Search

The transcriptional interplay of human RNA polymerase I (RNA Pol I), RNA Pol II, and RNA Pol III remains largely uncharacterized due to limited integrative genomic analyses for all three enzymes. To address this gap, we applied a uniform framework to quantify global RNA Pol I, RNA Pol II, and RNA Pol III occupancies and identify both canonical and noncanonical patterns of gene localization. Most notably, our survey captures unexpected RNA Pol III recruitment at promoters of specific protein-coding genes. We show that such RNA Pol III-occupied promoters are enriched for small nascent RNAs terminating in a run of 4 Ts-a hallmark of RNA Pol III termination indicative of constrained RNA Pol III transcription. Taken further, RNA Pol III disruption generally reduces the expression of RNA Pol III-occupied protein-coding genes, suggesting RNA Pol III recruitment and transcription enhance RNA Pol II activity. These findings resemble analogous patterns of RNA Pol II activity at RNA Pol III-transcribed genes, altogether uncovering a reciprocal form of crosstalk between RNA Pol II and RNA Pol III., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Pathogenic variants in subunits of RNA polymerase (Pol) III cause a spectrum of Polr3 -related neurodegenerative diseases including 4H leukodystrophy. Disease onset occurs from infancy to early adulthood and is associated with a variable range and severity of neurological and non-neurological features. The molecular basis of Polr3 -related disease pathogenesis is unknown. We developed a postnatal whole-body mouse model expressing pathogenic Polr3a mutations to examine the molecular mechanisms by which reduced Pol III transcription results primarily in central nervous system phenotypes. Polr3a mutant mice exhibit behavioral deficits, cerebral pathology and exocrine pancreatic atrophy. Transcriptome and immunohistochemistry analyses of cerebra during disease progression show a reduction in most Pol III transcripts, induction of innate immune and integrated stress responses and cell-type-specific gene expression changes reflecting neuron and oligodendrocyte loss and microglial activation. Earlier in the disease when integrated stress and innate immune responses are minimally induced, mature tRNA sequencing revealed a global reduction in tRNA levels and an altered tRNA profile but no changes in other Pol III transcripts. Thus, changes in the size and/or composition of the tRNA pool have a causal role in disease initiation. Our findings reveal different tissue- and brain region-specific sensitivities to a defect in Pol III transcription., Competing Interests: RM, EM, VC, ES, IW No competing interests declared, (© 2024, Moir, Merheb et al.)

RNA polymerase III (Pol III) transcribes tRNA genes using type II promoters. The internal control regions contain a Box A and a Box B, which are recognized by TFIIIC. The 5'-flanking regions of tRNA genes clearly play a role in the regulation of transcription, but consensus sequences in it have been found only in some plants and S. pombe ; although, the TATA binding protein (TBP) is a component of the TFIIIB complex in all eukaryotes. Archaea utilize an ortholog of the TBP. The goal of this work is the detection of the positions of intragenic and extragenic promoters of Pol III, which regulate the transcription of tRNA genes in eukaryotes and archaea. For this purpose, we analyzed textual and some structural, mechanical, and physicochemical properties of the DNA in the 5'-flanking regions of tRNA genes, as well as in 30 bp at the beginning of genes and 60 bp at the end of genes in organisms possessing the TBP or its analog (eukaryotes, archaea) and organisms not possessing the TBP (bacteria). Representative tRNA gene sets of 11 organisms were taken from the GtRNAdb database. We found that the consensuses of A- and B-boxes in organisms from all three domains are identical; although, they differ in the conservativism of some positions. Their location relative to the ends of tRNA genes is also identical. In contrast, the structural and mechanical properties of DNA in the 5'-flanking regions of tRNA genes differ not only between organisms from different domains, but also between organisms from the same domain. Well-expressed TBP binding positions are found only in S. pombe and A. thaliana . We discuss possible reasons for the variability of the 5'-flanking regions of tRNA genes.

Objective: POLR3B encodes the second largest subunit of RNA polymerase III, which is essential for transcription of small non-coding RNAs. Biallelic pathogenic variants in POLR3B are associated with an inherited hypomyelinating leukodystrophy. Recently, de novo heterozygous variants in POLR3B were reported in six individuals with ataxia, spasticity, and demyelinating peripheral neuropathy. Three of these individuals had epileptic seizures. The aim of this article is to precisely define the epilepsy phenotype associated with de novo heterozygous POLR3B variants., Methods: We used online gene-matching tools to identify 13 patients with de novo POLR3B variants. We systematically collected genotype and phenotype data from clinicians using two standardized proformas., Results: All 13 patients had novel POLR3B variants. Twelve of 13 variants were classified as pathogenic or likely pathogenic as per American College of Medical Genetics (ACMG) criteria. Patients presented with generalized myoclonic, myoclonic-atonic, atypical absence, or tonic-clonic seizures between the ages of six months and 4 years. Epilepsy was classified as epilepsy with myoclonic-atonic seizures (EMAtS) in seven patients and "probable EMAtS" in two more. Seizures were treatment resistant in all cases. Three patients became seizure-free. All patients had some degree of developmental delay or intellectual disability. In most cases developmental delay was apparent before the onset of seizures. Three of 13 cases were reported to have developmental stagnation or regression in association with seizure onset. Treatments for epilepsy that were reported by clinicians to be effective were: sodium valproate, which was effective in five of nine patients (5/9) who tried it; rufinamide (2/3); and ketogenic diet (2/3). Additional features were ataxia/incoordination (8/13); microcephaly (7/13); peripheral neuropathy (4/13), and spasticity/hypertonia (6/13)., Significance: POLR3B is a novel genetic developmental and epileptic encephalopathy (DEE) in which EMAtS is the predominant epilepsy phenotype. Ataxia, neuropathy, and hypertonia may be variously observed in these patients., (© 2024 The Author(s). Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

