Three-step mechanism of promoter escape by RNA polymerase II.

The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.

Persistent TFIIH binding to non-excised DNA damage causes cell and developmental failure.

Congenital nucleotide excision repair (NER) deficiency gives rise to several cancer-prone and/or progeroid disorders. It is not understood how defects in the same DNA repair pathway cause different disease features and severity. Here, we show that the absence of functional ERCC1-XPF or XPG endonucleases leads to stable and prolonged binding of the transcription/DNA repair factor TFIIH to DNA damage, which correlates with disease severity and induces senescence features in human cells. In vivo, in C. elegans, this prolonged TFIIH binding to non-excised DNA damage causes developmental arrest and neuronal dysfunction, in a manner dependent on transcription-coupled NER. NER factors XPA and TTDA both promote stable TFIIH DNA binding and their depletion therefore suppresses these severe phenotypical consequences. These results identify stalled NER intermediates as pathogenic to cell functionality and organismal development, which can in part explain why mutations in XPF or XPG cause different disease features than mutations in XPA or TTDA.

The role of Transcription Factor IIH complex in nucleotide excision repair.

DNA damage occurs throughout life from a variety of sources, and it is imperative to repair damage in a timely manner to maintain genome stability. Thus, DNA repair mechanisms are a fundamental part of life. Nucleotide excision repair (NER) plays an important role in the removal of bulky DNA adducts, such as cyclobutane pyrimidine dimers from ultraviolet light or DNA crosslinking damage from platinum-based chemotherapeutics, such as cisplatin. A main component for the NER pathway is transcription factor IIH (TFIIH), a multifunctional, 10-subunit protein complex with crucial roles in both transcription and NER. In transcription, TFIIH is a component of the pre-initiation complex and is important for promoter opening and the phosphorylation of RNA Polymerase II (RNA Pol II). During repair, TFIIH is important for DNA unwinding, recruitment of downstream repair factors, and verification of the bulky lesion. Several different disease states can arise from mutations within subunits of the TFIIH complex. Most strikingly are xeroderma pigmentosum (XP), XP combined with Cockayne syndrome (CS), and trichothiodystrophy (TTD). Here, we summarize the recruitment and functions of TFIIH in the two NER subpathways, global genomic (GG-NER) and transcription-coupled NER (TC-NER). We will also discuss how TFIIH's roles in the two subpathways lead to different genetic disorders.

The Link of mRNA and rRNA Transcription by PUF60/FIR through TFIIH/P62 as a Novel Therapeutic Target for Cancer.

The interaction between mRNA and ribosomal RNA (rRNA) transcription in cancer remains unclear. RNAP I and II possess a common N-terminal tail (NTT), RNA polymerase subunit RPB6, which interacts with P62 of transcription factor (TF) IIH, and is a common target for the link between mRNA and rRNA transcription. The mRNAs and rRNAs affected by FUBP1-interacting repressor (FIR) were assessed via RNA sequencing and qRT-PCR analysis. An FIR, a c-myc transcriptional repressor, and its splicing form FIRΔexon2 were examined to interact with P62. Protein interaction was investigated via isothermal titration calorimetry measurements. FIR was found to contain a highly conserved region homologous to RPB6 that interacts with P62. FIRΔexon2 competed with FIR for P62 binding and coactivated transcription of mRNAs and rRNAs. Low-molecular-weight chemical compounds that bind to FIR and FIRΔexon2 were screened for cancer treatment. A low-molecular-weight chemical, BK697, which interacts with FIRΔexon2, inhibited tumor cell growth with rRNA suppression. In this study, a novel coactivation pathway for cancer-related mRNA and rRNA transcription through TFIIH/P62 by FIRΔexon2 was proposed. Direct evidence in X-ray crystallography is required in further studies to show the conformational difference between FIR and FIRΔexon2 that affects the P62-RBP6 interaction.

TFIIH central activity in nucleotide excision repair to prevent disease.

