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.

To aggregate or not to aggregate - Is it a matter of the ribosome?

Neurodegenerative syndromes present as proteinopathies - does ribosomal infidelity contribute to the protein toxicity that is the driving force for neuronal cell loss? Intracellular and extracellular protein aggregates overwhelm the clearance capacity of cells and tissues. Proteins aggregate when hydrophobic residues are exposed. Hydrophobic residues become exposed when proteins are misfolded. Protein misfolding can originate from translational errors at the ribosome. Indeed, the most error-prone process in gene expression is translation at the ribosome. Recent evidence indicates that manipulating the ribosomal accuracy impacts on the lifespan of model organisms and a reduced translational accuracy is accompanied by neurodegeneration. The first hit in aging-associated neurodegenerative disease may be the well-documented decline of cellular buffering capacity by aging. A second hit that impacts on the quality of protein synthesis could be the driving force for the observed loss of proteostasis in neurodegeneration. This hypothesis provides an explanation for the late onset of most neurodegenerative diseases.

General loss of proteostasis links Huntington disease to Cockayne syndrome.

Cockayne syndrome (CS) is an autosomal recessive disorder of developmental delay, multiple organ system degeneration and signs of premature ageing. We show here, using the RNA-seq data from two CS mutant cell lines, that the CS key transcriptional signature displays significant enrichment of neurodegeneration terms, including genes relevant in Huntington disease (HD). By using deep learning approaches and two published RNA-Seq datasets, the CS transcriptional signature highly significantly classified and predicted HD and control samples. Neurodegeneration is one hallmark of CS disease, and fibroblasts from CS patients with different causative mutations display disturbed ribosomal biogenesis and a consecutive loss of protein homeostasis - proteostasis. Encouraged by the transcriptomic data, we asked whether this pathomechanism is also active in HD. In different HD cell-culture models, we showed that mutant Huntingtin impacts ribosomal biogenesis and function. This led to an error-prone protein synthesis and, as shown in different mouse models and human tissue, whole proteome instability, and a general loss of proteostasis.

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.

Cockayne syndrome group A and ferrochelatase finely tune ribosomal gene transcription and its response to UV irradiation.

CSA and CSB proteins are key players in transcription-coupled nucleotide excision repair (TC-NER) pathway that removes UV-induced DNA lesions from the transcribed strands of expressed genes. Additionally, CS proteins play relevant but still elusive roles in other cellular pathways whose alteration may explain neurodegeneration and progeroid features in Cockayne syndrome (CS). Here we identify a CS-containing chromatin-associated protein complex that modulates rRNA transcription. Besides RNA polymerase I (RNAP1) and specific ribosomal proteins (RPs), the complex includes ferrochelatase (FECH), a well-known mitochondrial enzyme whose deficiency causes erythropoietic protoporphyria (EPP). Impairment of either CSA or FECH functionality leads to reduced RNAP1 occupancy on rDNA promoter that is associated to reduced 47S pre-rRNA transcription. In addition, reduced FECH expression leads to an abnormal accumulation of 18S rRNA that in primary dermal fibroblasts from CS and EPP patients results in opposed rRNA amounts. After cell irradiation with UV light, CSA triggers the dissociation of the CSA-FECH-CSB-RNAP1-RPs complex from the chromatin while it stabilizes its binding to FECH. Besides disclosing a function for FECH within nucleoli, this study sheds light on the still unknown mechanisms through which CSA modulates rRNA transcription.

TFIIH is an elongation factor of RNA polymerase I.

TFIIH is a multisubunit factor essential for transcription initiation and promoter escape of RNA polymerase II and for the opening of damaged DNA double strands in nucleotide excision repair (NER). In this study, we have analyzed at which step of the transcription cycle TFIIH is essential for transcription by RNA polymerase I. We demonstrate that TFIIH associates with the rDNA promoter and gene-internal sequences and leaves the rDNA promoter in a complex with RNA polymerase I after start of transcription. Moreover, mutations in the TFIIH subunits XPB and XPD found in Cockayne syndrome impair the interaction of TFIIH with the rDNA, but do not influence initiation complex formation or promoter escape of RNA polymerase I, but preclude the productivity of the enzyme by reducing transcription elongation in vivo and in vitro. Our results implicate that reduced RNA polymerase I transcription elongation and ribosomal stress could be one factor contributing to the Cockayne syndrome phenotype.