tRNAs are evolutionarily ancient molecular decoders essential for protein translation. In eukaryotes, tRNAs and other short, noncoding RNAs are transcribed by RNA polymerase (Pol) III, an enzyme that promotes ageing in yeast, worms, and flies. Here, we show that a partial reduction in Pol III activity specifically disrupts tRNA levels. This effect is conserved across worms, flies, and mice, where computational models indicate that it impacts mRNA decoding. In all 3 species, reduced Pol III activity increases proteostatic resilience. In worms, it activates the unfolded protein response (UPR) and direct disruption of tRNA metabolism is sufficient to recapitulate this. In flies, decreasing Pol III's transcriptional initiation on tRNA genes by a loss-of-function in the TFIIIC transcription factor robustly extends lifespan, improves proteostatic resilience and recapitulates the broad-spectrum benefits to late-life health seen following partial Pol III inhibition. We provide evidence that a partial reduction in Pol III activity impacts translation, quantitatively or qualitatively, in both worms and flies, indicating a potential mode of action. Our work demonstrates a conserved and previously unappreciated role of tRNAs in animal ageing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Malik et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression in vivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: POLIII-related leukodystrophies are a group of recently recognized hereditary white matter diseases with a similar clinical and radiological phenotype. No Tunisian studies have been published about POLIII-related leukodystrophy due to POLR3A variants. The aim of this study was to contribute to the clinical, radiological, and genetic characterization of POLR3A-related leukodystrophy in a Tunisian cohort., Methods: We report six cases of genetically confirmed POLR3A-related leukodystrophy belonging to six unrelated Tunisian families, along with a review of previously published pediatric cases., Results: All patients were born to consanguineous marriages and originated from the North or the Center of Tunisia. Age at onset varied between 15 months and 6 years. The clinical phenotype was similar in all patients with cerebellar ataxia, tremor, and nystagmus being the key features. Brain imaging showed diffuse hypomyelination in all patients with progressive cerebellar atrophy in three patients. Molecular analysis identified the same bi-allelic NM_007055.4:c.2011T>C; p.(Trp671Arg) variant in the POLR3A gene in all patients., Conclusion: We hypothesize a founder effect for the identified variant given its recurrence in six unrelated individuals with a similar clinical phenotype. Given the apparent genetic homogeneity of Tunisian POLR3A patients, the recurrent variant should be directly targeted. This should facilitate diagnosis in index patients, and genetic counseling., (© 2024 The Author(s). Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Purpose: To report on a new phenotype in a patient carrying a novel, undescribed de novo variant in POLR3B, affected by generalized myoclonic epilepsy and neurodevelopmental disorder, without neuropathy. It is known that biallelic pathogenic variants in POLR3B cause hypomyelinating leukodystrophy-8, and heterozygous de novo variants are described in association to a phenotype characterized by predominantly demyelinating sensory-motor peripheral neuropathy, ataxia, spasticity, intellectual disability and epilepsy, in which the peripheral neuropathy is often the main clinical presentation., Methods: We collected clinical, electrophysiological and neuroimaging data from the affected subject and performed a Trio-Clinical Exome Sequencing., Results: We detected a de novo novel heterozygous missense variant c.1132A>G in POLR3B (NM_018082.6) that was considered as likely pathogenic following ACMG criteria. We also consulted our custom genomic database of a total of 1485 patients that were genetically analysed from 2018 for epilepsy, and found no other de novo variants in the POLR3B gene., Conclusion: We hypothesize a possible genotype-phenotype correlation, particularly regarding epilepsy. We also provide a review of the literature about the previously described POLR3B heterozygous patients, with particular attention to the epileptic phenotype, underlining the association between POLR3B and early onset myoclonic epilepsy, which can represent the main manifestation of the disease at its onset., Competing Interests: Declaration of competing interest None., (Copyright © 2024 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

