A novel reporter for helicase activity in translation uncovers DDX3X interactions.

DDX3X regulates the translation of a subset of human transcripts containing complex 5' untranslated regions (5' UTRs). In this study we developed the helicase activity reporter for translation (HART) which uses DDX3X-sensitive 5' UTRs to measure DDX3X mediated translational activity in cells. To directly measure RNA structure in DDX3X dependent mRNAs, we used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5' UTRs and then employed HART to investigate how sequence alterations influence DDX3X-sensitivity. Additionally, we identified residues 38-44 as potential mediators of DDX3X's interaction with the translational machinery. HART revealed that both DDX3X's association with the translational machinery as well as its helicase activity are required for its function in promoting the translation of DDX3X sensitive 5' UTRs. These findings suggest DDX3X plays a crucial role regulating translation through its interaction with the translational machinery during ribosome scanning, and establish the HART reporter as a robust, lentivirally-encoded, colorimetric measurement of DDX3X-dependent translation in cells.

IGHMBP2 deletion suppresses translation and activates the integrated stress response.

IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 up-regulation. With recent studies showing the integrated stress response (ISR) can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.

Protein-protein interactions with G3BPs drive stress granule condensation and gene expression changes under cellular stress.

Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these condensates is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs condense into SGs following stress-induced translational arrest. Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of different G3BP paralogs to stress granule formation and stress-induced gene expression changes is incompletely understood. Here, we identified key residues for G3BP condensation such as V11. This conserved amino acid is required for formation of the G3BP-Caprin-1 complex, hence promoting SG assembly. Total RNA sequencing and ribosome profiling revealed that disruption of G3BP condensation corresponds to changes in mRNA levels and ribosome engagement during the integrated stress response (ISR). Moreover, we found that G3BP2B preferentially condenses and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Together, this work suggests that stress granule assembly promotes changes in gene expression under cellular stress, which is differentially regulated by G3BP paralogs.

A ubiquitous GC content signature underlies multimodal mRNA regulation by DDX3X.

The road from transcription to protein synthesis is paved with many obstacles, allowing for several modes of post-transcriptional regulation of gene expression. A fundamental player in mRNA biology is DDX3X, an RNA binding protein that canonically regulates mRNA translation. By monitoring dynamics of mRNA abundance and translation following DDX3X depletion, we observe stabilization of translationally suppressed mRNAs. We use interpretable statistical learning models to uncover GC content in the coding sequence as the major feature underlying RNA stabilization. This result corroborates GC content-related mRNA regulation detectable in other studies, including hundreds of ENCODE datasets and recent work focusing on mRNA dynamics in the cell cycle. We provide further evidence for mRNA stabilization by detailed analysis of RNA-seq profiles in hundreds of samples, including a Ddx3x conditional knockout mouse model exhibiting cell cycle and neurogenesis defects. Our study identifies a ubiquitous feature underlying mRNA regulation and highlights the importance of quantifying multiple steps of the gene expression cascade, where RNA abundance and protein production are often uncoupled.

A Legionella toxin exhibits tRNA mimicry and glycosyl transferase activity to target the translation machinery and trigger a ribotoxic stress response.

A widespread strategy employed by pathogens to establish infection is to inhibit host-cell protein synthesis. Legionella pneumophila, an intracellular bacterial pathogen and the causative organism of Legionnaires' disease, secretes a subset of protein effectors into host cells that inhibit translation elongation. Mechanistic insights into how the bacterium targets translation elongation remain poorly defined. We report here that the Legionella effector SidI functions in an unprecedented way as a transfer-RNA mimic that directly binds to and glycosylates the ribosome. The 3.1 Å cryo-electron microscopy structure of SidI reveals an N-terminal domain with an 'inverted L' shape and surface-charge distribution characteristic of tRNA mimicry, and a C-terminal domain that adopts a glycosyl transferase fold that licenses SidI to utilize GDP-mannose as a sugar precursor. This coupling of tRNA mimicry and enzymatic action endows SidI with the ability to block protein synthesis with a potency comparable to ricin, one of the most powerful toxins known. In Legionella-infected cells, the translational pausing activated by SidI elicits a stress response signature mimicking the ribotoxic stress response, which is activated by elongation inhibitors that induce ribosome collisions. SidI-mediated effects on the ribosome activate the stress kinases ZAKα and p38, which in turn drive an accumulation of the protein activating transcription factor 3 (ATF3). Intriguingly, ATF3 escapes the translation block imposed by SidI, translocates to the nucleus and orchestrates the transcription of stress-inducible genes that promote cell death, revealing a major role for ATF3 in the response to collided ribosome stress. Together, our findings elucidate a novel mechanism by which a pathogenic bacterium employs tRNA mimicry to hijack a ribosome-to-nuclear signalling pathway that regulates cell fate.