The heterodecameric transcription factor IIH (TFIIH) functions in multiple cellular processes, foremost in nucleotide excision repair (NER) and transcription initiation by RNA polymerase II. TFIIH is essential for life and hereditary mutations in TFIIH cause the devastating human syndromes xeroderma pigmentosum, Cockayne syndrome or trichothiodystrophy, or combinations of these. In NER, TFIIH binds to DNA after DNA damage is detected and, using its translocase and helicase subunits XPB and XPD, opens up the DNA and checks for the presence of DNA damage. This central activity leads to dual incision and removal of the DNA strand containing the damage, after which the resulting DNA gap is restored. In this review, we discuss new structural and mechanistic insights into the central function of TFIIH in NER. Moreover, we provide an elaborate overview of all currently known patients and diseases associated with inherited TFIIH mutations and describe how our understanding of TFIIH function in NER and transcription can explain the different disease features caused by TFIIH deficiency.

When the +1 nucleosome gets in the way, TFIIH fails but does not fall.

In this issue of Molecular Cell, Abril-Garrido et al.1 used cryo-EM to uncover that the +1 nucleosome inhibits transcription by interfering with the function of the TFIIH translocase via mechanisms that depend on its position relative to the transcription start site.

Structural polymorphism of the PH domain in TFIIH.

The general transcription factor TFIIH is a multi-subunit complex involved in transcription, DNA repair, and cell cycle in eukaryotes. In the human p62 subunit and the budding yeast Saccharomyces cerevisiae Tfb1 subunit of TFIIH, the pleckstrin homology (PH) domain (hPH/scPH) recruits TFIIH to transcription-start and DNA-damage sites by interacting with an acidic intrinsically disordered region in transcription and repair factors. Whereas metazoan PH domains are highly conserved and adopt a similar structure, fungal PH domains are divergent and only the scPH structure is available. Here, we have determined the structure of the PH domain from Tfb1 of fission yeast Schizosaccharomyces pombe (spPH) by NMR. spPH holds an architecture, including the core and external backbone structures, that is closer to hPH than to scPH despite having higher amino acid sequence identity to scPH. In addition, the predicted target-binding site of spPH shares more amino acid similarity with scPH, but spPH contains several key residues identified in hPH as required for specific binding. Using chemical shift perturbation, we have identified binding modes of spPH to spTfa1, a homologue of hTFIIEα, and to spRhp41, a homologue of the repair factors hXPC and scRad4. Both spTfa1 and spRhp41 bind to a similar but distinct surface of spPH by modes that differ from those of target proteins binding to hPH and scPH, revealing that the PH domain of TFIIH interacts with its target proteins in a polymorphic manner in Metazoa, and budding and fission yeasts.

Key Risk Genes Identified From the Postmortem Brain of Patients With Major Depressive Disorder and Their Potential Clinical Applications.

BACKGROUND: Major depressive disorder (MDD) is a type of emotional dysfunction, and its pathogenesis has not been fully elucidated. Specifically, the key molecules in depression-related brain regions involved in this disease and their contributions to this disease are currently unclear. METHODS: GSE53987 and GSE54568 were selected from the Gene Expression Omnibus database. The data were standardized to identify the common differentially expressed genes (DEGs) in the cortex of MDD patients in the 2 datasets. The DEGs were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. The STRING database was used to build protein-protein interaction networks, and the cytoHubba plugin was used to identify hub genes. Furthermore, we selected another blood transcriptome dataset that included 161 MDD and 169 control samples to explore the changes in the screened hub genes. Mice were subjected to 4 weeks of chronic unpredictable mild stress to establish an animal model of depression, and the expression of these hub genes in tissues of the prefrontal cortex was then detected by quantitative real time polymerase chain reaction (qRT-PCR). We subsequently predicted the possible posttranscriptional regulatory networks and traditional Chinese medicine according to the hub genes using a few online databases. RESULTS: The analysis identified 147 upregulated genes and 402 downregulated genes were identified in the cortex of MDD patients compared with that of the controls. Enrichment analyses revealed that DEGs were predominantly enriched in synapse-related cell functions, linoleic acid metabolism, and other pathways. Protein-protein interaction analysis identified 20 hub genes based on the total score. The changes in KDM6B, CUX2, NAAA, PHKB, NFYA, GTF2H1, CRK, CCNG2, ACER3, and SLC4A2 in the peripheral blood of MDD patients were consistent with those in the brain. Furthermore, the prefrontal cortex of mice with depressive-like behaviors showed significantly increased Kdm6b, Aridb1, Scaf11, and Thoc2 expression and decreased Ccng2 expression compared with that of normal mice, which was consistent with the results found for the human brain. Potential therapeutic candidates, such as citron, fructus citri, leaves of Panax Notoginseng, sanchi flower, pseudoginseng, and dan-shen root, were selected via traditional Chinese medicine screening. CONCLUSIONS: This study identified several novel hub genes in specific brain regions involved in the pathogenesis of MDD, which may not only deepen our understanding of depression but may also provide new ideas for its diagnosis and treatment.

Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases.

Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH's global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.

Identification of two unannotated miRNAs in classic Hodgkin lymphoma cell lines.

MicroRNAs (miRNAs) are small non coding RNAs responsible for posttranscriptional regulation of gene expression. Even though almost 2000 precursors have been described so far, additional miRNAs are still being discovered in normal as well as malignant cells. Alike protein coding genes, miRNAs may acquire oncogenic properties in consequence of altered expression or presence of gain or loss of function mutations. In this study we mined datasets from miRNA expression profiling (miRNA-seq) of 7 classic Hodgkin Lymphoma (cHL) cell lines, 10 non-Hodgkin lymphoma (NHL) cell lines and 56 samples of germinal center derived B-cell lymphomas. Our aim was to discover potential novel cHL oncomiRs not reported in miRBase (release 22.1) and expressed in cHL cell lines but no other B-cell lymphomas. We identified six such miRNA candidates in cHL cell lines and verified the expression of two of them encoded at chr2:212678788-212678849 and chr5:168090507-168090561 (GRCh38). Interestingly, we showed that one of the validated miRNAs (located in an intron of the TENM2 gene) is expressed together with its host gene. TENM2 is characterized by hypomethylation and open chromatin around its TSS in cHL cell lines in contrast to NHL cell lines and germinal centre B-cells respectively. It indicates an epigenetic mechanism responsible for aberrant expression of both, the TENM2 gene and the novel miRNA in cHL cell lines. Despite the GO analysis performed with the input of the in silico predicted novel miRNA target genes did not reveal ontologies typically associated with cHL pathogenesis, it pointed to several interesting candidates involved in i.e. lymphopoiesis. These include the lymphoma related BCL11A gene, the IKZF2 gene involved in lymphocyte development or the transcription initiator GTF2H1.

A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair.

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

TFIIH mutations can impact on translational fidelity of the ribosome.

TFIIH is a complex essential for transcription of protein-coding genes by RNA polymerase II, DNA repair of UV-lesions and transcription of rRNA by RNA polymerase I. Mutations in TFIIH cause the cancer prone DNA-repair disorder xeroderma pigmentosum (XP) and the developmental and premature aging disorders trichothiodystrophy (TTD) and Cockayne syndrome. A total of 50% of the TTD cases are caused by TFIIH mutations. Using TFIIH mutant patient cells from TTD and XP subjects we can show that the stress-sensitivity of the proteome is reduced in TTD, but not in XP. Using three different methods to investigate the accuracy of protein synthesis by the ribosome, we demonstrate that translational fidelity of the ribosomes of TTD, but not XP cells, is decreased. The process of ribosomal synthesis and maturation is affected in TTD cells and can lead to instable ribosomes. Isolated ribosomes from TTD patients show an elevated error rate when challenged with oxidized mRNA, explaining the oxidative hypersensitivity of TTD cells. Treatment of TTD cells with N-acetyl cysteine normalized the increased translational error-rate and restored translational fidelity. Here we describe a pathomechanism that might be relevant for our understanding of impaired development and aging-associated neurodegeneration.

Impact of GTF2H1 and RAD54L2 polymorphisms on the risk of lung cancer in the Chinese Han population.

