RESUMO
Maf1 is a general and global negative regulator of RNA polymerase III (Pol III) transcription. Under repressive conditions, Maf1 binds directly to the Pol III complex and sequesters Pol III elements that are involved in transcription initiation. To further understand Pol III regulation, we searched for genetic bypass suppressors of a maf1 deletion mutant (maf1Δ) of Saccharomyces cerevisiae. Strains that carried maf1Δ were temperature-sensitive on media that contained nonfermentable carbon sources and showed the antisuppressor phenotype. Suppressors allowed colonies to grow at the restrictive temperature on glycerol media and partially complemented the antisuppressor phenotype of maf1Δ. DNA plasmids that were identified as overdose suppressors encoded N-terminal fragments of the largest Pol III subunit, C160 of various lengths. The shortest fragment, 372 amino acids long, the overdose of which partially complemented the antisuppressor phenotype and temperature-sensitive respiratory growth of maf1Δ, was named C160-NTF. In this study, we showed that the expression of HA-tagged C160-NTF resulted in accumulation of approximately 40kDa protein that was distributed throughout the yeast cell, in the cytoplasm and nucleus. The overdose of C160-NTF led to decrease of tRNA transcription in maf1Δ mutant cells, demonstrating functional suppression. Levels of newly synthesized individual tRNAs and Pol III occupancies on tRNA genes were decreased by C160-NTF in the maf1Δ mutant. Additionally, we analyzed the effect of C160-NTF overproduction and the presence of Maf1 on the associations among Pol III subunits. Previous structural analyzes of Pol III have indicated that the N-terminal region of C160 interacts with the C82-34-31 heterotrimeric Pol III subcomplex. We suggest that the negative effect of C160-NTF overdose on tRNA transcription is related to defective Pol III assembly, because overproduction of C160-NTF altered C160 interactions with C34 and C82 in the maf1Δ mutant.
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Short Interspersed Elements (SINEs) are eukaryotic retrotransposons transcribed by RNA polymerase III (pol III). Many mammalian SINEs (T+ SINEs) contain a polyadenylation signal (AATAAA), a pol III transcription terminator, and an A-rich tail in their 3'-end. The RNAs of such SINEs have the capacity for AAUAAA-dependent polyadenylation, which is unique to pol III-generated transcripts. The structure, evolution, and polyadenylation of the Ere SINE of ungulates (horses, rhinos, and tapirs) were investigated in this study. A bioinformatics analysis revealed the presence of up to ~4 × 105 Ere copies in representatives of all three families. These copies can be classified into two large subfamilies, EreA and EreB, the former distinguished by an additional 60 bp sequence. The 3'-end of numerous EreA and all EreB copies exhibit a 50 bp sequence designated as a terminal domain (TD). The Ere family can be further subdivided into subfamilies EreA_0TD, EreA_1TD, EreB_1TD, and EreB_2TD, depending on the presence and number of terminal domains (TDs). Only EreA_0TD copies can be assigned to T+ SINEs as they contain the AATAAA signal and the TCTTT transcription terminator. The analysis of young Ere copies identified by comparison with related perissodactyl genomes revealed that EreA_0TD and, to a much lesser extent, EreB_2TD have retained retrotranspositional activity in the recent evolution of equids and rhinoceroses. The targeted mutagenesis and transfection of HeLa cells were used to identify sequences in equine EreA_0TD that are critical for the polyadenylation of its pol III transcripts. In addition to AATAAA and the transcription terminator, two sites in the 3' half of EreA, termed the ß and τ signals, were found to be essential for this process. The evolution of Ere, with a particular focus on the emergence of T+ SINEs, as well as the polyadenylation signals are discussed in comparison with other T+ SINEs.