RNA polymerases must initiate and pause within a complex chromatin environment, surrounded by nucleosomes and other transcriptional machinery. This environment creates a spatial arrangement along individual chromatin fibers ripe for both competition and coordination, yet these relationships remain largely unknown owing to the inherent limitations of traditional structural and sequencing methodologies. To address this, we employed long-read chromatin fiber sequencing (Fiber-seq) in Drosophila to visualize RNA polymerase (Pol) within its native chromatin context with single-molecule precision along up to 30 kb fibers. We demonstrate that Fiber-seq enables the identification of individual Pol II, nucleosome, and transcription factor footprints, revealing Pol II pausing-driven destabilization of downstream nucleosomes. Furthermore, we demonstrate pervasive direct distance-dependent transcriptional coupling between nearby Pol II genes, Pol III genes, and transcribed enhancers, modulated by local chromatin architecture. Overall, transcription initiation reshapes surrounding nucleosome architecture and couples nearby transcriptional machinery along individual chromatin fibers., Competing Interests: Declaration of interests A.B.S. is a co-inventor on a patent relating to the Fiber-seq method (US17/995,058)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest Alexander Tonevitsky is an employee of Art Photonics GmbH., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 40 kDa 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Maf1, originally described as a repressor of RNA polymerase III (RNAP III) transcription in yeast, participates in multiple functions across eukaryotes. However, the knowledge about Maf1 in protozoan parasites is scarce. To initiate the study of Maf1 in Leishmania major, we generated a cell line that overexpresses this protein. Overexpression of Maf1 led to a significant reduction in the abundance of tRNAs, 5S rRNA, and U4 snRNA, demonstrating that Maf1 regulates RNAP III activity in L. major. To further explore the roles played by Maf1 in this microorganism, global transcriptomic and proteomic changes due to Maf1 overexpression were determined using RNA-sequencing and label-free quantitative mass spectrometry. Compared to wild-type cells, differential expression was observed for 1082 transcripts (615 down-regulated and 467 up-regulated) and 205 proteins (132 down-regulated and 73 up-regulated) in the overexpressing cells. A correlation of 44% was found between transcriptomic and proteomic results. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially expressed genes and proteins are mainly involved in transcription, cell cycle regulation, lipid metabolism and transport, ribosomal biogenesis, carbohydrate metabolism, autophagy, and cytoskeleton modification. Thus, our results suggest the involvement of Maf1 in the regulation of all these processes in L. major, as reported in other species, indicating that the functions performed by Maf1 were established early in eukaryotic evolution. Notably, our data also suggest the participation of L. major Maf1 in mRNA post-transcriptional control, a role that, to the best of our knowledge, has not been described in other organisms., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Single-cell RNA sequencing (scRNA-seq) has emerged as a versatile tool in biology, enabling comprehensive genomic-level characterization of individual cells. Currently, most scRNA-seq methods generate barcoded cDNAs by capturing the polyA tails of mRNAs, which exclude many non-coding RNAs (ncRNAs), especially those transcribed by RNA polymerase III (Pol III). Although previously thought to be expressed constitutively, Pol III-transcribed ncRNAs are expressed variably in healthy and disease states and play important roles therein, necessitating their profiling at the single-cell level. In this study, we developed a measurement protocol for nc886 as a model case and initial step for scRNA-seq for Pol III-transcribed ncRNAs. Specifically, we spiked in an oligo-tagged nc886-specific primer during the polyA tail capture process for the 5'scRNA-seq. We then produced sequencing libraries for standard 5' gene expression and oligo-tagged nc886 separately, to accommodate different cDNA sizes and ensure undisturbed transcriptome analysis. We applied this protocol in three cell lines that express high, low, and zero levels of nc886. Our results show that the identification of oligo tags exhibited limited target specificity, and sequencing reads of nc886 enabled the correction of non-specific priming. These findings suggest that gene-specific primers (GSPs) can be employed to capture RNAs lacking a polyA tail, with subsequent sequence verification ensuring accurate gene expression counting. Moreover, we embarked on an analysis of differentially expressed genes in cell line sub-clusters with differential nc886 expression, demonstrating variations in gene expression phenotypes. Collectively, the primer spike-in strategy allows combined analysis of ncRNAs and gene expression phenotype., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Hypomyelinating leukodystrophies (HLD) are a group of heterogeneous genetic disorders characterized by a deficit in myelin deposition during brain development. Specifically, 4H-Leukodystrophy is a recessive disease due to biallelic mutations in the POLR3A gene, which encodes one of the subunits forming the catalytic core of RNA polymerase III (PolIII). The disease also presents non-neurological signs such as hypodontia and hypogonadotropic hypogonadism. Here, we report the generation of a human induced pluripotent stem cell (hiPSC) line from fibroblasts of the first identified carrier of the biallelic POLR3A variants c.1802 T > A and c.4072G > A., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jessica Rosati reports financial support, article publishing charges, and equipment, drugs, or supplies were provided by Ministry of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Bud27 is a prefoldin-like protein that participates in transcriptional regulation mediated by the three RNA polymerases in Saccharomyces cerevisiae. Lack of Bud27 significantly affects RNA pol III transcription, although the involved mechanisms have not been characterized. Here, we show that Bud27 regulates the phosphorylation state of the RNA pol III transcriptional repressor, Maf1, influences its nuclear localization, and likely its activity. We demonstrate that Bud27 is associated with the Maf1 main phosphatase PP4 in vivo, and that this interaction is required for proper Maf1 dephosphorylation. Lack of Bud27 decreases the interaction among PP4 and Maf1, Maf1 dephosphorylation, and its nuclear entry. Our data uncover a new nuclear function of Bud27, identify PP4 as a novel Bud27 interactor and demonstrate the effect of this prefoldin-like protein on the posttranslational regulation of Maf1. Finally, our data reveal a broader effect of Bud27 on PP4 activity by influencing, at least, the phosphorylation of Rad53., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: POLR3-HLD or 4H leukodystrophy is an autosomal recessive disorder characterized by hypomyelination, hypodontia, and hypogonadotropic hypogonadism, and caused by variants in POLR3A , POLR3B , POLR1C , or POLR3K genes. Neurological and non-neurological clinical features and disease severity vary. While previous studies reference variable cognition, this is the first report of 4H detailing a comprehensive neuropsychological assessment. Method: The current study presents a 20-year-old, English-speaking, right-handed, non-Hispanic White female with 12 years of education with genetically confirmed 4H POLR3B -related leukodystrophy without hormonal replacement treatment. Results: At age 4, developmental delays, ataxia, hearing loss, and abnormal dentition were present. Imaging, endocrinology, and neurologic examinations revealed hypomyelination, reduced cerebellar volume, delayed bone age density, osteopenia, and evidence of adrenarche without signs of true puberty. Neuropsychological assessment at age 20 revealed global cognitive impairment with intellectual, attention, verbal memory retrieval, construction, executive (e.g. processing speed, sustained attention) and math computation deficits, along with behavioral dysregulation. Conclusion: We present the first detailed neuropsychological assessment of a patient with 4H leukodystrophy. The neuropsychological assessment revealed cognitive and behavioral dysexecutive deficits aligning with hypomyelination observed on imaging. Further longitudinal studies are needed to shed light on the neurobehavioral presentation associated with this disorder to assist care providers, patients, and their families.