Cilia defects upon loss of WDR4 are linked to proteasomal hyperactivity and ubiquitin shortage.

The WD repeat-containing protein 4 (WDR4) has repeatedly been associated with primary microcephaly, a condition of impaired brain and skull growth. Often, faulty centrosomes cause microcephaly, yet aberrant cilia may also be involved. Here, we show using a combination of approaches in human fibroblasts, zebrafish embryos and patient-derived cells that WDR4 facilitates cilium formation. Molecularly, we associated WDR4 loss-of-function with increased protein synthesis and concomitant upregulation of proteasomal activity, while ubiquitin precursor pools are reduced. Inhibition of proteasomal activity as well as supplementation with free ubiquitin restored normal ciliogenesis. Proteasome inhibition ameliorated microcephaly phenotypes. Thus, we propose that WDR4 loss-of-function impairs head growth and neurogenesis via aberrant cilia formation, initially caused by disturbed protein and ubiquitin homeostasis.

CEBP factors regulate telomerase reverse transcriptase promoter activity in whey acidic protein-T mice during mammary carcinogenesis.

Telomerase is activated in the majority of invasive breast cancers, but the time point of telomerase activation during mammary carcinogenesis is not clear. We have recently presented a transgenic mouse model to study human telomerase reverse transcriptase (TERT) gene expression in vivo (hTERTp-lacZ). In the present study, hTERTp-lacZxWAP-T bitransgenic mice were generated to analyze the mechanisms responsible for human and mouse TERT upregulation during tumor progression in vivo. We found that telomerase activity and TERT expression were consistently upregulated in SV40-induced invasive mammary tumors compared to normal and hyperplastic tissues and ductal carcinoma in situ (DCIS). Human and mouse TERT genes are regulated similarly in the breast tissue, involving the CEBP transcription factors. Loss of CEBP-α and induction of CEBP-ß expression correlated well with the activation of TERT expression in mouse mammary tumors. Transfection of CEBP-α into human or murine cells resulted in TERT repression, whereas knockdown of CEBP-α in primary human mammary epithelial cells resulted in reactivation of endogenous TERT expression and telomerase activity. Conversely, ectopic expression of CEBP-ß activated endogenous TERT gene expression. Moreover, ChIP and EMSA experiments revealed binding of CEBP-α and CEBP-ß to human TERT-promoter. This is the first evidence indicating that CEBP-α and CEBP-ß are involved in TERT gene regulation during carcinogenesis.

Ribosomal Dysfunction Is a Common Pathomechanism in Different Forms of Trichothiodystrophy.

Mutations in a broad variety of genes can provoke the severe childhood disorder trichothiodystrophy (TTD) that is classified as a DNA repair disease or a transcription syndrome of RNA polymerase II. In an attempt to identify the common underlying pathomechanism of TTD we performed a knockout/knockdown of the two unrelated TTD factors TTDN1 and RNF113A and investigated the consequences on ribosomal biogenesis and performance. Interestingly, interference with these TTD factors created a nearly uniform impact on RNA polymerase I transcription with downregulation of UBF, disturbed rRNA processing and reduction of the backbone of the small ribosomal subunit rRNA 18S. This was accompanied by a reduced quality of decoding in protein translation and the accumulation of misfolded and carbonylated proteins, indicating a loss of protein homeostasis (proteostasis). As the loss of proteostasis by the ribosome has been identified in the other forms of TTD, here we postulate that ribosomal dysfunction is a common underlying pathomechanism of TTD.