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shRNA-mediated strategy of miRNA overexpression based on RNA Polymerase III (Pol III) expression cassettes is widely used for miRNA functional studies. For some miRNAs, e.g., encoded in the genome as a part of a polycistronic miRNA cluster, it is most likely the only way for their individual stable overexpression. Here we have revealed that expression of miRNAs longer than 19 nt (e.g. 23 nt in length hsa-miR-93-5p) using such approach could be accompanied by undesired predominant generation of 5' end miRNA isoforms (5'-isomiRs). Extra U residues (up to five) added by Pol III at the 3' end of the transcribed shRNA during transcription termination could cause a shift in the Dicer cleavage position of the shRNA. This results in the formation of 5'-isomiRs, which have a significantly altered seed region compared to the initially encoded canonical hsa-miR-93-5p. We demonstrated that the commonly used qPCR method is insensitive to the formation of 5'-isomiRs and cannot be used to confirm miRNA overexpression. However, the predominant expression of 5'-isomiRs without three or four first nucleotides instead of the canonical isoform could be disclosed based on miRNA-Seq analysis. Moreover, mRNA sequencing data showed that the 5'-isomiRs of hsa-miR-93-5p presumably regulate their own mRNA targets. Thus, omitting miRNA-Seq analysis may lead to erroneous conclusions regarding revealed mRNA targets and possible molecular mechanisms in which studied miRNA is involved. Overall, the presented results show that structures of shRNAs for stable overexpression of miRNAs requires careful design to avoid generation of undesired 5'-isomiRs.
Assuntos
MicroRNAs , RNA Interferente Pequeno , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/metabolismo , Ribonuclease III/genética , RNA Polimerase III/metabolismo , RNA Polimerase III/genética , Células HEK293 , Isoformas de RNA/genética , Isoformas de RNA/metabolismoRESUMO
Enhancers are the key regulators of other DNA-based processes by virtue of their unique ability to generate nucleosome-depleted regions in a highly regulated manner. Enhancers regulate cell-type-specific transcription of tRNA genes by RNA polymerase III (Pol III). They are also responsible for the binding of the origin replication complex (ORC) to DNA replication origins, thereby regulating origin utilization, replication timing, and replication-dependent chromosome breaks. Additionally, enhancers regulate V(D)J recombination by increasing access of the recombination-activating gene (RAG) recombinase to target sites and by generating non-coding enhancer RNAs and localized regions of trimethylated histone H3-K4 recognized by the RAG2 PHD domain. Thus, enhancers represent the first step in decoding the genome, and hence they regulate biological processes that, unlike RNA polymerase II (Pol II) transcription, do not have dedicated regulatory proteins.
Assuntos
Replicação do DNA , Elementos Facilitadores Genéticos , RNA Polimerase III , Transcrição Gênica , Recombinação V(D)J , Animais , Humanos , Replicação do DNA/genética , Regulação da Expressão Gênica/genética , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Transcrição Gênica/genética , Recombinação V(D)J/genéticaRESUMO
Regulation of eukaryotic gene expression involves a dynamic interplay between the core transcriptional machinery, transcription factors, and chromatin organization and modification. While this applies to transcription by all RNA polymerase complexes, RNA polymerase III (RNAPIII) seems to be atypical with respect to its mechanisms of regulation. One distinctive feature of most RNAPIII transcribed genes is that they are devoid of nucleosomes, which relates to the high levels of transcription. Moreover, most of the regulatory sequences are not outside but within the transcribed open chromatin regions. Yet, several lines of evidence suggest that chromatin factors affect RNAPIII dynamics and activity and that gene sequence alone does not explain the observed regulation of RNAPIII. Here we discuss the role of chromatin modification and organization of RNAPIII transcribed genes and how they interact with the core transcriptional RNAPIII machinery and regulatory DNA elements in and around the transcribed genes.