While often undetected and untreated, persistent seasonal asymptomatic malaria infections remain a global public health problem. Despite the presence of parasites in the peripheral blood, no symptoms develop. Disease severity is correlated with the levels of infected red blood cells (iRBCs) adhering within blood vessels. Changes in iRBC adhesion capacity have been linked to seasonal asymptomatic malaria infections, however how this is occurring is still unknown. Here, we present evidence that RNA polymerase III (RNA Pol III) transcription in Plasmodium falciparum is downregulated in field isolates obtained from asymptomatic individuals during the dry season. Through experiments with in vitro cultured parasites, we have uncovered an RNA Pol III-dependent mechanism that controls pathogen proliferation and expression of a major virulence factor in response to external stimuli. Our findings establish a connection between P. falciparum cytoadhesion and a non-coding RNA family transcribed by Pol III. Additionally, we have identified P. falciparum Maf1 as a pivotal regulator of Pol III transcription, both for maintaining cellular homeostasis and for responding adaptively to external signals. These results introduce a novel perspective that contributes to our understanding of P. falciparum virulence. Furthermore, they establish a connection between this regulatory process and the occurrence of seasonal asymptomatic malaria infections., Competing Interests: GD, AC, AB, PN, FD, MS, DL, AC, AS No competing interests declared, (© 2024, Diffendall et al.)

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., Competing Interests: Declaration of interests I declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

While biallelic POLR3A loss-of-function variants are traditionally linked to hypomyelinating leukodystrophy, patients with a specific splice variant c.1909+22G>A manifest as adolescent-onset spastic ataxia without overt leukodystrophy. In this study, we reported eight new cases, POLR3A-related disorder with c.1909+22 variant. One of these patients showed expanded phenotypic spectrum of generalised dystonia and her sister remained asymptomatic except for hypodontia. Two patients with dystonic arm tremor responded to deep brain stimulation. In our systemic literature review, we found that POLR3A-related disorder with c.1909+22 variant has attenuated disease severity but frequency of dystonia and upper limb tremor did not differ among genotypes., (© 2024 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Organismal growth and lifespan are inextricably linked. Target of Rapamycin (TOR) signalling regulates protein production for growth and development, but if reduced, extends lifespan across species. Reduction in the enzyme RNA polymerase III, which transcribes tRNAs and 5S rRNA, also extends longevity. Here, we identify a temporal genetic relationship between TOR and Pol III in Caenorhabditis elegans , showing that they collaborate to regulate progeny production and lifespan. Interestingly, the lifespan interaction between Pol III and TOR is only revealed when TOR signaling is reduced, specifically in adulthood, demonstrating the importance of timing to control TOR regulated developmental versus adult programs. In addition, we show that Pol III acts in C. elegans muscle to promote both longevity and healthspan and that reducing Pol III even in late adulthood is sufficient to extend lifespan. This demonstrates the importance of Pol III for lifespan and age-related health in adult C. elegans ., (© 2024 Malik et al.)

Defects in tRNA biogenesis are associated with multiple neurological disorders, yet our understanding of these diseases has been hampered by an inability to determine tRNA expression in individual cell types within a complex tissue. Here, we developed a mouse model in which RNA polymerase III is conditionally epitope tagged in a Cre-dependent manner, allowing us to accurately profile tRNA expression in any cell type in vivo. We investigated tRNA expression in diverse nervous system cell types, revealing dramatic heterogeneity in the expression of tRNA genes between populations. We found that while maintenance of levels of tRNA isoacceptor families is critical for cellular homeostasis, neurons are differentially vulnerable to insults to distinct tRNA isoacceptor families. Cell-type-specific translatome analysis suggests that the balance between tRNA availability and codon demand may underlie such differential resilience. Our work provides a platform for investigating the complexities of mRNA translation and tRNA biology in the brain., Competing Interests: Declaration of interests S.L.A. is a member of the scientific advisory board of Tevard Biosciences., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2024 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Ovarian cancer, among all gynecologic malignancies, exhibits the highest incidence and mortality rate, primarily because it is often presents with non-specific or no symptoms during its early stages. For the advancement of Ovarian Cancer Diagnosis, it is crucial to identify the potential molecular signatures that could significantly differentiate between healthy and ovarian cancerous tissues and can be used further as a diagnostic biomarker for detecting ovarian cancer. In this study, we investigated the genome-wide methylation patterns in ovarian cancer patients using Methylated DNA Immunoprecipitation (MeDIP-Seq) followed by NGS. Identified differentially methylated regions (DMRs) were further validated by targeted bisulfite sequencing for CpG site-specific methylation profiles. Furthermore, expression validation of six genes by Quantitative Reverse Transcriptase-PCR was also performed. Out of total 120 differentially methylated genes (DMGs), 68 genes were hypermethylated, and 52 were hypomethylated in their promoter region. After analysis, we identified the top 6 hub genes, namely POLR3B, PLXND1, GIGYF2, STK4, BMP2 and CRKL. Interestingly we observed Non-CpG site methylation in the case of POLR3B and CRKL which was statistically significant in discriminating ovarian cancer samples from normal controls. The most significant pathways identified were focal adhesion, the MAPK signaling pathway, and the Ras signaling pathway. Expression analysis of hypermethylated genes was correlated with the downregulation of the genes. POLR3B and GIGYF2 turned out to be the novel genes associated with the carcinogenesis of EOC. Our study demonstrated that methylation profiling through MeDIP-sequencing has effectively identified six potential hub genes and pathways that might exacerbate our understanding of underlying molecular mechanisms of ovarian carcinogenesis., (© 2024. The Author(s).)

