CTD Code Research Articles

Suppression of inositol pyrophosphate toxicosis and hyper-repression of the fission yeast PHO regulon by loss-of-function mutations in chromatin remodelers Snf22 and Sol1.

Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes pho1, pho84, and tgp1) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP8 levels derepress the PHO regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP8 are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP8 toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP8 toxicity is alleviated by deletion of snf22+, the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating snf22-(D996A-E997A) allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of snf22+ hyper-repressed pho1 expression in phosphate-replete cells; suppressed the pho1 derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious prt lncRNA termination); and delayed pho1 induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the tgp1 and pho1 promoters, but not for the promoters that drive the synthesis of the PHO-repressive lncRNAs. Transcription profiling of snf22∆ and snf22-(D996A-E997A) cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic asp1-STF alleles derepress the PHO genes via the action of IP8 as an agonist of precocious lncRNA 3'-processing/termination. IP8 toxicosis is alleviated by mutations of the Pol2 CTD and the 3'-processing/termination machinery that dampen the impact of toxic IP8 levels on termination. In this study, a forward genetic screen revealed that IP8 toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the PHO mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of PHO-activating transcription factor Pho7 to its binding sites in the PHO mRNA promoters.

Duf89 abets lncRNA control of fission yeast phosphate homeostasis via its antagonism of precocious lncRNA transcription termination.

Fission yeast phosphate homeostasis gene pho1 is actively repressed during growth in phosphate-rich medium by transcription in cis of a long noncoding (lnc) RNA from the 5' flanking prt(nc-pho1) gene. Pho1 expression is: (i) derepressed by genetic maneuvers that favor precocious lncRNA 3'-processing and termination, in response to DSR and PAS signals in prt; and (ii) hyperrepressed in genetic backgrounds that dampen 3'-processing/termination efficiency. Governors of 3'-processing/termination include the RNA polymerase CTD code, the CPF (cleavage and polyadenylation factor) complex, termination factors Seb1 and Rhn1, and the inositol pyrophosphate signaling molecule 1,5-IP8 Here, we present genetic and biochemical evidence that fission yeast Duf89, a metal-dependent phosphatase/pyrophosphatase, is an antagonist of precocious 3'-processing/termination. We show that derepression of pho1 in duf89Δ cells correlates with squelching the production of full-length prt lncRNA and is erased or attenuated by: (i) DSR/PAS mutations in prt; (ii) loss-of-function mutations in components of the 3'-processing and termination machinery; (iii) elimination of the CTD Thr4-PO4 mark; (iv) interdicting CTD prolyl isomerization by Pin1; (v) inactivating the Asp1 kinase that synthesizes IP8; and (vi) loss of the putative IP8 sensor Spx1. The findings that duf89Δ is synthetically lethal with pho1-derepressive mutations CTD-S7A and aps1Δ-and that this lethality is rescued by CTD-T4A, CPF/Rhn1/Pin1 mutations, and spx1Δ-implicate Duf89 more broadly as a collaborator in cotranscriptional regulation of essential fission yeast genes. The duf89-D252A mutation, which abolishes Duf89 phosphohydrolase activity, phenocopied duf89 +, signifying that duf89Δ phenotypes are a consequence of Duf89 protein absence, not absence of Duf89 catalysis.

Transcriptional interference at tandem lncRNA and protein-coding genes: an emerging theme in regulation of cellular nutrient homeostasis.