Assuntos
Cromatina , Regulação da Expressão Gênica , RNA Polimerase III , Transcrição Gênica , RNA Polimerase III/metabolismo , Cromatina/metabolismo , Humanos , Animais , Nucleossomos/metabolismo , Histonas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Hypomyelinating leukodystrophy (HLD) is a rare genetic heterogeneous disease that can affect myelin development in the central nervous system. This study aims to analyze the clinical phenotype and genetic function of a family with HLD-7 caused by POLR3A mutation. The proband (IV6) in this family mainly showed progressive cognitive decline, dentin dysplasia, and hypogonadotropic hypogonadism. Her three old brothers (IV1, IV2, and IV4) also had different degrees of ataxia, dystonia, or dysarthria besides the aforementioned manifestations. Their brain magnetic resonance imaging showed bilateral periventricular white matter atrophy, brain atrophy, and corpus callosum atrophy and thinning. The proband and her two living brothers (IV2 and IV4) were detected to carry a homozygous mutation of the POLR3A (NM_007055.4) gene c. 2300G > T (p.Cys767Phe), and her consanguineous married parents (III1 and III2) were p.Cys767Phe heterozygous carriers. In the constructed POLR3A wild-type and p.Cys767Phe mutant cells, it was seen that overexpression of wild-type POLR3A protein significantly enhanced Pol III transcription of 5S rRNA and tRNA Leu-CAA. However, although the mutant POLR3A protein overexpression was increased compared to the wild-type protein overexpression, it did not show the expected further enhancement of Pol III function. On the contrary, Pol III transcription function was frustrated (POLR3A, BC200, and tRNA Leu-CAA expression decreased), and MBP and 18S rRNA expressions were decreased. This study indicates that the POLR3A p.Cys767Phe variant caused increased expression of mutated POLR3A protein and abnormal expression of Pol III transcripts, and the mutant POLR3A protein function was abnormal.
Assuntos
Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central , Masculino , Feminino , Humanos , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Mutação , Fenótipo , Atrofia , RNA de Transferência , RNA Polimerase III/genética , RNA Polimerase III/metabolismoRESUMO
The genetic pathways that modulate ageing in multicellular organisms are typically highly conserved across wide evolutionary distances. Recently RNA polymerase III (Pol III) was shown to promote ageing in yeast, C. elegans and D. melanogaster. In this study we investigated the role of Pol III in mammalian ageing using C57BL/6N mice heterozygous for Pol III (Polr3b+/-). We identified sexually dimorphic, organ-specific beneficial as well as detrimental effects of the Polr3b+/- mutation on health. Female Polr3b+/- mice displayed improved bone health during ageing, but their ability to maintain an effective gut barrier function was compromised and they were susceptible to idiopathic dermatitis (ID). In contrast, male Polr3b+/- mice were lighter than wild-type (WT) males and had a significantly improved gut barrier function in old age. Several metabolic parameters were affected by both age and sex, but no genotype differences were detected. Neither male nor female Polr3b+/- mice were long-lived compared to WT controls. Overall, we find no evidence that a reduced Pol III activity extends mouse lifespan but we do find some potential organ- and sex-specific benefits for old-age health.
Assuntos
Envelhecimento , Heterozigoto , Longevidade , RNA Polimerase III , Animais , Feminino , Masculino , Camundongos , Envelhecimento/genética , Longevidade/genética , Camundongos Endogâmicos C57BL , RNA Polimerase III/genética , RNA Polimerase III/metabolismoRESUMO
In all cases tested, TFIIIB is responsible for recruiting pol III to its genetic templates. In mammalian cells, RB binds TFIIIB and prevents its interactions with both promoter DNA and pol III, thereby suppressing transcription. As TFIIIB is not recruited to its target genes when bound by RB, the mechanism predicts that pol III-dependent templates will not be occupied by RB; this contrasts with the situation at most genes controlled by RB, where it can be tethered by promoter-bound sequence-specific DNA-binding factors such as E2F. Contrary to this prediction, however, ChIP-seq data reveal the presence of RB in multiple cell types and the related protein p130 at many loci that rely on pol III for their expression, including RMRP, RN7SL, and a variety of tRNA genes. The sets of genes targeted varies according to cell type and growth state. In such cases, recruitment of RB and p130 can be explained by binding of E2F1, E2F4 and/or E2F5. Genes transcribed by pol III had not previously been identified as common targets of E2F family members. The data provide evidence that E2F may allow for the selective regulation of specific non-coding RNAs by RB, in addition to its influence on overall pol III output through its interaction with TFIIIB.