RNA molecular recording with an engineered RNA deaminase.

RNA deaminases are powerful tools for base editing and RNA molecular recording. However, the enzymes used in currently available RNA molecular recorders such as TRIBE, DART or STAMP have limitations due to RNA structure and sequence dependence. We designed a platform for directed evolution of RNA molecular recorders. We engineered an RNA A-to-I deaminase (an RNA adenosine base editor, rABE) that has high activity, low bias and low background. Using rABE, we present REMORA (RNA-encoded molecular recording in adenosines), wherein deamination by rABE writes a molecular record of RNA-protein interactions. By combining rABE with the C-to-U deaminase APOBEC1 and long-read RNA sequencing, we measured binding by two RNA-binding proteins on single messenger RNAs. Orthogonal RNA molecular recording of mammalian Pumilio proteins PUM1 and PUM2 shows that PUM1 competes with PUM2 for a subset of sites in cells. Furthermore, we identify transcript isoform-specific RNA-protein interactions driven by isoform changes distal to the binding site. The genetically encodable RNA deaminase rABE enables single-molecule identification of RNA-protein interactions with cell type specificity.

Nucleolar dynamics are determined by the ordered assembly of the ribosome.

Ribosome biogenesis is coordinated within the nucleolus, a biomolecular condensate that exhibits dynamic material properties that are thought to be important for nucleolar function. However, the relationship between ribosome assembly and nucleolar dynamics is not clear. Here, we screened 364 genes involved in ribosome biogenesis and RNA metabolism for their impact on dynamics of the nucleolus, as measured by automated, high-throughput fluorescence recovery after photobleaching (FRAP) of the nucleolar scaffold protein NPM1. This screen revealed that gene knockdowns that caused accumulation of early rRNA intermediates were associated with nucleolar rigidification, while accumulation of late intermediates led to increased fluidity. These shifts in dynamics were accompanied by distinct changes in nucleolar morphology. We also found that genes involved in mRNA processing impact nucleolar dynamics, revealing connections between ribosome biogenesis and other RNA processing pathways. Together, this work defines mechanistic ties between ribosome assembly and the biophysical features of the nucleolus, while establishing a toolbox for understanding how molecular dynamics impact function across other biomolecular condensates.

Determinants of DDX3X sensitivity uncovered using a helicase activity in translation reporter.

DDX3X regulates the translation of a subset of human transcripts containing complex 5' untranslated regions (5' UTRs). In this study we developed the helicase activity reporter for translation (HART) which uses DDX3X-sensitive 5' UTRs to measure DDX3X mediated translational activity in cells. To dissect the structural underpinnings of DDX3X dependent translation, we first used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5' UTRs and then employed HART to investigate how their perturbation impacts DDX3X-sensitivity. Additionally, we identified residues 38-44 as potential mediators of DDX3X's interaction with the translational machinery. HART revealed that both DDX3X's association with the ribosome complex as well as its helicase activity are required for its function in promoting the translation of DDX3X-sensitive 5' UTRs. These findings suggest DDX3X plays a crucial role regulating translation through its interaction with the translational machinery during ribosome scanning, and establish the HART reporter as a robust, lentivirally encoded measurement of DDX3X-dependent translation in cells.

The variant landscape and function of DDX3X in cancer and neurodevelopmental disorders.

RNA molecules rely on proteins across their life cycle. DDX3X encodes an X-linked DEAD-box RNA helicase with a Y-linked paralog, DDX3Y. DDX3X is central to the RNA life cycle and is implicated in many conditions, including cancer and the neurodevelopmental disorder DDX3X syndrome. DDX3X-linked conditions often exhibit sex differences, possibly due to differences between expression or function of the X- and Y-linked paralogs DDX3X and DDX3Y. DDX3X-related diseases have different mutational landscapes, indicating different roles of DDX3X. Understanding the role of DDX3X in normal and disease states will inform the understanding of DDX3X in disease. We review the function of DDX3X and DDX3Y, discuss how mutation type and sex bias contribute to human diseases involving DDX3X, and review possible DDX3X-targeting treatments.