Intranuclear inclusions of polyQ-expanded ATXN1 sequester RNA molecules.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease caused by a trinucleotide (CAG) repeat expansion in the ATXN1 gene. It is characterized by the presence of polyglutamine (polyQ) intranuclear inclusion bodies (IIBs) within affected neurons. In order to investigate the impact of polyQ IIBs in SCA1 pathogenesis, we generated a novel protein aggregation model by inducible overexpression of the mutant ATXN1(Q82) isoform in human neuroblastoma SH-SY5Y cells. Moreover, we developed a simple and reproducible protocol for the efficient isolation of insoluble IIBs. Biophysical characterization showed that polyQ IIBs are enriched in RNA molecules which were further identified by next-generation sequencing. Finally, a protein interaction network analysis indicated that sequestration of essential RNA transcripts within ATXN1(Q82) IIBs may affect the ribosome resulting in error-prone protein synthesis and global proteome instability. These findings provide novel insights into the molecular pathogenesis of SCA1, highlighting the role of polyQ IIBs and their impact on critical cellular processes.

A Comparative Assessment of Replication Stress Markers in the Context of Telomerase.

Aberrant replication stress (RS) is a source of genome instability and has serious implications for cell survival and tumourigenesis. Therefore, the detection of RS and the identification of the underlying molecular mechanisms are crucial for the understanding of tumourigenesis. Currently, three protein markers-p33-phosphorylated replication protein A2 (pRPA2), γ-phosphorylated H2AX (γ-H2AX), and Tumor Protein P53 Binding Protein 1 (53BP1)-are frequently used to detect RS. However, to our knowledge, there is no report that compares their suitability for the detection of different sources of RS. Therefore, in this study, we evaluate the suitability of pRPA2, γ-H2AX, and 53BP1 for the detection of RS caused by different sources of RS. In addition, we examine their suitability as markers of the telomerase-mediated alleviation of RS. For these purposes, we use here telomerase-negative human fibroblasts (BJ) and their telomerase-immortalized counterparts (BJ-hTERT). Replication stress was induced by the ectopic expression of the oncogenic RAS mutant RASG12V (OI-RS), by the knockdown of ploidy-control genes ORP3 or MAD2 (AI-RS), and by treatment with hydrogen peroxide (ROS-induced RS). The level of RS was determined by immunofluorescence staining for pRPA2, γ-H2AX, and 53BP1. Evaluation of the staining results revealed that pRPA2- and γ-H2AX provide a significant and reliable assessment of OI-RS and AI-RS compared to 53BP1. On the other hand, 53BP1 and pRPA2 proved to be superior to γ-H2AX for the evaluation of ROS-induced RS. Moreover, the data showed that among the tested markers, pRPA2 is best suited to evaluate the telomerase-mediated suppression of all three types of RS. In summary, the data indicate that the choice of marker is important for the evaluation of RS activated through different conditions.

Antigen-specific induction of osteopontin contributes to the chronification of allergic contact dermatitis.

Allergic contact dermatitis is a T cell-mediated immune response, which in its relapsing chronic form is of high socioeconomic impact. The phosphoglycoprotein osteopontin (OPN) has chemotactic and Th1 cytokine functions and in various models is essential for robust T cell-mediated immunity. Here we demonstrate that OPN is abundantly expressed by both effector T cells and keratinocytes in allergic contact dermatitis lesions. T cells from nickel-allergic donors secrete high levels of OPN following antigen-specific stimulation. OPN may substitute for missing IFN-gamma secretion in T effector cells because low IFN-gamma-producing T cell clones secrete high levels of OPN, and OPN down-modulates their interleukin-4 expression. Furthermore, interferon-gamma from T effector cells augments OPN in allergic contact dermatitis by inducing OPN in keratinocytes, which in turn polarizes dendritic cells and attracts inflammatory cells. In the murine contact hypersensitivity (CHS) model for allergic contact dermatitis, OPN is strongly induced in antigen-specific proliferating T cells, and OPN null mice display a reduced chronic CHS inflammatory response due to a decreased influx of effector T cells. Importantly, because of its function for chronic allergic contact dermatitis, OPN may well be a therapeutic target, because anti-OPN antibody treatment in part suppresses established chronic CHS.