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Transcription is a tightly regulated, complex, and essential cellular process in all living organisms. Transcription is comprised of three steps, transcription initiation, elongation, and termination. The distinct transcription initiation and termination mechanisms of eukaryotic RNA polymerases I, II, and III (Pols I, II, and III) have long been appreciated. Recent methodological advances have empowered high-resolution investigations of the Pols' transcription elongation mechanisms. Here, we review the kinetic similarities and differences in the individual steps of Pol I-, II-, and III-catalyzed transcription elongation, including NTP binding, bond formation, pyrophosphate release, and translocation. This review serves as an important summation of Saccharomyces cerevisiae (yeast) Pol I, II, and III kinetic investigations which reveal that transcription elongation by the Pols is governed by distinct mechanisms. Further, these studies illustrate how basic, biochemical investigations of the Pols can empower the development of chemotherapeutic compounds.
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Tratamento Farmacológico , RNA Polimerase III , RNA Polimerase II , RNA Polimerase I , Saccharomyces cerevisiae , Elongação da Transcrição Genética , Biocatálise/efeitos dos fármacos , Cinética , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Saccharomyces cerevisiae/enzimologia , Elongação da Transcrição Genética/efeitos dos fármacosRESUMO
Terminal repeat retrotransposons in miniature (TRIMs) are short non-autonomous long terminal repeat (LTR) retrotransposons found from various eukaryotes. Cassandra is a unique TRIM lineage which contains a 5S rRNA-derived sequence in its LTRs. Here, two new groups of TRIMs, designated Helenus and Ajax, are reported based on bioinformatics analysis and the usage of Repbase. Helenus is found from fungi, animals, and plants, and its LTRs contain a tRNA-like sequence. It includes two LTRs and between them, a primer-binding site (PBS) and polypurine tract (PPT) exist. Fungal and plant Helenus generate 5 bp target site duplications (TSDs) upon integration, while animal Helenus generates 4 bp TSDs. Ajax includes a 5S rRNA-derived sequence in its LTR and is found from two nemertean genomes. Ajax generates 5 bp TSDs upon integration. These results suggest that despite their unique promoters, Helenus and Ajax are TRIMs whose transposition is dependent on autonomous LTR retrotransposon. These TRIMs can originate through an insertion of SINE in an LTR of TRIM. The discovery of Helenus and Ajax suggests the presence of TRIMs with a promoter for RNA polymerase III derived from a small RNA gene, which is here collectively termed TRIMp3.
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Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.
