Clinical phenotype and genetic function analysis of a family with hypomyelinating leukodystrophy-7 caused by POLR3A mutation.

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.

Comprehensive DNA methylation profiling by MeDIP-NGS identifies potential genes and pathways for epithelial ovarian cancer.

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.

HSP70 binds to specific non-coding RNA and regulates human RNA polymerase III.

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.

A novel de novo variant in POLR3B gene associated with a primary axonal involvement of the largest nerve fibers.

BACKGROUND AND AIMS: POLR3B gene encodes a subunit of RNA polymerase III (Pol III). Biallelic mutations in POLR3B are associated with leukodystrophies, but recently de novo heterozygous mutations have been described in early onset peripheral demyelinating neuropathies with or without central involvement. Here, we report the first Italian case carrying a de novo variant in POLR3B with a pure neuropathy phenotype and primary axonal involvement of the largest nerve fibers. METHODS: Nerve conduction studies, sympathetic skin response, dynamic sweat test, tactile and thermal quantitative sensory testing and brain magnetic resonance imaging were performed according to standard procedures. Histopathological examination was performed on skin and sural nerve biopsies. Molecular analysis of the proband and his relatives was performed with Next Generation Sequencing. The impact of the identified variant on the overall protein structure was evaluated through rotamers method. RESULTS: Since his early adolescence, the patient presented with signs of polyneuropathy with severe distal weakness, atrophy, and reduced sensation. Neurophysiological studies showed a sensory-motor axonal polyneuropathy, with confirmed small fiber involvement. In addition, skin biopsy and sural nerve biopsy showed predominant large fibers involvement. A trio's whole exome sequencing revealed a novel de novo variant p.(Arg1046Cys) in POLR3B, which was classified as Probably Pathogenic. Molecular modeling data confirmed a deleterious effect of the variant on protein structure. INTERPRETATION: Neurophysiological and morphological findings suggest a primary axonal involvement of the largest nerve fibers in POLR3B-related neuropathies. A partial loss of function mechanism is proposed for both neuropathy and leukodystrophy phenotypes.

nc886, an RNA Polymerase III-Transcribed Noncoding RNA Whose Expression Is Dynamic and Regulated by Intriguing Mechanisms.

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.

A novel variant of the POLR3A gene in a Chinese patient with POLR3-related leukodystrophy.

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.

The Pol III transcriptome: Basic features, recurrent patterns, and emerging roles in cancer.

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.

STAT3 promotes RNA polymerase III-directed transcription by controlling the miR-106a-5p/TP73 axis.

Deregulation of Pol III products causes a range of diseases, including neural diseases and cancers. However, the factors and mechanisms that modulate Pol III-directed transcription remain to be found, although massive advances have been achieved. Here, we show that STAT3 positively regulates the activities of Pol III-dependent transcription and cancer cell growth. RNA-seq analysis revealed that STAT3 inhibits the expression of TP73, a member of the p53 family. We found that TP73 is not only required for the regulation of Pol III-directed transcription mediated by STAT3 but also independently suppresses the synthesis of Pol III products. Mechanistically, TP73 can disrupt the assembly of TFIIIB subunits and inhibit their occupancies at Pol III target loci by interacting with TFIIIB subunit TBP. MiR-106a-5p can activate Pol III-directed transcription by targeting the TP73 mRNA 3' UTR to reduce TP 73 expression. We show that STAT3 activates the expression of miR-106a-5p by binding to the miRNA promoter, indicating that the miR-106a-5p links STAT3 with TP73 to regulate Pol III-directed transcription. Collectively, these findings indicate that STAT3 functions as a positive regulator in Pol III-directed transcription by controlling the miR-106a-5p/TP73 axis.

TFIIB-related factor 1 is a nucleolar protein that promotes RNA polymerase I-directed transcription and tumour cell growth.