Tandem transcription interference occurs when the act of transcription from an upstream promoter suppresses utilization of a co-oriented downstream promoter. Because eukaryal genomes are liberally interspersed with transcription units specifying long non-coding (lnc) RNAs, there are many opportunities for lncRNA synthesis to negatively affect a neighboring protein-coding gene. Here, I review two eukaryal systems in which lncRNA interference with mRNA expression underlies a regulated biological response to nutrient availability. Budding yeast SER3 is repressed under serine-replete conditions by transcription of an upstream SRG1 lncRNA that traverses the SER3 promoter and elicits occlusive nucleosome rearrangements. SER3 is de-repressed by serine withdrawal, which leads to shut-off of SRG1 synthesis. The fission yeast phosphate homeostasis (PHO) regulon comprises three phosphate acquisition genes – pho1, pho84, and tgp1 – that are repressed under phosphate-replete conditions by 5′ flanking lncRNAs prt, prt2, and nc-tgp1, respectively. lncRNA transcription across the PHO mRNA promoters displaces activating transcription factor Pho7. PHO mRNAs are transcribed during phosphate starvation when lncRNA synthesis abates. The PHO regulon is de-repressed in phosphate-replete cells by genetic manipulations that favor ‘precocious’ lncRNA 3′-processing/termination upstream of the mRNA promoters. PHO lncRNA termination is governed by the Pol2 CTD code and is subject to metabolite control by inositol pyrophosphates.

Inositol pyrophosphates impact phosphate homeostasis via modulation of RNA 3' processing and transcription termination.

Fission yeast phosphate acquisition genes pho1, pho84, and tgp1 are repressed in phosphate-rich medium by transcription of upstream lncRNAs. Here, we show that phosphate homeostasis is subject to metabolite control by inositol pyrophosphates (IPPs), exerted through the 3′-processing/termination machinery and the Pol2 CTD code. Increasing IP8 (via Asp1 IPP pyrophosphatase mutation) de-represses the PHO regulon and leads to precocious termination of prt lncRNA synthesis. pho1 de-repression by IP8 depends on cleavage-polyadenylation factor (CPF) subunits, termination factor Rhn1, and the Thr4 letter of the CTD code. pho1 de-repression by mutation of the Ser7 CTD letter depends on IP8. Simultaneous inactivation of the Asp1 and Aps1 IPP pyrophosphatases is lethal, but this lethality is suppressed by mutations of CPF subunits Ppn1, Swd22, Ssu72, and Ctf1 and CTD mutation T4A. Failure to synthesize IP8 (via Asp1 IPP kinase mutation) results in pho1 hyper-repression. Synthetic lethality of asp1Δ with Ppn1, Swd22, and Ssu72 mutations argues that IP8 plays an important role in essential 3′-processing/termination events, albeit in a manner genetically redundant to CPF. Transcriptional profiling delineates an IPP-responsive regulon composed of genes overexpressed when IP8 levels are increased. Our results establish a novel role for IPPs in cell physiology.

Tyr1 phosphorylation of RNA polymerase II CTD directs and expands the CTD code.

The C‐terminal domain of RNA polymerase II (CTD) acts as a platform to recruit factors that facilitate gene expression, RNA processing, and integrate transcription with the essential cellular processes of DNA repair and histone modification. This domain is composed of a species‐specific number of repeats of the consensus amino acid heptad YSPTSPS, classically numbered as Tyr1‐Ser2‐Pro3‐Thr4‐Ser5‐Pro6‐Ser7. The targeted recruitment of these myriad factors is accomplished by post‐translational modification (PTM) of these heptads, specifically phosphorylation of Tyr1, Ser2, Ser5, and Ser7. Although the contribution of these marks in isolation is extensively studied, the ability of these marks to co‐exist or influence one another has not been rigorously investigated.We take the first steps to characterize “cross‐talk” between phosphorylation marks and reveal a previously unknown role for Tyr1 phosphorylation in diversifying and directing CTD modification events. Tyr1 phosphorylation has been previously implicated in RNA polymerase II stability, antisense and enhancer transcription, anti‐termination, and as an indicator of DNA damage. However, the mechanism through which Tyr1 influences this diverse array of functions is unknown. Using a combination of mass spectrometry, molecular biology, and targeted cellular assays we find that Tyr1 phosphorylation generates a chemically diverse CTD landscape and can influence the specificity of downstream modification enzymes, resulting in novel and uncharacterized marks in the CTD code. We provide direct evidence for combinatorial CTD phosphorylation events, expand the lexicon of CTD coding marks, and demonstrate unexplored mechanisms to precisely control eukaryotic transcription and transcription coupled‐processes.Support or Funding InformationThis work is supported by grants from the National Institutes of Health (R01 GM104896 to Y.J.Z. and R21EB018391 to J.S.B.) and Welch Foundation (F‐1778 to Y.J.Z. and F‐1155 to J.S.B.). Voyager DE‐PROT MALDI MS data was collected in the University of Texas at Austin Proteomics Facility. Funding from the UT System for support of the UT System Proteomics Core Facility Network is gratefully acknowledged.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Human TFIIH Kinase CDK7 Regulates Transcription-Associated Epigenetic Modifications