Assuntos
Proteínas de Choque Térmico HSP70 , Neoplasias , Humanos , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , RNA , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , RNA de Transferência/genética , RNA não Traduzido/genéticaRESUMO
RNA polymerase III-related leukodystrophy (POLR3-related leukodystrophy) is a rare, genetically determined hypomyelinating disease arising from biallelic pathogenic variants in genes encoding subunits of RNA polymerase III (Pol III). Here, we describe the first reported case of POLR3-related leukodystrophy caused by biallelic pathogenic variants in POLR3D, encoding the RPC4 subunit of Pol III. The individual, a female, demonstrated delays in walking and expressive and receptive language as a child and later cognitively plateaued. Additional neurological features included cerebellar signs (e.g., dysarthria, ataxia, and intention tremor) and dysphagia, while non-neurological features included hypodontia, hypogonadotropic hypogonadism, and dysmorphic facial features. Her MRI was notable for diffuse hypomyelination with myelin preservation of early myelinating structures, characteristic of POLR3-related leukodystrophy. Exome sequencing revealed the biallelic variants in POLR3D, a missense variant (c.541C > T, p.P181S) and an intronic splice site variant (c.656-6G > A, p.?). Functional studies of the patient's fibroblasts demonstrated significantly decreased RNA-level expression of POLR3D, along with reduced expression of other Pol III subunit genes. Notably, Pol III transcription was also shown to be aberrant, with a significant decrease in 7SK RNA and several distinct tRNA genes analyzed. Affinity purification coupled to mass spectrometry of the POLR3D p.P181S variant showed normal assembly of Pol III subunits yet altered interaction of Pol III with the PAQosome chaperone complex, indicating the missense variant is likely to alter complex maturation. This work identifies biallelic pathogenic variants in POLR3D as a novel genetic cause of POLR3-related leukodystrophy, expanding the molecular spectrum associated with this disease, and proposes altered tRNA homeostasis as a factor in the underlying biology of this hypomyelinating disorder.
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Transcription of tRNA genes by RNA polymerase III (RNAPIII) is tuned by signaling cascades. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulators have not been described. Decoding the local chromatin proteome of a native tRNA gene in yeast revealed reprogramming of the RNAPIII transcription machinery upon nutrient perturbation. Among the dynamic proteins, we identified Fpt1, a protein of unknown function that uniquely occupied RNAPIII-regulated genes. Fpt1 binding at tRNA genes correlated with the efficiency of RNAPIII eviction upon nutrient perturbation and required the transcription factors TFIIIB and TFIIIC but not RNAPIII. In the absence of Fpt1, eviction of RNAPIII was reduced, and the shutdown of ribosome biogenesis genes was impaired upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and tuning the physiological response to changing metabolic demands.
Assuntos
RNA Polimerase III , Proteínas de Saccharomyces cerevisiae , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismo , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Transcrição GênicaRESUMO
Precursor tRNAs are transcribed with flanking and intervening sequences known to be processed by specific ribonucleases. Here, we show that transcription complexes of RNA polymerase III assembled on tRNA genes comprise RNase P that cleaves precursor tRNA and subsequently degrades the excised 5' leader. Degradation is based on a 3'-5' exoribonucleolytic activity carried out by the protein subunit Rpp14, as determined by biochemical and reverse genetic analyses. Neither reconstituted nor purified RNase P displays this magnesium ion-dependent, processive exoribonucleolytic activity. Markedly, knockdown of Rpp14 by RNA interference leads to a wide-ranging inhibition of cleavage of flanking and intervening sequences of various precursor tRNAs in extracts and cells. This study reveals that RNase P controls tRNA splicing complex and RNase Z for ordered maturation of nascent precursor tRNAs by transcription complexes.
Assuntos
Precursores de RNA , Ribonuclease P , Humanos , Ribonuclease P/genética , Ribonuclease P/metabolismo , Precursores de RNA/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , RNA de Transferência/metabolismo , Ribonucleases/metabolismo , Splicing de RNARESUMO
Short Interspersed Elements (SINEs) are common in the genomes of most multicellular organisms. They are transcribed by RNA polymerase III from an internal promoter comprising boxes A and B. As transcripts of certain SINEs from mammalian genomes can be polyadenylated, such transcripts should contain the AATAAA sequence as well as those called ß- and τ-signals. One of the goals of this work was to evaluate how autonomous and independent other SINE parts are ß- and τ-signals. Extended regions outside of ß- and τ-signals were deleted from SINEs B2 and Ves and the derived constructs were used to transfect HeLa cells in order to evaluate the relative levels of their transcripts as well as their polyadenylation efficiency. If the deleted regions affected boxes A and B, the 5'-flanking region of the U6 RNA gene with the external promoter was inserted upstream. Such substitution of the internal promoter in B2 completely restored its transcription. Almost all tested deletions/substitutions did not reduce the polyadenylation capacity of the transcripts, indicating a weak dependence of the function of ß- and τ-signals on the neighboring sequences. A similar analysis of B2 and Ves constructs containing a 55-bp foreign sequence inserted between ß- and τ-signals showed an equal polyadenylation efficiency of their transcripts compared to those of constructs without the insertion. The acquired poly(A)-tails significantly increased the lifetime and thus the cellular level of such transcripts. The data obtained highlight the potential of B2 and Ves SINEs as cassettes for the expression of relatively short sequences for various applications.