Eukaryotic RNA polymerase I (Pol I) products play fundamental roles in ribosomal assembly, protein synthesis, metabolism and cell growth. Abnormal expression of both Pol I transcription-related factors and Pol I products causes a range of diseases, including ribosomopathies and cancers. However, the factors and mechanisms governing Pol I-dependent transcription remain to be elucidated. Here, we report that transcription factor IIB-related factor 1 (BRF1), a subunit of transcription factor IIIB required for RNA polymerase III (Pol III)-mediated transcription, is a nucleolar protein and modulates Pol I-mediated transcription. We showed that BRF1 can be localized to the nucleolus in several human cell types. BRF1 expression correlates positively with Pol I product levels and tumour cell growth in vitro and in vivo. Pol III transcription inhibition assays confirmed that BRF1 modulates Pol I-directed transcription in an independent manner rather than through a Pol III product-to-45S pre-rRNA feedback mode. Mechanistically, BRF1 binds to the Pol I transcription machinery components and can be recruited to the rDNA promoter along with them. Additionally, alteration of BRF1 expression affects the recruitment of Pol I transcription machinery components to the rDNA promoter and the expression of TBP and TAF1A. These findings indicate that BRF1 modulates Pol I-directed transcription by controlling the expression of selective factor 1 subunits. In summary, we identified a novel role of BRF1 in Pol I-directed transcription, suggesting that BRF1 can independently regulate both Pol I- and Pol III-mediated transcription and act as a key coordinator of Pol I and Pol III.

IMMUNOEDITING IN AUTOIMMUNE RHEUMATIC DISEASES.

The striking association of specific autoantibodies with distinct disease phenotypes and trajectories in human autoimmune rheumatic diseases provides a powerful opportunity to interrogate disease mechanism. In scleroderma, a subgroup of patients with autoantibodies to POLR3 have coincident onset of cancer and scleroderma. The majority of these patients have genetic changes (somatic mutations and loss of heterozygosity) in the POLR3A gene in their matched cancers, coupled with immune responses directed against the mutated and wild type autoantigen. In some individuals with scleroderma or dermatomyositis where specific immune responses mark a high risk of emergent cancer, cancer does not emerge. Such patients have a broader immune response that targets additional autoantigens, suggesting that the breadth and magnitude of the immune response regulates cancer, and that the rheumatic diseases provide a unique window into natural immunoediting of cancer in humans. This has implications for prediction and therapy in both autoimmunity and cancer.

Biogenesis of telomerase RNA from a protein-coding mRNA precursor.

Telomerase is a eukaryotic ribonucleoprotein (RNP) enzyme that adds DNA repeats onto chromosome ends to maintain genomic stability and confer cellular immortality in cancer and stem cells. The telomerase RNA (TER) component is essential for telomerase catalytic activity and provides the template for telomeric DNA synthesis. The biogenesis of TERs is extremely divergent across eukaryotic kingdoms, employing distinct types of transcription machinery and processing pathways. In ciliates and plants, TERs are transcribed by RNA polymerase III (Pol III), while animal and ascomycete fungal TERs are transcribed by RNA Pol II and share biogenesis pathways with small nucleolar RNA (snoRNA) and small nuclear RNA (snRNA), respectively. Here, we report an unprecedented messenger RNA (mRNA)-derived biogenesis pathway for the 1,291 nucleotide TER from the basidiomycete fungus Ustilago maydis. The U. maydis TER (UmTER) contains a 5'-monophosphate, distinct from the 5' 2,2,7-trimethylguanosine (TMG) cap common to animal and ascomycete fungal TERs. The mature UmTER is processed from the 3'-untranslated region (3'-UTR) of a larger RNA precursor that possesses characteristics of mRNA including a 5' 7-methyl-guanosine (m7G) cap, alternative splicing of introns, and a poly(A) tail. Moreover, this mRNA transcript encodes a protein called Early meiotic induction protein 1 (Emi1) that is conserved across dikaryotic fungi. A recombinant UmTER precursor expressed from an mRNA promoter is processed correctly to yield mature UmTER, confirming an mRNA-processing pathway for producing TER. Our findings expand the plethora of TER biogenesis mechanisms and demonstrate a pathway for producing a functional long noncoding RNA from a protein-coding mRNA precursor.

Transcriptional response to VZV infection is modulated by RNA polymerase III in lung epithelial cell lines.