Compromised nonsense-mediated RNA decay results in truncated RNA-binding protein production upon DUX4 expression.

Nonsense-mediated RNA decay (NMD) degrades transcripts carrying premature termination codons. NMD is thought to prevent the synthesis of toxic truncated proteins. However, whether loss of NMD results in widespread production of truncated proteins is unclear. A human genetic disease, facioscapulohumeral muscular dystrophy (FSHD), features acute inhibition of NMD upon expression of the disease-causing transcription factor, DUX4. Using a cell-based model of FSHD, we show production of truncated proteins from physiological NMD targets and find that RNA-binding proteins are enriched for aberrant truncations. The NMD isoform of one RNA-binding protein, SRSF3, is translated to produce a stable truncated protein, which is detected in FSHD patient-derived myotubes. Ectopic expression of truncated SRSF3 confers toxicity, and its downregulation is cytoprotective. Our results delineate the genome-scale impact of NMD loss. This widespread production of potentially deleterious truncated proteins has implications for FSHD biology as well as other genetic diseases where NMD is therapeutically modulated.

A ubiquitous GC content signature underlies multimodal mRNA regulation by DDX3X.

The road from transcription to protein synthesis is paved with many obstacles, allowing for several modes of post-transcriptional regulation of gene expression. A fundamental player in mRNA biology is DDX3X, an RNA binding protein that canonically regulates mRNA translation. By monitoring dynamics of mRNA abundance and translation following DDX3X depletion, we observe stabilization of translationally suppressed mRNAs. We use interpretable statistical learning models to uncover GC content in the coding sequence as the major feature underlying RNA stabilization. This result corroborates GC content-related mRNA regulation detectable in other studies, including hundreds of ENCODE datasets and recent work focusing on mRNA dynamics in the cell cycle. We provide further evidence for mRNA stabilization by detailed analysis of RNA-seq profiles in hundreds of samples, including a Ddx3x conditional knockout mouse model exhibiting cell cycle and neurogenesis defects. Our study identifies a ubiquitous feature underlying mRNA regulation and highlights the importance of quantifying multiple steps of the gene expression cascade, where RNA abundance and protein production are often uncoupled.

IGHMBP2 deletion suppresses translation and activates the integrated stress response.

IGHMBP2 is a non-essential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 upregulation. With recent studies showing the ISR can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.

Lysate and cell-based assays to probe the translational role of RNA helicases.

Translation initiation is the first step in protein synthesis, during which the small subunit of the ribosome scans the 5' untranslated region (5'UTR) of an mRNA to identify a start codon and commence translation elongation. By unwinding and modulating secondary structures and other RNA features present in the 5'UTR, RNA helicases can regulate ribosome scanning and start codon selection. This chapter presents an approach to measure the effect of RNA helicases on mRNA translation initiation. 5'UTR luciferase reporters are transcribed in vitro and employed in either of two assays. The in vitro assay translates the reporters in a cell-free whole-cell lysate system, which allows for greater biochemical manipulation and tighter control over confounding effects. In the alternative cell-based approach, the reporters are transfected and translated in living cells, which provides a more physiological setup. Either method can be used to investigate how the perturbation of a helicase, such as changes in protein levels or mutations, affects translation initiation at the 5'UTR level. The chapter also discusses alternative approaches, troubleshooting, and further applications of these methods. These assays will provide insights on the role of helicases and other translational factors as regulators of the proteome both in physiological and diseased settings.

Aberrant cortical development is driven by impaired cell cycle and translational control in a DDX3X syndrome model.

Mutations in the RNA helicase, DDX3X, are a leading cause of Intellectual Disability and present as DDX3X syndrome, a neurodevelopmental disorder associated with cortical malformations and autism. Yet, the cellular and molecular mechanisms by which DDX3X controls cortical development are largely unknown. Here, using a mouse model of Ddx3x loss-of-function we demonstrate that DDX3X directs translational and cell cycle control of neural progenitors, which underlies precise corticogenesis. First, we show brain development is sensitive to Ddx3x dosage; complete Ddx3x loss from neural progenitors causes microcephaly in females, whereas hemizygous males and heterozygous females show reduced neurogenesis without marked microcephaly. In addition, Ddx3x loss is sexually dimorphic, as its paralog, Ddx3y, compensates for Ddx3x in the developing male neocortex. Using live imaging of progenitors, we show that DDX3X promotes neuronal generation by regulating both cell cycle duration and neurogenic divisions. Finally, we use ribosome profiling in vivo to discover the repertoire of translated transcripts in neural progenitors, including those which are DDX3X-dependent and essential for neurogenesis. Our study reveals invaluable new insights into the etiology of DDX3X syndrome, implicating dysregulated progenitor cell cycle dynamics and translation as pathogenic mechanisms.