Spastic Ataxias (SA) are a group of neurodegenerative disorders with combined pyramidal and cerebellar system affection, leading to an overlap phenotype between Hereditary Spastic Paraplegias (HSP) and Cerebellar Ataxias (CA). Here we describe the generation of iPSCs from three unrelated patients with an ultra-rare subtype of SA caused by compound heterozygous mutations in POLR3A, that encodes the largest subunit of RNA polymerase III. iPSCs were reprogrammed from normal human dermal fibroblasts (NHDFs) using episomal reprogramming with integration-free plasmid vectors: HIHRSi004-A, derived from a 44 year-old male carrying the mutations c.1909 + 22G > A/c.3944_3945delTG, HIHRSi005-A obtained from a 66 year-old male carrying the mutations c.1909 + 22G > A/c.1531C > T, and HIHRSi006-A from a 27 year-old male carrying the mutations c.1909 + 22G > A/c.2472_2472delC (ENST00000372371.8)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s).)

RNA polymerase III synthesizes a wide range of noncoding RNAs shorter than 400 nucleotides in length. These RNAs are involved in protein synthesis (tRNA, 5S rRNA, and 7SL RNA), maturation, and splicing of different types of RNA (RPR, MRP RNA, and U6 snRNA), regulation of transcription (7SK RNA), replication (Y RNA), and intracellular transport (vault RNA). BC200 and BC1 RNA genes are transcribed by RNA polymerase III in neurons only where these RNAs regulate protein synthesis. Mutations in the regulatory elements of the genes transcribed by RNA polymerase III as well as in transcription factors of this RNA polymerase are associated with the development of a number of diseases, primarily oncological and neurological. In this regard, the mechanisms of regulation of the expression of the genes containing various RNA polymerase III promoters were actively studied. This review describes the structural and functional classification of polymerase III promoters, as well as the factors involved in the regulation of promoters of different types. A number of examples demonstrate the role of the described factors in the pathogenesis of human diseases.

General transcription factor IIIC subunit 5 (GTF3C5) encodes transcription factor IIIC63 (TFIIIC63). It binds to DNA to recruit another transcription factor, TFIIIB, and RNA polymerase III (Pol III) to mediate the transcription of small noncoding RNAs, such as tRNAs. Here, we report four individuals from three families presenting with a multisystem developmental disorder phenotype with biallelic variants in GTF3C5. The overlapping features include growth retardation, developmental delay, intellectual disability, dental anomalies, cerebellar malformations, delayed bone age, skeletal anomalies, and facial dysmorphism. Using lymphoblastoid cell lines (LCLs) from two affected individuals, we observed a reduction in TFIIIC63 protein levels compared to control LCLs. Genome binding of TFIIIC63 protein is also reduced in LCL from one of the affected individuals. Additionally, approximately 40% of Pol III binding regions exhibited reduction in the level of Pol III occupancy in the mutant genome relative to the control, while approximately 54% of target regions showed comparable levels of Pol III occupancy between the two, indicating partial impairment of Pol III occupancy in the mutant genome. Yeasts with subject-specific variants showed temperature sensitivity and impaired growth, supporting the notion that the identified variants have deleterious effects. gtf3c5 mutant zebrafish showed developmental defects, including a smaller body, head, and eyes. Taken together, our data show that GTF3C5 plays an important role in embryonic development, and that biallelic variants in this gene cause a multisystem developmental disorder. Our study adds GTF3C5-related disorder to the growing list of genetic disorders associated with Pol III transcription machinery., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Wiedemann-Rautenstrauch Syndrome (WRS; MIM 264090) is an extremely rare and highly heterogeneous syndrome that is inherited in a recessive fashion. The patients have hallmark features such as prenatal and postnatal growth retardation, short stature, a progeroid appearance, hypotonia, facial dysmorphology, hypomyelination leukodystrophy, and mental impairment. Biallelic disease-causing variants in the RNA polymerase III subunit A (POLR3A) have been associated with WRS. Here, we report the first identified cases of WRS syndrome with novel phenotypes in three consanguineous families (two Omani and one Saudi) characterized by biallelic variants in POLR3A. Using whole-exome sequencing, we identified one novel homozygous missense variant (NM_007055: c.2456C>T; p. Pro819Leu) in two Omani families and one novel homozygous variant (c.1895G>T; p Cys632Phe) in Saudi family that segregates with the disease in the POLR3A gene. In silico homology modeling of wild-type and mutated proteins revealed a substantial change in the structure and stability of both proteins, demonstrating a possible effect on function. By identifying the homozygous variants in the exon 14 and 18 of the POLR3A gene, our findings will contribute to a better understanding of the phenotype-genotype relationship and molecular etiology of WRS syndrome., (© 2024 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