Ancestral RNA polymerase III (Pol III) is a multi-subunit polymerase responsible for transcription of short non-coding RNA, such as double-stranded short interspersed nuclear elements (SINEs). Although SINE ncRNAs are generally transcriptionally repressed, they can be induced in response to viral infections and can stimulate immune signaling pathways. Indeed, mutations in RNA Pol III have been associated with poor antiviral interferon response following infection with varicella zoster virus (VZV). In this study, we probed the role of Pol III transcripts in the detection and initial immune response to VZV by characterizing the transcriptional response following VZV infection of wild type A549 lung epithelial cells as well as A549 cells lacking specific RNA sensors MAVS and TLR3, or interferon-stimulated genes RNase L and PKR in presence or absence of functional RNA Pol III. Multiple components of the antiviral sensing and interferon signaling pathways were involved in restricting VZV replication in lung epithelial cells thus suggesting an innate defense system with built-in redundancy. In addition, RNA Pol III silencing altered the antiviral transcriptional program indicating that it plays an essential role in the sensing of VZV infection.

Circ-POLR3A accelerates TGF-ß2-induced promotion in cell viability, migration, and invasion of lens epithelial cells via miR-31/TXNIP signaling cascade.

Posterior capsular opacification (PCO) is the major complication after cataract surgery and can result in secondary vision loss. Circular RNAs (circRNAs) are reported to play critical regulatory roles in multiple cell biological processes. The most common working mechanism of circRNAs is by acting as microRNA sponges. Here, we analyzed the role and mechanism of circRNA RNA polymerase III subunit A (POLR3A) in PCO. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell motility was assessed by transwell and wound healing assays. Dual-luciferase reporter and RNA-pull-down assays were performed to verify the interaction between microRNA-31 (miR-31) and circ-POLR3A or thioredoxin interacting protein (TXNIP). PCO cell model was established by treating SRA01/04 cells with transforming growth factor-ß2 (TGF-ß2). We found that TGF-ß2 enhanced SRA01/04 cell viability, migration, and invasion abilities. Circ-POLR3A expression was upregulated in PCO tissues and TGF-ß2-induced SRA01/04 cells. TGF-ß2 promoted the viability and motility of SRA01/04 cells largely by upregulating circ-POLR3A. Circ-POLR3A negatively regulated the miR-31 level by directly interacting with it. Circ-POLR3A absence-induced influences in TGF-ß2-induced SRA01/04 cells were partly reversed by silencing miR-31. miR-31 is directly bound to the 3'-untranslated region of TXNIP. TXNIP overexpression largely attenuated miR-31 overexpression-mediated effects in TGF-ß2-induced SRA01/04 cells. Circ-POLR3A could elevate the protein expression of TXNIP by sponging miR-31. Exosomes were involved in mediating the delivery of circ-POLR3A in SRA01/04 cells. In conclusion, circ-POLR3A contributed to TGF-ß2-induced promotion of cell viability, migration, and invasion of SRA01/04 cells by targeting miR-31/TXNIP axis.

A cancer-associated RNA polymerase III identity drives robust transcription and expression of snaR-A noncoding RNA.

RNA polymerase III (Pol III) includes two alternate isoforms, defined by mutually exclusive incorporation of subunit POLR3G (RPC7α) or POLR3GL (RPC7ß), in mammals. The contributions of POLR3G and POLR3GL to transcription potential has remained poorly defined. Here, we discover that loss of subunit POLR3G is accompanied by a restricted repertoire of genes transcribed by Pol III. Particularly sensitive is snaR-A, a small noncoding RNA implicated in cancer proliferation and metastasis. Analysis of Pol III isoform biases and downstream chromatin features identifies loss of POLR3G and snaR-A during differentiation, and conversely, re-establishment of POLR3G gene expression and SNAR-A gene features in cancer contexts. Our results support a model in which Pol III identity functions as an important transcriptional regulatory mechanism. Upregulation of POLR3G, which is driven by MYC, identifies a subgroup of patients with unfavorable survival outcomes in specific cancers, further implicating the POLR3G-enhanced transcription repertoire as a potential disease factor.

MAP kinases are involved in RNA polymerase III regulation upon LPS treatment in macrophages.