Nuclear actin and myosin I are required for RNA polymerase I transcription.

The presence of actin and nuclear myosin I (NMI) in the nucleus suggests a role for these motor proteins in nuclear functions. We have investigated the role of actin and nuclear myosin I (NMI) in the transcription of ribosomal RNA genes (rDNA). Both proteins are associated with rDNA and are required for RNA polymerase I (Pol I) transcription. Microinjection of antibodies against actin or NMI, as well as short interfering RNA-mediated depletion of NMI, decreased Pol I transcription in vivo, whereas overexpression of NMI augmented pre-rRNA synthesis. In vitro, recombinant NMI activated Pol I transcription, and antibodies to NMI or actin inhibited Pol I transcription both on naked DNA and pre-assembled chromatin templates. Whereas actin associated with Pol I, NMI bound to Pol I through the transcription-initiation factor TIF-IA. The association with Pol I requires phosphorylation of TIF-IA at Ser 649 by RSK kinase, indicating a role for NMI in the growth-dependent regulation of rRNA synthesis.

Cockayne Syndrome-Associated CSA and CSB Mutations Impair Ribosome Biogenesis, Ribosomal Protein Stability, and Global Protein Folding.

Cockayne syndrome (CS) is a developmental disorder with symptoms that are typical for the aging body, including subcutaneous fat loss, alopecia, and cataracts. Here, we show that in the cells of CS patients, RNA polymerase I transcription and the processing of the pre-rRNA are disturbed, leading to an accumulation of the 18S-E intermediate. The mature 18S rRNA level is reduced, and isolated ribosomes lack specific ribosomal proteins of the small 40S subunit. Ribosomal proteins are susceptible to unfolding and the CS cell proteome is heat-sensitive, indicating misfolded proteins and an error-prone translation process in CS cells. Pharmaceutical chaperones restored impaired cellular proliferation. Therefore, we provide evidence for severe protein synthesis malfunction, which together with a loss of proteostasis constitutes the underlying pathophysiology in CS.

MicroRNA-708 is a novel regulator of the Hoxa9 program in myeloid cells.

MicroRNAs (miRNAs) are commonly deregulated in acute myeloid leukemia (AML), affecting critical genes not only through direct targeting, but also through modulation of downstream effectors. Homeobox (Hox) genes balance self-renewal, proliferation, cell death, and differentiation in many tissues and aberrant Hox gene expression can create a predisposition to leukemogenesis in hematopoietic cells. However, possible linkages between the regulatory pathways of Hox genes and miRNAs are not yet fully resolved. We identified miR-708 to be upregulated in Hoxa9/Meis1 AML inducing cell lines as well as in AML patients. We further showed Meis1 directly targeting miR-708 and modulating its expression through epigenetic transcriptional regulation. CRISPR/Cas9 mediated knockout of miR-708 in Hoxa9/Meis1 cells delayed disease onset in vivo, demonstrating for the first time a pro-leukemic contribution of miR-708 in this context. Overexpression of miR-708 however strongly impeded Hoxa9 mediated transformation and homing capacity in vivo through modulation of adhesion factors and induction of myeloid differentiation. Taken together, we reveal miR-708, a putative tumor suppressor miRNA and direct target of Meis1, as a potent antagonist of the Hoxa9 phenotype but an effector of transformation in Hoxa9/Meis1. This unexpected finding highlights the yet unexplored role of miRNAs as indirect regulators of the Hox program during normal and aberrant hematopoiesis.

A novel activity enhances promoter escape of RNA polymerase I.