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., Competing Interests: Declaration of interests S.L. and P.A. are employees of AstraZeneca. F.A.-G. is employee of Cytena GmbH., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Several covalent modifications are found associated with the transcriptionally active chromatin regions constituted by the genes transcribed by RNA polymerase (pol) II. Pol III-transcribed genes code for the small, stable RNA species, which participate in many cellular processes, essential for survival. Pol III transcription is repressed under most of the stress conditions by its negative regulator Maf1. We found that most of the histone acetylations increase with starvation-induced repression on several genes transcribed by the yeast pol III. On one of these genes, SNR6 (coding for the U6snRNA), a strongly positioned nucleosome in the gene upstream region plays regulatory role under repression. On this nucleosome, the changes in H3K9 and H3K14 acetylations show different dynamics. During repression, acetylation levels on H3K9 show steady increase whereas H3K14 acetylation increases with a peak at 40 min after which levels reduce. Both the levels settle by 2 hr to a level higher than the active state, which revert to normal levels with nutrient repletion. The increase in H3 acetylations is seen in the mutants reported to show reduced SNR6 transcription but not in the maf1Δ cells. This increase on a regulatory nucleosome may be part of the signaling mechanisms, which prepare cells for the stress-related quick repression as well as reactivation. The contrasting association of the histone acetylations with pol II and pol III transcription may be an important consideration to make in research studies focused on drug developments targeting histone modifications., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Aneeshkumar Gopalakrishnan Arimbasseri reports financial support was provided by Council of Scientific & Industrial Research. Ashutosh Shukla reports financial support was provided by Council of Scientific & Industrial Research. Ashis Kumar Pradhan reports financial support was provided by Council of Scientific & Industrial Research., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: Foramen magnum (FM) meningiomas pose significant surgical challenges and have high morbidity and mortality rates. This study aimed to investigate the distribution of clinically actionable mutations in FM meningiomas and identify clinical characteristics associated with specific mutational profiles., Methods: The authors conducted targeted next-generation sequencing of 62 FM meningiomas from three international institutions, covering all relevant meningioma genes (AKT1, KLF4, NF2, POLR2A, PIK3CA, SMO, TERT promoter, and TRAF7). Patients with a radiation-induced meningioma or neurofibromatosis type 2 (NF2) were excluded from the study. Additionally, patient and tumor characteristics, including age, sex, radiological features, and tumor location, were retrospectively collected and evaluated., Results: The study cohort consisted of 46 female and 16 male patients. Clinically significant driver mutations were detected in 58 patients (93.5%). The most commonly observed alteration was TRAF7 mutations (26, 41.9%), followed by AKT1E17K mutations (19, 30.6%). Both mutations were significantly associated with an anterolateral tumor location relative to the brainstem (p = 0.0078). NF2 mutations were present in 11 cases (17.7%) and were associated with posterior tumor location, in contrast to tumors with TRAF7 and AKT1E17K mutations. Other common mutations in FM meningiomas included POLR2A mutations (8, 12.9%; 6 POLR2AQ403K and 2 POLR2AH439_L440del), KLF4K409Q mutations (7, 11.3%), and PIK3CA mutations (4, 6.5%; 2 PIK3CAH1047R and 2 PIK3CAE545K). POLR2A and KLF4 mutations exclusively occurred in female patients and showed no significant association with specific tumor locations. All tumors harboring AKT1E17K and POLR2A mutations displayed meningothelial histology. Ten tumors exhibited intratumoral calcification, which was significantly more frequent in NF2-mutant compared with AKT1-mutant FM meningiomas (p = 0.047)., Conclusions: These findings provide important insights into the molecular genetics and clinicopathological characteristics of FM meningiomas. The identification of specific genetic alterations associated with tumor location, volume, calcification, histology, and sex at diagnosis may have implications for personalized treatment strategies in the future.

Ischaemic stroke is a leading cause of death and life-long disability due to neuronal cell death resulting from interruption of glucose and oxygen supplies. RNA polymerase III (Pol III)-dependent transcription plays a central role in protein synthesis that is necessary for normal cerebral neuronal functions, and the survival and recovery under pathological conditions. Notably, Pol III transcription is highly sensitive to ischaemic stress that is known to rapidly shut down Pol III transcriptional activity. However, its precise role in ischaemic stroke, especially during the acute and recovery phases, remains poorly understood. The microenvironment within the ischaemic brain undergoes dynamic changes in different phases after stroke. Emerging evidence highlights the distinct roles of Pol III transcription in neuroprotection during the acute phase and repair during the recovery phase of stroke. Additionally, investigations into the mTOR-MAF1 signalling pathway, a conserved regulator of Pol-III transcription, reveal its therapeutic potential in enhancing acute phase neuroprotection and recovery phase repair.

Transfer RNAs are essential for translating genetic information into proteins. The human genome contains hundreds of predicted tRNA genes, many in multiple copies. How their expression is regulated to control tRNA repertoires is unknown. Here we combined quantitative tRNA profiling and chromatin immunoprecipitation with sequencing to measure tRNA expression following the differentiation of human induced pluripotent stem cells into neuronal and cardiac cells. We find that tRNA transcript levels vary substantially, whereas tRNA anticodon pools, which govern decoding rates, are more stable among cell types. Mechanistically, RNA polymerase III transcribes a wide range of tRNA genes in human induced pluripotent stem cells but on differentiation becomes constrained to a subset we define as housekeeping tRNAs. This shift is mediated by decreased mTORC1 signalling, which activates the RNA polymerase III repressor MAF1. Our data explain how tRNA anticodon pools are buffered to maintain decoding speed across cell types and reveal that mTORC1 drives selective tRNA expression during differentiation., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests B.F.P. is an owner of and has a financial interest in Peconic, which uses the ChIP-exo technology (U.S. Patent 20100323361A1) implemented in this study and could potentially benefit from the outcomes of this research., (Copyright © 2023 Elsevier Inc. All rights reserved.)

RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Wiedemann-Rautenstrauch syndrome (neonatal progeroid syndrome) is an ultra-orphan disease from the group of premature aging syndromes with an autosomal recessive type of inheritance associated with mutations in the POLR3A, POLR3B, and POLR3GL genes encoding RNA polymerase III. The incidence of the disease is currently unknown. We present the first clinical description in Russian Federation of a patient 7 years 6 months old with Wiedemann-Rautenstrauch syndrome (compound heterozygous mutations in POLR3A gene) with progeroid features, adentia, growth retardation (height SDS -3,41, height velocity SDS -2,47), underweight (BMI SDS -6,20), and generalized lipodystrophy. The article presents the observation of the patient for 1.5 years, the world experience of dynamic follow-up of patients with neonatal progeroid syndrome, differential diagnosis, as well as recommendations for the management of patients with this syndrome. Given the lack of specific treatment to date, patients are observed by a multidisciplinary team of physicians.