Macrophages are transcriptionally highly dynamic cell type, rapidly adapting to a changing environment to execute innate immune functions. Activation of macrophages with lipopolysaccharides (LPS), a major component of the outer membrane of most Gram-negative bacteria, induces rapid transcriptional changes and within a few hours transcription of several hundred genes is altered. Within these genes are tRNAs, which are synthesised by RNA Polymerase (Pol) III, and whose expression is rapidly upregulated in response to LPS. However, the mechanisms that govern Pol III activation are not fully elucidated. LPS engage the Toll-like receptor (TLR) 4 and induce various signalling pathways, including mitogen-activated protein kinases (MAPK). MAPKs are serine/threonine kinases that catalyse the phosphorylation of transcription factors, protein kinases, and many other substrates including functional proteins, play a central role in mediating cellular responses to extracellular signals, including inflammatory cues. Here we show that ERK and p38 MAP kinases contribute to the activation of Pol III in macrophages stimulated with LPS. We also demonstrate that MAP kinases effector MSK1/2 kinases are involved in tRNA upregulation. Our data show that ERK, p38, and MSK kinases are required for upregulation of Pol III activity in macrophages stimulated by LPS. The possible modes of their action are discussed.

Early Growth Response 1 Strengthens Pol-III-Directed Transcription and Transformed Cell Proliferation by Controlling PTEN/AKT Signalling Activity.

RNA polymerase III (Pol III) products play essential roles in ribosome assembly, protein synthesis, and cell survival. Deregulation of Pol-III-directed transcription is closely associated with tumorigenesis. However, the regulatory pathways or factors controlling Pol-III-directed transcription remain to be investigated. In this study, we identified a novel role of EGR1 in Pol-III-directed transcription. We found that Filamin A (FLNA) silencing stimulated EGR1 expression at both RNA and protein levels. EGR1 expression positively correlated with Pol III product levels and cell proliferation activity. Mechanistically, EGR1 downregulation dampened the occupancies of Pol III transcription machinery factors at the loci of Pol III target genes. Alteration of EGR1 expression did not affect the expression of p53, c-MYC, and Pol III general transcription factors. Instead, EGR1 activated RhoA expression and inhibited PTEN expression in several transformed cell lines. We found that PTEN silencing, rather than RhoA overexpression, could reverse the inhibition of Pol-III-dependent transcription and cell proliferation caused by EGR1 downregulation. EGR1 could positively regulate AKT phosphorylation levels and is required for the inhibition of Pol-III-directed transcription mediated by FLNA. The findings from this study indicate that EGR1 can promote Pol-III-directed transcription and cell proliferation by controlling the PTEN/AKT signalling pathway.

Manipulation of RNA polymerase III by Herpes Simplex Virus-1.

RNA polymerase III (Pol III) transcribes noncoding RNA, including transfer RNA (tRNA), and is commonly targeted during cancer and viral infection. We find that Herpes Simplex Virus-1 (HSV-1) stimulates tRNA expression 10-fold. Perturbation of host tRNA synthesis requires nuclear viral entry, but not synthesis of specific viral transcripts. tRNA with a specific codon bias were not targeted-rather increased transcription was observed from euchromatic, actively transcribed loci. tRNA upregulation is linked to unique crosstalk between the Pol II and III transcriptional machinery. While viral infection results in depletion of Pol II on host mRNA promoters, we find that Pol II binding to tRNA loci increases. Finally, we report Pol III and associated factors bind the viral genome, which suggests a previously unrecognized role in HSV-1 gene expression. These findings provide insight into mechanisms by which HSV-1 alters the host nuclear environment, shifting key processes in favor of the pathogen.

POLR3A variants in hereditary spastic paraparesis and ataxia: clinical, genetic, and neuroradiological findings in a cohort of Italian patients.