CDK7 phosphorylates the RNA polymerase II (pol II) and activates the P-TEFb-associated kinase, CDK9, but its regulatory roles remain obscure. Here, using human CDK7 analog-sensitive (CDK7as) cells, we observed reduced capping enzyme recruitment, increased pol II promoterproximal pausing, and defective termination at gene 3'-ends upon CDK7 inhibition. Surprisingly, we also noted CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5'-ends and H3K36me3 was displaced toward gene 3'-ends in CDK7as cells. Mass spectrometry identified factors that bound TFIIH-phosphorylated vs. P-TEFb-phosphorylated (vs. unmodified); capping enzymes and H3K4 methyltransferase complexes, SETD1A/B, selectively bound phosphorylated and the H3K36 methyltransferase SETD2 specifically bound P-TEFb-phosphorylated CTD. Moreover, TFIIH phosphorylated stimulated SETD1A/B activity toward nucleosomes, revealing a mechanistic basis for CDK7 regulation of H3K4me3 spreading. Collectively, these results implicate a CDK7 dependent CTD code that regulates epigenetic marks in addition to RNA processing and pol II pausing.

Poly(A) site choice and Pol2 CTD Serine-5 status govern lncRNA control of phosphate-responsive tgp1 gene expression in fission yeast.

Expression of fission yeast glycerophosphate transporter Tgp1 is repressed in phosphate-rich medium and induced during phosphate starvation. Repression is enforced by transcription of the nc-tgp1 locus upstream of tgp1 to produce a long noncoding (lnc) RNA. Here we identify two essential elements of the nc-tgp1 promoter: a TATA box −30TATATATA−23 and a HomolD box −64CAGTCACA−57, mutations of which inactivate the nc-tgp1 promoter and de-repress the downstream tgp1 promoter under phosphate-replete conditions. The nc-tgp1 lncRNA poly(A) site maps to nucleotide +1636 of the transcription unit, which coincides with the binding site for Pho7 (1632TCGGACATTCAA1643), the transcription factor that drives tgp1 expression. Overlap between the lncRNA template and the tgp1 promoter points to transcriptional interference as the simplest basis for lncRNA repression. We identify a shorter RNA derived from the nc-tgp1 locus, polyadenylated at position +508, well upstream of the tgp1 promoter. Mutating the nc-tgp1-short RNA polyadenylation signal abolishes de-repression of the downstream tgp1 promoter elicited by Pol2 CTD Ser5Ala phospho-site mutation. Ser5 mutation favors utilization of the short RNA poly(A) site, thereby diminishing transcription of the lncRNA that interferes with the tgp1 promoter. Mutating the nc-tgp1-short RNA polyadenylation signal attenuates induction of the tgp1 promoter during phosphate starvation. Polyadenylation site choice governed by CTD Ser5 status adds a new level of lncRNA control of gene expression and reveals a new feature of the fission yeast CTD code.