Background: RNA polymerase III-related or 4H leukodystrophy (POLR3-HLD) is an autosomal recessive hypomyelinating leukodystrophy characterized by neurological dysfunction, hypodontia and hypogonadotropic hypogonadism. The disease is caused by biallelic pathogenic variants in POLR3A , POLR3B , POLR1C or POLR3K . Craniofacial abnormalities reminiscent of Treacher Collins syndrome have been originally described in patients with POLR3-HLD caused by biallelic pathogenic variants in POLR1C . To date, no published studies have appraised in detail the craniofacial features of patients with POLR3-HLD. In this work, the specific craniofacial characteristics of patients with POLR3-HLD associated with biallelic pathogenic variants in POLR3A , POLR3B and POLR1C are described., Methods: The craniofacial features of 31 patients with POLR3-HLD were evaluated, and potential genotype-phenotype associations were evaluated., Results: Various craniofacial abnormalities were recognized in this patient cohort, with each individual presenting at least one craniofacial abnormality. The most frequently identified features included a flat midface (61.3%), a smooth philtrum (58.0%) and a pointed chin (51.6%). In patients with POLR3B biallelic variants, a thin upper lip was frequent. Craniofacial anomalies involving the forehead were most commonly associated with biallelic variants in POLR3A and POLR3B while a higher proportion of patients with POLR1C biallelic variants demonstrated bitemporal narrowing., Conclusion: Through this study, we demonstrated that craniofacial abnormalities are common in patients with POLR3-HLD. This report describes in detail the dysmorphic features of POLR3-HLD associated with biallelic variants in POLR3A , POLR3B and POLR1C ., Competing Interests: Competing interests: LTT currently manages sponsored clinical trials at the site level for Ionis Pharmaceuticals (Alexander disease clinical trial 2021–present), Passage Bio (Krabbe disease and GM1 gangliosidosis clinical trials, 2021–present) and Teva Pharmaceuticals (chronic and episodic migraine clinical trials, 2022–present). He also manages a GM1 gangliosidosis natural history study sponsored by the University of Pennsylvania with funding from Passage Bio. NIW is advisor and/or co-investigator for trials in Metachromatic Leukodystrophy (Shire/Takeda, Orchard, Evidera) and other leukodystrophies (Ionis, PassageBio, Vigil Neuro, Sana Biotech), without personal payment. AV receives research grants or in-kind research support without any personal compensation from Takeda, Passage Bio, Sanofi, Affynia, Orchard Therapeutics, Eli Lilly, ISD therapeutics, Illumina, Myrtelle, Homology, Sana and Ionis. She is a site investigator for the Ionis clinical trial in Alexander’s disease, SHP611 in Metachromatic leukodystrophy of Shire/Takeda and Passage Bio gene therapy in Krabbe. She serves on the scientific advisory board of the ELA foundation, the ULF Foundation and the Yaya Foundation Scientific and Clinical Advisory Council. She is a member of the Vanishing White Matter Consortium, the H-ABC Clinical Advisory Board. She receives grant funding from this RDCRN NCATS/NINDS (U01 NS106845, U54TR002823 and R21 NS123477. GB is/was a consultant for Passage Bio Inc (2020–2022) and Ionis (2019). She is/was a site investigator for the Alexander’s disease trial of Ionis (2021–present), Metachromatic leukodystrophy of Shire/Takeda (2020–2021), Krabbe and GM1 gene therapy trials of Passage Bio (2021–present), GM1 natural history study sponsored by the University of Pennsylvania with funding from Passage Bio (2021–present) and Adrenoleukodystrophy/Hematopoietic stem cell transplantation natural history study of Bluebird Bio (2019), a site sub-investigator for the MPS II gene therapy trial of Regenxbio (2021–present) and the MPS II clinical trial of Denali (2022–present). She has received an unrestricted educational grant from Takeda (2021–2022). She serves on the scientific advisory board of the Pelizaeus-Merzbacher Foundation, the Yaya Foundation Scientific and Clinical Advisory Council and is the Chair of the Medical and Scientific Advisory Board of the United Leukodystrophy Foundation. She is a member of the Vanishing White Matter Consortium, the H-ABC Clinical Advisory Board and the Chair of the POLR3-related (4H) Leukodystrophy Consortium. She is on the editorial boards of Neurology Genetics, Frontiers in Neurology – Neurogenetics and Journal of Medical Genetics., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

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 β- and τ-signals. One of the goals of this work was to evaluate how autonomous and independent other SINE parts are β- and τ-signals. Extended regions outside of β- 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 β- and τ-signals on the neighboring sequences. A similar analysis of B2 and Ves constructs containing a 55-bp foreign sequence inserted between β- 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.

Competing Interests: None

The RNA polymerase III (Pol III) transcriptome is universally comprised of short, highly structured noncoding RNA (ncRNA). Through RNA-protein interactions, the Pol III transcriptome actuates functional activities ranging from nuclear gene regulation (7SK), splicing (U6, U6atac), and RNA maturation and stability (RMRP, RPPH1, Y RNA), to cytoplasmic protein targeting (7SL) and translation (tRNA, 5S rRNA). In higher eukaryotes, the Pol III transcriptome has expanded to include additional, recently evolved ncRNA species that effectively broaden the footprint of Pol III transcription to additional cellular activities. Newly evolved ncRNAs function as riboregulators of autophagy (vault), immune signaling cascades (nc886), and translation (Alu, BC200, snaR). Notably, upregulation of Pol III transcription is frequently observed in cancer, and multiple ncRNA species are linked to both cancer progression and poor survival outcomes among cancer patients. In this review, we outline the basic features and functions of the Pol III transcriptome, and the evidence for dysregulation and dysfunction for each ncRNA in cancer. When taken together, recurrent patterns emerge, ranging from shared functional motifs that include molecular scaffolding and protein sequestration, overlapping protein interactions, and immunostimulatory activities, to the biogenesis of analogous small RNA fragments and noncanonical miRNAs, augmenting the function of the Pol III transcriptome and further broadening its role in cancer. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Processing of Small RNAs RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications., (© 2023 The Authors. WIREs RNA published by Wiley Periodicals LLC.)