Mutations in POLR3A are characterized by high phenotypic heterogeneity, with manifestations ranging from severe childhood-onset hypomyelinating leukodystrophic syndromes to milder and later-onset gait disorders with central hypomyelination, with or without additional non-neurological signs. Recently, a milder phenotype consisting of late-onset spastic ataxia without hypomyelinating leukodystrophy has been suggested to be specific to the intronic c.1909 + 22G > A mutation in POLR3A. Here, we present 10 patients from 8 unrelated families with POLR3A-related late-onset spastic ataxia, all harboring the c.1909 + 22G > A variant. Most of them showed an ataxic-spastic picture, two a "pure" cerebellar phenotype, and one a "pure" spastic presentation. The non-neurological findings typically associated with POLR3A mutations were absent in all the patients. The main findings on brain MRI were bilateral hyperintensity along the superior cerebellar peduncles on FLAIR sequences, observed in most of the patients, and cerebellar and/or spinal cord atrophy, found in half of the patients. Only one patient exhibited central hypomyelination. The POLR3A mutations present in this cohort were the c.1909 + 22G > A splice site variant found in compound heterozygosity with six additional variants (three missense, two nonsense, one splice) and, in one patient, with a novel large deletion involving exons 14-18. Interestingly, this patient had the most "complex" presentation among those observed in our cohort; it included some neurological and non-neurological features, such as seizures, neurosensory deafness, and lipomas, that have not previously been reported in association with late-onset POLR3A-related disorders, and therefore further expand the phenotype.

Pol3Base: a resource for decoding the interactome, expression, evolution, epitranscriptome and disease variations of Pol III-transcribed ncRNAs.

RNA polymerase III (Pol III) transcribes hundreds of non-coding RNA genes (ncRNAs), which involve in a variety of cellular processes. However, the expression, functions, regulatory networks and evolution of these Pol III-transcribed ncRNAs are still largely unknown. In this study, we developed a novel resource, Pol3Base (http://rna.sysu.edu.cn/pol3base/), to decode the interactome, expression, evolution, epitranscriptome and disease variations of Pol III-transcribed ncRNAs. The current release of Pol3Base includes thousands of regulatory relationships between ∼79 000 ncRNAs and transcription factors by mining 56 ChIP-seq datasets. By integrating CLIP-seq datasets, we deciphered the interactions of these ncRNAs with >240 RNA binding proteins. Moreover, Pol3Base contains ∼9700 RNA modifications located within thousands of Pol III-transcribed ncRNAs. Importantly, we characterized expression profiles of ncRNAs in >70 tissues and 28 different tumor types. In addition, by comparing these ncRNAs from human and mouse, we revealed about 4000 evolutionary conserved ncRNAs. We also identified ∼11 403 tRNA-derived small RNAs (tsRNAs) in 32 different tumor types. Finally, by analyzing somatic mutation data, we investigated the mutation map of these ncRNAs to help uncover their potential roles in diverse diseases. This resource will help expand our understanding of potential functions and regulatory networks of Pol III-transcribed ncRNAs.

tRNA biogenesis and specific aminoacyl-tRNA synthetases regulate senescence stability under the control of mTOR.

Oncogenes or chemotherapy treatments trigger the induction of suppressive pathways such as apoptosis or senescence. Senescence was initially defined as a definitive arrest of cell proliferation but recent results have shown that this mechanism is also associated with cancer progression and chemotherapy resistance. Senescence is therefore much more heterogeneous than initially thought. How this response varies is not really understood, it has been proposed that its outcome relies on the secretome of senescent cells and on the maintenance of their epigenetic marks. Using experimental models of senescence escape, we now described that the stability of this proliferative arrest relies on specific tRNAs and aminoacyl-tRNA synthetases. Following chemotherapy treatment, the DNA binding of the type III RNA polymerase was reduced to prevent tRNA transcription and induce a complete cell cycle arrest. By contrast, during senescence escape, specific tRNAs such as tRNA-Leu-CAA and tRNA-Tyr-GTA were up-regulated. Reducing tRNA transcription appears necessary to control the strength of senescence since RNA pol III inhibition through BRF1 depletion maintained senescence and blocked the generation of escaping cells. mTOR inhibition also prevented chemotherapy-induced senescence escape in association with a reduction of tRNA-Leu-CAA and tRNA-Tyr-GTA expression. Further confirming the role of the tRNA-Leu-CAA and tRNA-Tyr-GTA, results showed that their corresponding tRNA ligases, LARS and YARS, were necessary for senescence escape. This effect was specific since the CARS ligase had no effect on persistence. By contrast, the down-regulation of LARS and YARS reduced the emergence of persistent cells and this was associated with the modulation of E2F1 target genes expression. Overall, these findings highlight a new regulation of tRNA biology during senescence and suggest that specific tRNAs and ligases contribute to the strength and heterogeneity of this tumor suppressive pathway.