During development, a complex network of genes ensures that the brain develops in the right way. In particular, they control how special 'progenitor' cells multiply and mature to form neurons during a process known as neurogenesis. Genetic mutations that interfere with neurogenesis can lead to disability and defects such as microcephaly, where children are born with abnormally small brains. DDX3X syndrome is a recently identified condition characterised by intellectual disability, delayed acquisition of movement and language skills, low muscle tone and, frequently, a diagnosis of autism spectrum disorder. It emerges when certain mutations are present in the DDX3X gene, which helps to control the process by which proteins are built in a cell (also known as translation). The syndrome affects girls more often than boys, potentially because DDX3X is carried on the X chromosome. Many of the disease-causing mutations in the DDX3X gene also reduce the levels of DDX3X protein. However, exactly what genes DDX3X controls and how its loss impairs brain development remain poorly understood. To address this problem, Hoye et al. set out to investigate the role of Ddx3x in mice neurogenesis. Experiments with genetically altered mice confirmed that complete loss of the gene indeed caused severe reduction in brain size at birth; just as in humans with mild microcephaly, this was only present in affected females. Further genetic studies revealed the reason for this: the closely related Ddx3y gene, which is only present on the Y (male) chromosome, helped to compensate for the loss of Ddx3x in the male mice. Next, the effect of the loss of just one copy of Ddx3x on neurogenesis was examined by following how progenitor cells developed. This likely reflects DDX3X levels in patients with the syndrome. Loss of the gene made the cells divide more slowly and produce fewer mature nerve cells, suggesting that smaller brain size and brain malformations caused by mutations in DDX3X could be due to impaired neurogenesis. Finally, a set of further biochemical and genetic experiments revealed a key set of genes that are under the control of the DDX3X protein. These results shed new light on how a molecular actor which helps to control translation is a key part of normal brain development. This understanding could one day help improve clinical management or treatments for DDX3X syndrome and related neurological disorders.

SRSF1 governs progenitor-specific alternative splicing to maintain adult epithelial tissue homeostasis and renewal.

Alternative splicing generates distinct mRNA variants and is essential for development, homeostasis, and renewal. Proteins of the serine/arginine (SR)-rich splicing factor family are major splicing regulators that are broadly required for organ development as well as cell and organism viability. However, how these proteins support adult organ function remains largely unknown. Here, we used the continuously growing mouse incisor as a model to dissect the functions of the prototypical SR family protein SRSF1 during tissue homeostasis and renewal. We identified an SRSF1-governed alternative splicing network that is specifically required for dental proliferation and survival of progenitors but dispensable for the viability of differentiated cells. We also observed a similar progenitor-specific role of SRSF1 in the small intestinal epithelium, indicating a conserved function of SRSF1 across adult epithelial tissues. Thus, our findings define a regulatory mechanism by which SRSF1 specifically controls progenitor-specific alternative splicing events to support adult tissue homeostasis and renewal.

Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance.

Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant that achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-electron microscopy structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.