Background: POLR3-related leukodystrophy is a group of rare neurodegenerative disorders characterized by degeneration of the white matter with different combinations of major clinical features., Case: An 18-year-old lady was admitted for no menstruation since childhood. She gradually developed slight symptoms, such as choking after drinking water and unsteady walking in the last 2 years. Furthermore, her test scores and response capability were far lower than that of her peers. Physical examination revealed her to be of a slightly short stature, with stiff expressions and bilateral breast enlargement. She revealed clumsy movements when examined for ataxia, with an SARA score of 9., Findings: The laboratory data revealed a decreased level of estradiol, FSH, and LH, with a MoCA score of 7. Conventional karyotype analysis revealed a 46 XX 9qh + karyotype. Ultrasound indicated primordial uterus (19 × 11 × 10 mm). Brain MRI showed bilateral cerebral hemisphere myelin dysplasia, brain atrophy, thin corpus callosum, and small pituitary gland with uneven reinforcement and enlarged ventricles. Exome sequencing exhibited two missense mutations in the POLR3A gene (c.3013C > T and c.1757C > T), which were inherited from her mother and father, respectively., Conclusion: Collectively, we identified novel compound heterozygous mutations of the POLR3A gene that caused POLR3A-related hypomyelinating leukodystrophy with hypogonadism in the patient combined with the clinical presentation, MRI brain pattern, and medical exome sequencing., Teaching Points: The complexity of clinical phenotypes and heterogeneity of genotypes raise new challenges in genetic diagnoses. This study will further aid our understanding of POLR3A-related leukodystrophy and promote further analysis of phenotype-genotype correlations of related diseases., (© 2023. Fondazione Società Italiana di Neurologia.)

RNA polymerase III (Pol III) plays a vital role in transcription and as a viral-DNA sensor, but how it is assembled and distributed within cells remains poorly understood. Here, we show that Pol III is assembled with chaperones in the cytoplasm and forms transcription-dependent protein clusters upon transport into the nucleus. The largest subunit (RPC1) depletion through an auxin-inducible degron leads to rapid degradation and disassembly of Pol III complex in the nucleus and cytoplasm, respectively. This generates a pool of partially assembled Pol III intermediates, which can be rapidly mobilized into the nucleus upon the restoration of RPC1. Our study highlights the critical role of subcellular localization in determining Pol III's fate and provides insight into the dynamic regulation of nuclear Pol III levels and the origin of cytoplasmic Pol III complexes involved in mediating viral immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Aims: Interferon (IFN) signaling is critical to the pathogenesis of alcohol-associated hepatitis (AH), yet the mechanisms for activation of this system are elusive. We hypothesize that host-derived 5S rRNA pseudogene (RNA5SP) transcripts regulate IFN production and modify immunity in AH., Approach and Results: Mining of transcriptomic datasets revealed that in patients with severe alcohol-associated hepatitis (sAH), hepatic expression of genes regulated by IFNs was perturbed and gene sets involved in IFN production were enriched. RNA5SP transcripts were also increased and correlated with expression of type I IFNs. Interestingly, inflammatory mediators upregulated in sAH, but not in other liver diseases, were positively correlated with certain RNA5SP transcripts. Real-time quantitative PCR demonstrated that RNA5SP transcripts were upregulated in peripheral blood mononuclear cells (PBMCs) from patients with sAH. In sAH livers, increased 5S rRNA and reduced nuclear MAF1 (MAF1 homolog, negative regulator of RNA polymerase III) protein suggested a higher activity of RNA polymerase III (Pol III); inhibition of Pol III reduced RNA5SP expression in monocytic THP-1 cells. Expression of several RNA5SP transcript-interacting proteins was downregulated in sAH, potentially unmasking transcripts to immunosensors. Indeed, siRNA knockdown of interacting proteins potentiated the immunostimulatory activity of RNA5SP transcripts. Molecular interaction and cell viability assays demonstrated that RNA5SP transcripts adopted Z-conformation and contributed to ZBP1-mediated caspase-independent cell death., Conclusions: Increased expression and binding availability of RNA5SP transcripts was associated with hepatic IFN production and inflammation in sAH. These data identify RNA5SP transcripts as a potential target to mitigate inflammation and hepatocellular injury in AH., (Copyright © 2023 American Association for the Study of Liver Diseases.)

nc886 is a medium-sized non-coding RNA that is transcribed by RNA polymerase III (Pol III) and plays diverse roles in tumorigenesis, innate immunity, and other cellular processes. Although Pol III-transcribed ncRNAs were previously thought to be expressed constitutively, this concept is evolving, and nc886 is the most notable example. The transcription of nc886 in a cell, as well as in human individuals, is controlled by multiple mechanisms, including its promoter CpG DNA methylation and transcription factor activity. Additionally, the RNA instability of nc886 contributes to its highly variable steady-state expression levels in a given situation. This comprehensive review discusses nc886's variable expression in physiological and pathological conditions and critically examines the regulatory factors that determine its expression levels.