BACKGROUND: Repair pathway genes play an important role in the development of lung cancer. The study aimed to assess the correlation between single nucleotide polymorphisms (SNPs) in DNA repair gene (GTF2H1 and RAD54L2) and the risk of lung cancer. METHODS: Five SNPs in GTF2H1 and four SNPs in RAD54L2 in 506 patients with lung cancer and 510 age-and gender-matched healthy controls were genotyped via the Agena MassARRAY platform. The influence of GTF2H1 and RAD54L2 polymorphisms on lung cancer susceptibility was assessed using logistic regression analysis by calculating odds ratios (ORs) and their corresponding 95% confidence intervals (CIs). RESULTS: RAD54L2 rs9864693 GC genotype increased the risk of lung cancer (OR = 1.33, 95%CI: 1.01-1.77, p = 0.045). Stratified analysis found that associations of RAD54L2 rs11720298, RAD54L2 rs4687592, RAD54L2 rs9864693 and GTF2H1 rs4150667 with lung cancer risk were found in subjects aged ≤ 59 years. Precisely, a protective effect of RAD54L2 rs11720298 on the occurrence of lung cancer was observed in non-smokers and drinkers. GTF2H1 rs4150667 was associated with a decreased risk of lung cancer in subjects with BMI ≤ 24 kg/m2. RAD54L2 rs4687592 was associated with an increased risk of lung cancer in drinkers. In addition, GTF2H1 rs3802967 was associated with a reduced risk of lung squamous cell carcinoma. CONCLUSION: Our study first revealed that RAD54L2 rs9864693 was associated with an increased risk of lung cancer in the Chinese Han population. This study may increase the understanding of the effect of RAD54L2 and GTF2H1 polymorphisms on lung cancer occurrence.

TFIIH stabilization recovers the DNA repair and transcription dysfunctions in thermo-sensitive trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare hereditary disease whose prominent feature is brittle hair. Additional clinical signs are physical and neurodevelopmental abnormalities and in about half of the cases hypersensitivity to UV radiation. The photosensitive form of TTD (PS-TTD) is most commonly caused by mutations in the ERCC2/XPD gene encoding a subunit of the transcription/DNA repair complex TFIIH. Here we report novel ERCC2/XPD mutations affecting proper protein folding, which generate thermo-labile forms of XPD associated with thermo-sensitive phenotypes characterized by reversible aggravation of TTD clinical signs during episodes of fever. In patient cells, the newly identified XPD variants result in thermo-instability of the whole TFIIH complex and consequent temperature-dependent defects in DNA repair and transcription. Improving the protein folding process by exposing patient cells to low temperature or to the chemical chaperone glycerol allowed rescue of TFIIH thermo-instability and a concomitant recovery of the complex activities. Besides providing a rationale for the peculiar thermo-sensitive clinical features of these new cases, the present findings demonstrate how variations in the cellular concentration of mutated TFIIH impact the cellular functions of the complex and underlie how both quantitative and qualitative TFIIH alterations contribute to TTD clinical features.

Systematic mutagenesis of TFIIH subunit p52/Tfb2 identifies residues required for XPB/Ssl2 subunit function and genetic interactions with TFB6.

TFIIH is an evolutionarily conserved complex that plays central roles in both RNA polymerase II (pol II) transcription and DNA repair. As an integral component of the pol II preinitiation complex, TFIIH regulates pol II enzyme activity in numerous ways. The TFIIH subunit XPB/Ssl2 is an ATP-dependent DNA translocase that stimulates promoter opening prior to transcription initiation. Crosslinking-mass spectrometry and cryo-EM results have shown a conserved interaction network involving XPB/Ssl2 and the C-terminal Hub region of the TFIIH p52/Tfb2 subunit, but the functional significance of specific residues is unclear. Here, we systematically mutagenized the HubA region of Tfb2 and screened for growth phenotypes in a TFB6 deletion background in Saccharomyces cerevisiae. We identified six lethal and 12 conditional mutants. Slow growth phenotypes of all but three conditional mutants were relieved in the presence of TFB6, thus identifying a functional interaction between Tfb2 HubA mutants and Tfb6, a protein that dissociates Ssl2 from TFIIH. Our biochemical analysis of Tfb2 mutants with severe growth phenotypes revealed defects in Ssl2 association, with similar results in human cells. Further characterization of these tfb2 mutant cells revealed defects in GAL gene induction, and reduced occupancy of TFIIH and pol II at GAL gene promoters, suggesting that functionally competent TFIIH is required for proper pol II recruitment to preinitiation complexes in vivo. Consistent with recent structural models of TFIIH, our results identify key residues in the p52/Tfb2 HubA domain that are required for stable incorporation of XPB/Ssl2 into TFIIH and for pol II transcription.