Human TFIIH Kinase CDK7 Regulates Transcription-Associated Chromatin Modifications

CDK7 phosphorylates the RNA polymerase II (pol II) C-terminal domain CTD and activates the P-TEFb-associated kinase CDK9, but its regulatory roles remain obscure. Here, using human CDK7 analog-sensitive (CDK7as) cells, we observed reduced capping enzyme recruitment, increased pol II promoter-proximal pausing, and defective termination at gene 3' ends upon CDK7 inhibition. We also noted that CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5' ends and H3K36me3 was displacedtoward gene 3' ends in CDK7as cells. Mass spectrometry identified factors that bound TFIIH-phosphorylated versus P-TEFb-phosphorylated CTD (versus unmodified); capping enzymes and H3K4 methyltransferase complexes, SETD1A/B, selectively bound phosphorylated CTD, and the H3K36 methyltransferase SETD2 specifically bound P-TEFb-phosphorylated CTD. Moreover, TFIIH-phosphorylated CTD stimulated SETD1A/B activity toward nucleosomes, revealing a mechanistic basis for CDK7 regulation of H3K4me3 spreading. Collectively, these results implicate a CDK7-dependent "CTD code" that regulates chromatin marks in addition to RNA processing and pol II pausing.

Nature's Imitation Game: Decipher the Combinatorial CTD Code for Eukaryotic Transcription

In eukaryotes, the C‐terminal domain of RNA polymerase II (CTD) orchestrates the temporal and spatial control of transcription and is involved in the epigenetic regulation of gene expression. Errors in CTD regulation can result in cell death, cancer and severe developmental defects. The CTD executes its function as a transcription template to recruite transcription regulators through various post‐translational modifications on its heptad repeats. Recently, novel modifications on CTD have been identified, setting the stage for the possibility of combinatorial mechanisms for transcription regulation. We focused on the eukaryotic phosphatases in the CTD regulation and how they affect the function of CTD binding proteins. Determination of the structures of these CTD phosphatases guide our structure‐function investigation. Using a collection of chemical biology techniques including analytical chemistry, spectroscopy, enzymology and structural biology, we dissect the multiple layers of regulation in CTD‐mediated transcription. In particular, we developed an innovative method of mass spectromety, Ultraviolet photodissociation (UVPD), to map the site of modification in S. cerevisiae and Drosophila CTDs. Overall, we probe how the identities and phosphorylation states of individual residues in the consensus heptad repeats of CTD result in differentiated post‐translational modification states within CTD that lead to various transcription outcomes.Support or Funding InformationThe work is funded by National Institute for Health, Welch Foundation and Alzheimer's Drug Discovery Foundation.

Mapping the Phosphorylation Pattern of Drosophila melanogaster RNA Polymerase II Carboxyl-Terminal Domain Using Ultraviolet Photodissociation Mass Spectrometry.

Phosphorylation of the C-terminal domain of RNA polymerase II (CTD) plays an essential role in eukaryotic transcription by recruiting transcriptional regulatory factors to the active polymerase. However, the scarcity of basic residues and repetitive nature of the CTD sequence impose a huge challenge for site-specific characterization of phosphorylation, hindering our understanding of this crucial biological process. Herein, we apply LC-UVPD-MS methods to analyze post-translational modification along native sequence CTDs. Application of our method to the Drosophila melanogaster CTD reveals the phosphorylation pattern of this model organism for the first time. The divergent nature of fly CTD allows us to derive rules defining how flanking residues affect phosphorylation choice by CTD kinases. Our data support the use of LC-UVPD-MS to decipher the CTD code and determine rules that program its function.

Pol II CTD Code Light

In this issue of Molecular Cell, Schüller et al. (2016) and Suh et al. (2016) describe genetic and mass spectrometry methodologies for mapping phosphorylation sites on the tandem repeats of the RNA polymerase II CTD. The results suggest that the CTD Code may be simpler than expected.

