptosis induced by dl355. IVa2 mRNA, the only ARE-mRNA among transcripts of adenovirus was augmented by ethanol treatment. These data indicate that the enhancement of ARE-mRNA stabilization as a result of ethanol treatment upregulates the oncolytic activity of dl355 and suggests that the combined use of an oncolytic adenovirus and ethanol treatment may be a good strategy for cancer therapy.

Multi-omics: Differential expression of IFN-γ results in distinctive mechanistic features linking chronic inflammation, gut dysbiosis, and autoimmune diseases.

Low grade, chronic inflammation is a critical risk factor for immunologic dysfunction including autoimmune diseases. However, the multiplicity of complex mechanisms and lack of relevant murine models limit our understanding of the precise role of chronic inflammation. To address these hurdles, we took advantage of multi-omics data and a unique murine model with a low but chronic expression of IFN-γ, generated by replacement of the AU-rich element (ARE) in the 3' UTR region of IFN-γ mRNA with random nucleotides. Herein, we demonstrate that low but differential expression of IFN-γ in mice by homozygous or heterozygous ARE replacement triggers distinctive gut microbial alterations, of which alteration is female-biased with autoimmune-associated microbiota. Metabolomics data indicates that gut microbiota-dependent metabolites have more robust sex-differences than microbiome profiling, particularly those involved in fatty acid oxidation and nuclear receptor signaling. More importantly, homozygous ARE-Del mice have dramatic changes in tryptophan metabolism, bile acid and long-chain lipid metabolism, which interact with gut microbiota and nuclear receptor signaling similarly with sex-dependent metabolites. Consistent with these findings, nuclear receptor signaling, encompassing molecules such as PPARs, FXR, and LXRs, was detectable as a top canonical pathway in comparison of blood and tissue-specific gene expression between female homozygous vs heterozygous ARE-Del mice. Further analysis implies that dysregulated autophagy in macrophages is critical for breaking self-tolerance and gut homeostasis, while pathways interact with nuclear receptor signaling to regulate inflammatory responses. Overall, pathway-based integration of multi-omics data provides systemic and cellular insights about how chronic inflammation driven by IFN-γ results in the development of autoimmune diseases with specific etiopathological features.