Phosphorylation of XPD drives its mitotic role independently of its DNA repair and transcription functions.

The helicase XPD is known as a key subunit of the DNA repair/transcription factor TFIIH. However, here, we report that XPD, independently to other TFIIH subunits, can localize with the motor kinesin Eg5 to mitotic spindles and the midbodies of human cells. The XPD/Eg5 partnership is promoted upon phosphorylation of Eg5/T926 by the kinase CDK1, and conversely, it is reduced once Eg5/S1033 is phosphorylated by NEK6, a mitotic kinase that also targets XPD at T425. The phosphorylation of XPD does not affect its DNA repair and transcription functions, but it is required for Eg5 localization, checkpoint activation, and chromosome segregation in mitosis. In XPD-mutated cells derived from a patient with xeroderma pigmentosum, the phosphomimetic form XPD/T425D or even the nonphosphorylatable form Eg5/S1033A specifically restores mitotic chromosome segregation errors. These results thus highlight the phospho-dependent mitotic function of XPD and reveal how mitotic defects might contribute to XPD-related disorders.

Drugs repurposing: An approach to identify new hits against anticancer drug target TFIIH subunit p8.

Drug repositioning is one of the most effective approaches towards drug discovery and development. It involves the identification of new therapeutic indications of existing drugs. The present study evaluated several drugs for their ability to modulate activity of the p8 subunit of TFIIH complex. Negative modulation of p8 subunit activity disrupts protein-protein interactions (PPIs) among the subunits of TFIIH complex, and thereby the TFIIH-associated functions. TFIIH complex has key role in the transcription and nucleotide excision repair activity in cancerous cells. TFIIH complex has emerged as a privileged drug target in anticancer research. Out of 60 drugs, amlopipine (13), diltiazem (16), gemfibrozil (19), levocitrizine dihydrochloride (20), losartan potassium (22), clorthalidone (24), and escitalopram (28) showed interactions with subunit p8 in the ligand-protein binding and chemical shift perturbation studies. The Kd values were found to be between 0.25 and 1 mM. These drugs also caused thermal destabilization of the subunit p8 by negatively shifting the melting temperature by ≥ 2 °C. Molecular docking studies indicated the interaction of these drugs with important residues of p8-p52 complex, such as Glu48, Lys51, Glu496, and Glu455 via non-covalent interactions. This study has thereby identified 7 drugs that can be investigated further as potential anticancer drugs.

Ssl2/TFIIH function in transcription start site scanning by RNA polymerase II in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, RNA polymerase II (Pol II) selects transcription start sites (TSSs) by a unidirectional scanning process. During scanning, a preinitiation complex (PIC) assembled at an upstream core promoter initiates at select positions within a window ~40-120 bp downstream. Several lines of evidence indicate that Ssl2, the yeast homolog of XPB and an essential and conserved subunit of the general transcription factor (GTF) TFIIH, drives scanning through its DNA-dependent ATPase activity, therefore potentially controlling both scanning rate and scanning extent (processivity). To address questions of how Ssl2 functions in promoter scanning and interacts with other initiation activities, we leveraged distinct initiation-sensitive reporters to identify novel ssl2 alleles. These ssl2 alleles, many of which alter residues conserved from yeast to human, confer either upstream or downstream TSS shifts at the model promoter ADH1 and genome-wide. Specifically, tested ssl2 alleles alter TSS selection by increasing or narrowing the distribution of TSSs used at individual promoters. Genetic interactions of ssl2 alleles with other initiation factors are consistent with ssl2 allele classes functioning through increasing or decreasing scanning processivity but not necessarily scanning rate. These alleles underpin a residue interaction network that likely modulates Ssl2 activity and TFIIH function in promoter scanning. We propose that the outcome of promoter scanning is determined by two functional networks, the first being Pol II activity and factors that modulate it to determine initiation efficiency within a scanning window, and the second being Ssl2/TFIIH and factors that modulate scanning processivity to determine the width of the scanning widow.