Writing and Reading the CTD Code: In Vitro Reconstruction of Post‐Translational Modifications on RNA Polymerase II

Information flow within the cell is filtered through a vast array of codes. Vital biological data is compiled at the DNA, RNA, and protein levels with additional information relayed through an ever‐expanding repertoire of post‐transcriptional and post‐translational modifications (PTM). The PTM state of proteins can impact their recognition, structure, and function as well as relay important signals to influence cellular events, as found in histone code. A newly characterized coding system in eukaryotic transcription is “CTD code.” The C‐terminal Domain of RNA Polymerase II (CTD) is a unique structure composed of repeats of the consensus heptad YSPTSPS. These residues are subject to a variety of PTMs and specific factors are recruited depending on the PTM state of CTD. Particular PTM marks are linked to transcriptional events including initiation, elongation, and termination. The PTM state of CTD is generated and altered through the action of PTM enzymes such as kinases and phosphatases. Current technologies allow the determination of isolated and simplistic PTM marks along the CTD but lack the potential for the investigation of a combinatorial code or the identification of novel PTM marks. To overcome this we propose a bottom up approach to in vitro reconstruct the CTD code. We treat CTD substrate with PTM enzymes and characterize resultant modifications using a combination of molecular biology and mass spectrometry. Our novel mass spectrometry methodologies allow for heptad level resolution and characterization of individual and combinatorial PTM marks along CTD. Using this interdisciplinary cipher we can determine the lexicon for CTD code and better understand the impact of PTM on cellular communication.

Investigating the Structural Basis of the CTD Code

The goal of this project is to study the structure and function of the C‐terminal domain of RNA Polymerase II (CTD). The CTD, which contains tandem repeats of the consensus heptad sequence YSPTSPS, orchestrates transcription by recruiting trans‐acting regulatory factors, directed by a set of post‐translational modifications known as the CTD code. Since the discovery of the CTD code, much effort has gone into the identification of novel modifications, the determination of their effects on specific CTD interactions, and the structural characterization of transcription factors in complex with the CTD. Much less is known about the CTD in its unbound state and of the possible structural effects of the post‐translational modifications that constitute the code. Recently, we have begun biophysical characterization of the unmodified and hyperphosphorylated CTD using a combination of techniques, including 13C Direct‐Detect Nuclear Magnetic Resonance (NMR) spectroscopy and Mass Spectrometry. Preliminary results suggest that the CTD exists in an extended conformation that cannot be adequately described by the previously proposed beta–spiral model. The models we have developed serve as novel structural hypothesis, relevant for determination of the structural basis for the CTD code and the elucidation of the molecular mechanisms of eukaryotic transcription.This work is funded by NSF CAREER award (MCB‐0953918) to Scott A. Showalter.

CTD Code: a Combinatorial Code for Eukaryotic Transcription

In eukaryotes, the C‐terminal domain of RNA polymerase II (CTD) orchestrates the temporal and spatial control of transcription and is involved in the epigenetic regulation of gene expression. Errors in CTD regulation can result in cell death, cancer and severe developmental defects. The CTD executes its function as transcription template through various post‐translational modifications on its heptad repeat sequences. Recently, novel modifications on new regulatory sites of CTD have been identified, setting the stage for the possibility of combinatorial mechanisms for transcription regulation. We focused on two well‐characterized modification of CTD, namely serine phosphorylation and prolyl isomerization, and discuss the interplay between the enzymes regulating these modification states. To address this question, we developed chemical compounds that can be used to mimic the cis and trans isomer states of proline residues in the CTD and therefore probe the selectivity of CTD binding proteins that are sensitive for this subtle variation. Our results established that the selectivity of prolyl isomerization state of CTD by phosphatases results in different regulatory effects and can lead to differentiated CTD phosphorylation states and eventually, transcription outcome. In particular, the phosphatases that specially recognize the minor species of cis proline will display enhanced activity upon prolyl isomerase regulation which is essential for accurate transcription termination. The application of such chemical probes can help us understand the molecular mechanism of the interplay between different post‐translational modifications with prolyl isomerization state and how that affects the conformational status of CTD in transcription.