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Poliadenilação , RNA Polimerase III , Animais , Humanos , Poliadenilação/genética , RNA Polimerase III/genética , Células HeLa , Elementos Nucleotídeos Curtos e Dispersos/genética , Regiões Promotoras Genéticas , Mamíferos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
RNA polymerase III (RNAP III) holoenzyme activity and the processing of its products have been linked to several metabolic dysfunctions in lower and higher eukaryotes. Alterations in the activity of RNAP III-driven synthesis of non-coding RNA cause extensive changes in glucose metabolism. Increased RNAP III activity in the S. cerevisiae maf1Δ strain is lethal when grown on a non-fermentable carbon source. This lethal phenotype is suppressed by reducing tRNA synthesis. Neither the cause of the lack of growth nor the underlying molecular mechanism have been deciphered, and this area has been awaiting scientific explanation for a decade. Our previous proteomics data suggested mitochondrial dysfunction in the strain. Using model mutant strains maf1Δ (with increased tRNA abundance) and rpc128-1007 (with reduced tRNA abundance), we collected data showing major changes in the TCA cycle metabolism of the mutants that explain the phenotypic observations. Based on 13C flux data and analysis of TCA enzyme activities, the present study identifies the flux constraints in the mitochondrial metabolic network. The lack of growth is associated with a decrease in TCA cycle activity and downregulation of the flux towards glutamate, aspartate and phosphoenolpyruvate (PEP), the metabolic intermediate feeding the gluconeogenic pathway. rpc128-1007, the strain that is unable to increase tRNA synthesis due to a mutation in the C128 subunit, has increased TCA cycle activity under non-fermentable conditions. To summarize, cells with non-optimal activity of RNAP III undergo substantial adaptation to a new metabolic state, which makes them vulnerable under specific growth conditions. Our results strongly suggest that balanced, non-coding RNA synthesis that is coupled to glucose signaling is a fundamental requirement to sustain a cell's intracellular homeostasis and flexibility under changing growth conditions. The presented results provide insight into the possible role of RNAP III in the mitochondrial metabolism of other cell types.
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Mitocôndrias , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Mitocôndrias/metabolismo , Homeostase , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA não Traduzido/metabolismoRESUMO
In vertebrates, SMARCAD1 participates in transcriptional regulation, heterochromatin maintenance, DNA repair, and replication. The molecular basis underlying its involvement in these processes is not well understood. We identified the RNA polymerase III general transcription factor TFIIIC as an interaction partner of native SMARCAD1 in mouse and human models using endogenous co-immunoprecipitations. TFIIIC has dual functionality, acting as a general transcription factor and as a genome organizer separating chromatin domains. We found that its partnership with SMARCAD1 is conserved across different mammalian cell types, from somatic to pluripotent cells. Using purified proteins, we confirmed that their interaction is direct. A gene expression analysis suggested that SMARCAD1 is dispensable for TFIIIC function as an RNA polymerase III transcription factor in mouse ESCs. The distribution of TFIIIC and SMARCAD1 in the ESC genome is distinct, and unlike in yeast, SMARCAD1 is not enriched at active tRNA genes. Further analysis of SMARCAD1-binding partners in pluripotent and differentiated mammalian cells reveals that SMARCAD1 associates with several factors that have key regulatory roles in chromatin organization, such as cohesin, laminB, and DDX5. Together, our work suggests for the first time that the SMARCAD1 enzyme participates in genome organization in mammalian nuclei through interactions with architectural proteins.