In eukaryotic organisms such as yeast, the process of converting genes into proteins begins with the transcription of DNA sequences into mRNA molecules. An enzyme called RNA Polymerase II (Pol II) is responsible for creating new strands of mRNA, but a variety of other so called transcription factors is also needed to kickstart the transcription process. These transcription factors are delivered to genes, where they attach to specific sequences, or promoters, which sit at the beginning of each gene. Once these transcription factors are in place, the double stranded DNA is unzipped to provide access to the DNA that will serve as the template for transcription. In budding yeast, Pol II and another specific transcription factor, known as TFIIH, work together to scan these promoter sequences to find the appropriate start sites of mRNA synthesis. However, several aspects of this process, such as how TFIIH works in promoter scanning, how far its scanning functions can extend, and how its activity is controlled, are currently poorly understood. Zhao et al. have investigated these questions in budding yeast. Using a range of genetic and genomic techniques, Zhao et al. found that certain sections of TFIIH were involved in choosing specific transcription start sites of mRNA synthesis during promoter scanning. These sections were identical in different eukaryotic organisms from yeast to humans, suggesting that these regions may be important for tuning or controlling the activity of TFIIH. Moreover, in yeast, the activity of TFIIH determines how far the scanning unit was able to move along the promoter DNA. Finally, Zhao et al. found that the initiation by promoter scanning was regulated by two distinct networks. The first network controlled how well mRNA synthesis could be initiated at individual transcription start sites; and the second network ­ driven by TFIIH ­ controlled which promoter sequences could be scanned to initiate transcription. This research provides an in-depth look into the early steps of the process of converting DNA into mRNA. The biological machinery used to initiate and control this action is highly conserved between yeast and humans, suggesting that the mechanisms for controlling the activity of these factors could be similar, even if their initiation processes may differ.

Nucleolar TFIIE plays a role in ribosomal biogenesis and performance.

Ribosome biogenesis is a highly energy-demanding process in eukaryotes which requires the concerted action of all three RNA polymerases. In RNA polymerase II transcription, the general transcription factor TFIIH is recruited by TFIIE to the initiation site of protein-coding genes. Distinct mutations in TFIIH and TFIIE give rise to the degenerative disorder trichothiodystrophy (TTD). Here, we uncovered an unexpected role of TFIIE in ribosomal RNA synthesis by RNA polymerase I. With high resolution microscopy we detected TFIIE in the nucleolus where TFIIE binds to actively transcribed rDNA. Mutations in TFIIE affects gene-occupancy of RNA polymerase I, rRNA maturation, ribosomal assembly and performance. In consequence, the elevated translational error rate with imbalanced protein synthesis and turnover results in an increase in heat-sensitive proteins. Collectively, mutations in TFIIE-due to impaired ribosomal biogenesis and translational accuracy-lead to a loss of protein homeostasis (proteostasis) which can partly explain the clinical phenotype in TTD.

Reduced activity of intestinal surface Na+/H+ exchanger NHE3 is a key factor for induction of diarrhea after PEDV infection in neonatal piglets.

Porcine epidemic diarrhea virus (PEDV; family Coronaviridae, genus Alphacoronavirus) causes acute diarrhea and vomiting, dehydration, and high mortality in neonatal piglets. Despite extensive research focusing on the pathogenesis of PEDV infection, the molecular pathogenesis of PEDV-induced diarrhea in piglets remains unclear. Na+/H+ exchanger 3 (NHE3), the main exchanger of electroneutral sodium in intestinal epithelial cells, is closely associated with the occurrence of diarrhea. To date, there is no study on whether diarrhea caused by PEDV infection is related to the activity of NHE3. In the present study, it was found that the expression level of cell membrane protein NHE3 significantly decreased after PEDV infection, whereas the total level of protein expression was not significantly changed. The Na+/H+ transport rate and the mRNA abundance of NHE3 decreased; the NHE3 activity decreased gradually with increasing infection time. In vivo, after PEDV infection of newborn piglets, rupture of intestinal villi and interstitial degeneration of intestinal epithelial cells in different intestinal segments were observed by hematoxylin-eosin staining. Immunohistochemical and immunofluorescence methods were used to observe the decreased expression of NHE3 protein on the membrane of intestinal epithelial cells in the jejunum and ileum. Taken together, these data indicate that PEDV infection reduces NHE3 activity in intestinal epithelial cells, hindering Na+ transport and thus causing diarrhea.