Antibiotics treat or prevent infections by killing bacteria or slowing down their growth. A large proportion of these drugs do this by disrupting an essential piece of cellular machinery called the ribosome which the bacteria need to make proteins. However, over the course of the treatment, some bacteria may gain genetic alterations that allow them to resist the effects of the antibiotic. Antibiotic resistance is a major threat to global health, and understanding how it emerges and spreads is an important area of research. Recent studies have discovered populations of resistant bacteria carrying a gene for a protein named chloramphenicol-florfenicol resistance, or Cfr for short. Cfr inserts a small modification in to the ribosome that prevents antibiotics from inhibiting the production of proteins, making them ineffective against the infection. To date, Cfr has been found to cause resistance to eight different classes of antibiotics. Identifying which mutations enhance its activity and protect bacteria is vital for designing strategies that fight antibiotic resistance. To investigate how the gene for Cfr could mutate and make bacteria more resistant, Tsai et al. performed a laboratory technique called directed evolution, a cyclic process which mimics natural selection. Genetic changes were randomly introduced in the gene for the Cfr protein and bacteria carrying these mutations were treated with tiamulin, an antibiotic rendered ineffective by the modification Cfr introduces into the ribosome. Bacteria that survived were then selected and had more mutations inserted. By repeating this process several times, Tsai et al. identified 'super' variants of the Cfr protein that lead to greater resistance. The experiments showed that these variants boosted resistance by increasing the proportion of ribosomes that contained the protective modification. This process was facilitated by mutations that enabled higher levels of Cfr protein to accumulate in the cell. In addition, the current study allowed, for the first time, direct visualization of how the Cfr modification disrupts the effect antibiotics have on the ribosome. These findings will make it easier for clinics to look out for bacteria that carry these 'super' resistant mutations. They could also help researchers design a new generation of antibiotics that can overcome resistance caused by the Cfr protein.

DDX3X and DDX3Y are redundant in protein synthesis.

DDX3 is a DEAD-box RNA helicase that regulates translation and is encoded by the X- and Y-linked paralogs DDX3X and DDX3Y While DDX3X is ubiquitously expressed in human tissues and essential for viability, DDX3Y is male-specific and shows lower and more variable expression than DDX3X in somatic tissues. Heterozygous genetic lesions in DDX3X mediate a class of developmental disorders called DDX3X syndrome, while loss of DDX3Y is implicated in male infertility. One possible explanation for female-bias in DDX3X syndrome is that DDX3Y encodes a polypeptide with different biochemical activity. In this study, we use ribosome profiling and in vitro translation to demonstrate that the X- and Y-linked paralogs of DDX3 play functionally redundant roles in translation. We find that transcripts that are sensitive to DDX3X depletion or mutation are rescued by complementation with DDX3Y. Our data indicate that DDX3X and DDX3Y proteins can functionally complement each other in the context of mRNA translation in human cells. DDX3Y is not expressed in a large fraction of the central nervous system. These findings suggest that expression differences, not differences in paralog-dependent protein synthesis, underlie the sex-bias of DDX3X-associated diseases.

DDX3 depletion represses translation of mRNAs with complex 5' UTRs.

DDX3 is an RNA chaperone of the DEAD-box family that regulates translation. Ded1, the yeast ortholog of DDX3, is a global regulator of translation, whereas DDX3 is thought to preferentially affect a subset of mRNAs. However, the set of mRNAs that are regulated by DDX3 are unknown, along with the relationship between DDX3 binding and activity. Here, we use ribosome profiling, RNA-seq, and PAR-CLIP to define the set of mRNAs that are regulated by DDX3 in human cells. We find that while DDX3 binds highly expressed mRNAs, depletion of DDX3 particularly affects the translation of a small subset of the transcriptome. We further find that DDX3 binds a site on helix 16 of the human ribosomal rRNA, placing it immediately adjacent to the mRNA entry channel. Translation changes caused by depleting DDX3 levels or expressing an inactive point mutation are different, consistent with different association of these genetic variant types with disease. Taken together, this work defines the subset of the transcriptome that is responsive to DDX3 inhibition, with relevance for basic biology and disease states where DDX3 is altered.

Control of ribosomal protein synthesis by the Microprocessor complex.

Ribosome biogenesis in eukaryotes requires the coordinated production and assembly of 80 ribosomal proteins and four ribosomal RNAs (rRNAs), and its rate must be synchronized with cellular growth. Here, we showed that the Microprocessor complex, which mediates the first step of microRNA processing, potentiated the transcription of ribosomal protein genes by eliminating DNA/RNA hybrids known as R-loops. Nutrient deprivation triggered the nuclear export of Drosha, a key component of the Microprocessor complex, and its subsequent degradation by the E3 ubiquitin ligase Nedd4, thereby reducing ribosomal protein production and protein synthesis. In mouse erythroid progenitors, conditional deletion of Drosha led to the reduced production of ribosomal proteins, translational inhibition of the mRNA encoding the erythroid transcription factor Gata1, and impaired erythropoiesis. This phenotype mirrored the clinical presentation of human "ribosomopathies." Thus, the Microprocessor complex plays a pivotal role in synchronizing protein synthesis capacity with cellular growth rate and is a potential drug target for anemias caused by ribosomal insufficiency.