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DNA Helicases , Fatores Genéricos de Transcrição , Fatores de Transcrição TFIII , Animais , Humanos , Camundongos , Cromatina/genética , DNA Helicases/genética , Mamíferos , RNA Polimerase III , Fatores de Transcrição TFIII/genéticaRESUMO
Abnormally elevated expression of tRNA is a common feature of breast tumours. Rather than a uniform increase in all tRNAs, some are deregulated more strongly than others. Elevation of particular tRNAs has been associated with poor prognosis for patients, and experimental models have demonstrated the ability of some tRNAs to promote proliferation or metastasis. Each tRNA isoacceptor is encoded redundantly by multiple genes, which are commonly dispersed across several chromosomes. An unanswered question is whether the consistently high expression of a tRNA in a cancer type reflects the consistent activation of the same members of a gene family, or whether different family members are activated from one patient to the next. To address this question, we interrogated ChIP-seq data to determine which tRNA genes were active in individual breast tumours. This revealed that distinct sets of tRNA genes become activated in individual cancers, whereas there is much less variation in the expression patterns of families. Several pathways have been described that are likely to contribute to increases in tRNA gene transcription in breast tumours, but none of these can adequately explain the observed variation in the choice of genes between tumours. Current models may therefore lack at least one level of regulation.
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RNA polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. Here, we use cryoelectron microscopy (cryo-EM) to visualize the S. cerevisiae complex of TFIIIA and TFIIIC bound to the promoter. Gene-specific factor TFIIIA interacts with DNA and acts as an adaptor for TFIIIC-promoter interactions. We also visualize DNA binding of TFIIIB subunits, Brf1 and TBP (TATA-box binding protein), which results in the full-length 5S rRNA gene wrapping around the complex. Our smFRET study reveals that the DNA within the complex undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. Our findings provide new insights into the transcription initiation complex assembly on the 5S rRNA promoter and allow us to directly compare Pol III and Pol II transcription adaptations.
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Fatores de Transcrição , Transcrição Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Transcrição TFIIIB/genética , Fator de Transcrição TFIIIB/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Microscopia Crioeletrônica , DNA/metabolismoRESUMO
Introduction: RNA polymerase III (Pol III) is a critical enzymatic complex tasked with the transcription of ubiquitous non-coding RNAs including 5S rRNA and all tRNA genes. Despite the constitutive nature of this enzyme, hypomorphic biallelic pathogenic variants in genes encoding subunits of Pol III lead to tissue-specific features and cause a hypomyelinating leukodystrophy, characterized by a severe and permanent deficit in myelin. The pathophysiological mechanisms in POLR3- related leukodystrophy and specifically, how reduced Pol III function impacts oligodendrocyte development to account for the devastating hypomyelination seen in the disease, remain poorly understood. Methods: In this study, we characterize how reducing endogenous transcript levels of leukodystrophy-associated Pol III subunits affects oligodendrocyte maturation at the level of their migration, proliferation, differentiation, and myelination. Results: Our results show that decreasing Pol III expression altered the proliferation rate of oligodendrocyte precursor cells but had no impact on migration. Additionally, reducing Pol III activity impaired the differentiation of these precursor cells into mature oligodendrocytes, evident at both the level of OL-lineage marker expression and on morphological assessment, with Pol III knockdown cells displaying a drastically more immature branching complexity. Myelination was hindered in the Pol III knockdown cells, as determined in both organotypic shiverer slice cultures and co-cultures with nanofibers. Analysis of Pol III transcriptional activity revealed a decrease in the expression of distinct tRNAs, which was significant in the siPolr3a condition. Discussion: In turn, our findings provide insight into the role of Pol III in oligodendrocyte development and shed light on the pathophysiological mechanisms of hypomyelination in POLR3-related leukodystrophy.