We have characterized a novel transcriptional activity from HeLa cells that is required for ribosomal gene transcription by RNA polymerase I. This activity has a native molecular mass of 16 kDa and does not bind to conventional chromatographic resins. Single-round and immobilized-template experiments revealed that initiation complex formation is independent of the novel activity. Functional studies showed that it stimulates the transition from initiation to elongation, promoter escape. Thus the novel activity does not resemble the mouse initiation/elongation factor TIF-IC but is a true novel entity.

Nucleolar and Ribosomal Dysfunction-A Common Pathomechanism in Childhood Progerias?

The nucleolus organizes around the sites of transcription by RNA polymerase I (RNA Pol I). rDNA transcription by this enzyme is the key step of ribosome biogenesis and most of the assembly and maturation processes of the ribosome occur co-transcriptionally. Therefore, disturbances in rRNA transcription and processing translate to ribosomal malfunction. Nucleolar malfunction has recently been described in the classical progeria of childhood, Hutchinson-Gilford syndrome (HGPS), which is characterized by severe signs of premature aging, including atherosclerosis, alopecia, and osteoporosis. A deregulated ribosomal biogenesis with enlarged nucleoli is not only characteristic for HGPS patients, but it is also found in the fibroblasts of "normal" aging individuals. Cockayne syndrome (CS) is also characterized by signs of premature aging, including the loss of subcutaneous fat, alopecia, and cataracts. It has been shown that all genes in which a mutation causes CS, are involved in rDNA transcription by RNA Pol I. A disturbed ribosomal biogenesis affects mitochondria and translates into ribosomes with a reduced translational fidelity that causes endoplasmic reticulum (ER) stress and apoptosis. Therefore, it is speculated that disease-causing disturbances in the process of ribosomal biogenesis may be more common than hitherto anticipated.

Small molecular antioxidants effectively protect from PUVA-induced oxidative stress responses underlying fibroblast senescence and photoaging.

Exposure of human fibroblasts to 8-methoxypsoralen plus ultraviolet-A irradiation (PUVA) results in stress-induced cellular senescence in fibroblasts. We here studied the role of the antioxidant defense system in the accumulation of reactive oxygen species (ROS) and the effect of the antioxidants alpha-tocopherol, N-acetylcysteine, and alpha-lipoic acid on PUVA-induced cellular senescence. PUVA treatment induced an immediate and increasing generation of intracellular ROS. Supplementation of PUVA-treated fibroblasts with alpha-tocopherol (alpha-Toc), N-acetylcysteine (NAC), or alpha-lipoic acid (alpha-LA) abrogated the increased ROS generation and rescued fibroblasts from the ROS-dependent changes into the cellular senescence phenotype, such as cytoplasmic enlargement, enhanced expression of senescence-associated-beta-galactosidase and matrix-metalloproteinase-1, hallmarks of photoaging and intrinsic aging. PUVA treatment disrupted the integrity of cellular membranes and impaired homeostasis and function of the cellular antioxidant system with a significant decrease in glutathione and hydrogen peroxide-detoxifying enzymes activities. Supplementation with NAC, alpha-LA, and alpha-Toc counteracted these changes. Our data provide causal evidence that (i) oxidative stress due to an imbalance in the overall cellular antioxidant capacity contributes to the induction and maintenance of the PUVA-induced fibroblast senescence and that (ii) low molecular antioxidants protect effectively against these deleterious alterations.

Loss of Proteostasis Is a Pathomechanism in Cockayne Syndrome.

Retarded growth and neurodegeneration are hallmarks of the premature aging disease Cockayne syndrome (CS). Cockayne syndrome proteins take part in the key step of ribosomal biogenesis, transcription of RNA polymerase I. Here, we identify a mechanism originating from a disturbed RNA polymerase I transcription that impacts translational fidelity of the ribosomes and consequently produces misfolded proteins. In cells from CS patients, the misfolded proteins are oxidized by the elevated reactive oxygen species (ROS) and provoke an unfolded protein response that represses RNA polymerase I transcription. This pathomechanism can be disrupted by the addition of pharmacological chaperones, suggesting a treatment strategy for CS. Additionally, this loss of proteostasis was not observed in mouse models of CS.