CTD Code: a Combinatorial Code for Eukaryotic Transcription

In eukaryotes, the C-terminal domain of RNA polymerase II (CTD) orchestrates the temporal and spatial control of transcription and is involved in the epigenetic regulation of gene expression. Errors in CTD regulation can result in cell death, cancer and severe developmental defects. The CTD executes its function as transcription modulator through various post-translational modifications on its heptad repeat sequences. Recently, novel modifications on new regulatory sites of CTD have been identified, setting the stage for the possibility of combinatorial mechanisms for transcription regulation. We focused on two well-characterized modification of CTD, namely serine phosphorylation and prolyl isomerization, and discuss the interplay between the enzymes regulating these modification states. Our results established that the selectivity of prolyl isomerization state of CTD on phosphatases can lead to differentiated outcome for the CTD phosphorylation state and therefore, transcription. To further investigate the prolyl selectivity, we developed chemical compounds that can be used to probe such subtle structural variation in the CTD binding proteins. These compounds closely mimic the cis or trans proline state and can be effectively recognized by CTD phosphatases. The application of such chemical probes can help us understand the molecular mechanism of the interplay between phosphorylation and prolyl isomerization state and how that affect the conformational status of CTD in transcription temporally and spatially.

Emerging roles for RNA polymerase II CTD in Arabidopsis

Post-translational modifications of the carboxy-terminal domain of the largest subunit of RNA polymerase II (RNAPII CTD) provide recognition marks to coordinate recruitment of numerous nuclear factors controlling transcription, cotranscriptional RNA processing, chromatin remodeling, and RNA export. Compared with the progress in yeast and mammals, deciphering the regulatory roles of position-specific combinatorial CTD modifications, the so-called CTD code, is still at an early stage in plants. In this review, we discuss some of the recent advances in understanding of the molecular mechanisms controlling the deposition and recognition of RNAPII CTD marks in plants during the transcriptional cycle and highlight some intriguing differences between regulatory components characterized in yeast, mammals, and plants.

Expanding the CTD code

Expanding the CTD code

Modification and biological role of histone

Histone is one of critical components of chromatin, which amino acid residues at the N-terminus can be covalently modified. Histone modification (HM) can change the chromatin conformation and induce transcription or gene silencing. Not only can HM control gene expression, but also participate in cell division, cell apoptosis and memory formation by recruiting protein complex and affecting downstream proteins. HM can also have the impact on immune system and inflammatory reaction. In addition, lots of recent studies have indicated that histone code (or HM) is related to the CTD code, circadian clock and DNA repair, implying the significance of HM. The domains of protein complex can never be replaced, because they play a mediating role during the formation and deciphering of histone code, as well as the modification cascade and the recruitment of protein complex. Therefore, these domains are very important to comprehend the histone code. Because of the widespread use of analytical techniques, such as mass-spectrometry, new domains will be discovered. Herein, our review focuses on the basic concept, recent progress and hot points of the histone code study.

Gene Control during Transcription Elongation

Gene Control during Transcription Elongation

Bio-molecular architects: a scaffold provided by the C-terminal domain of eukaryotic RNA polymerase II.

In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and temporally by the C-terminal domain of RNA polymerase II (CTD). The CTD provides a dynamic platform to recruit different regulators of the transcription apparatus. Different posttranslational modifications are precisely applied to specific sites of the CTD to coordinate transcription process. Regulators of the RNA polymerase II must identify specific sites in the CTD for cellular survival, metabolism, and development. Even though the CTD is disordered in the eukaryotic RNA polymerase II crystal structures due to its intrinsic flexibility, recent advances in the complex structural analysis of the CTD with its binding partners provide essential clues for understanding how selectivity is achieved for individual site recognition. The recent discoveries of the interactions between the CTD and histone modification enzymes disclose an important role of the CTD in epigenetic control of the eukaryotic gene expression. The intersection of the CTD code with the histone code discloses an intriguing yet complicated network for eukaryotic transcriptional regulation.