Gene-Specific Control of tRNA Expression by RNA Polymerase II.

Increasing evidence suggests that tRNA levels are dynamically and specifically regulated in response to internal and external cues to modulate the cellular translational program. However, the molecular players and the mechanisms regulating the gene-specific expression of tRNAs are still unknown. Using an inducible auxin-degron system to rapidly deplete RPB1 (the largest subunit of RNA Pol II) in living cells, we identified Pol II as a direct gene-specific regulator of tRNA transcription. Our data suggest that Pol II transcription robustly interferes with Pol III function at specific tRNA genes. This activity was further found to be essential for MAF1-mediated repression of a large set of tRNA genes during serum starvation, indicating that repression of tRNA genes by Pol II is dynamically regulated. Hence, Pol II plays a direct and central role in the gene-specific regulation of tRNA expression.

Phase-separation antagonists potently inhibit transcription and broadly increase nucleosome density.

Biomolecular condensates are self-organized membraneless bodies involved in many critical cellular activities, including ribosome biogenesis, protein synthesis, and gene transcription. Aliphatic alcohols are commonly used to study biomolecular condensates, but their effects on transcription are unclear. Here, we explore the impact of the aliphatic dialcohol, 1,6-hexanediol (1,6-HD), on Pol II transcription and nucleosome occupancy in budding yeast. As expected, 1,6-HD, a reagent effective in disrupting biomolecular condensates, strongly suppressed the thermal stress-induced transcription of Heat Shock Factor 1-regulated genes that have previously been shown to physically interact and coalesce into intranuclear condensates. Surprisingly, the isomeric dialcohol, 2,5-HD, typically used as a negative control, abrogated Heat Shock Factor 1-target gene transcription under the same conditions. Each reagent also abolished the transcription of genes that do not detectably coalesce, including Msn2/Msn4-regulated heat-inducible genes and constitutively expressed housekeeping genes. Thus, at elevated temperature (39 °C), HDs potently inhibit the transcription of disparate genes and as demonstrated by chromatin immunoprecipitation do so by abolishing occupancy of RNA polymerase in chromatin. Concurrently, histone H3 density increased at least twofold within all gene coding and regulatory regions examined, including quiescent euchromatic loci, silent heterochromatic loci, and Pol III-transcribed loci. Our results offer a caveat for the use of HDs in studying the role of condensates in transcriptional control and provide evidence that exposure to these reagents elicits a widespread increase in nucleosome density and a concomitant loss of both Pol II and Pol III transcription.

Chl1, an ATP-Dependent DNA Helicase, Inhibits DNA:RNA Hybrids Formation at DSB Sites to Maintain Genome Stability in S. pombe.

As an ATP-dependent DNA helicase, human ChlR1/DDX11 (Chl1 in yeast) can unwind both DNA:RNA and DNA:DNA substrates in vitro. Studies have demonstrated that ChlR1 plays a vital role in preserving genome stability by participating in DNA repair and sister chromatid cohesion, whereas the ways in which the biochemical features of ChlR1 function in DNA metabolism are not well understood. Here, we illustrate that Chl1 localizes to double-strand DNA break (DSB) sites and restrains DNA:RNA hybrid accumulation at these loci. Mutation of Chl1 strongly impairs DSB repair capacity by homologous recombination (HR) and nonhomologous end-joining (NHEJ) pathways, and deleting RNase H further reduces DNA repair efficiency, which indicates that the enzymatic activities of Chl1 are needed in Schizosaccharomyces pombe. In addition, we found that the Rpc37 subunit of RNA polymerase III (RNA Pol III) interacts directly with Chl1 and that deletion of Chl1 has no influence on the localization of Rpc37 at DSB site, implying the role of Rpc37 in the recruitment of Chl1 to this site.

Clinical consequences of the presence of anti-RNA Pol III antibodies in systemic sclerosis.

INTRODUCTION: Anti-RNA polymerase III (a-RNA Pol III) antibodies are marker antibodies in patients with systemic sclerosis (SSc). AIM: To assess the prevalence of a-RNA Pol III in patients with SSc and to identify the differences in the disease picture in SSc patients with and without a-RNA Pol III antibodies. MATERIAL AND METHODS: The study was performed in 126 SSc patients. The subtype of SSc, incidence of internal organ involvement, malignancy, death and serological profiles were determined in the entire group. The study groups were studied according to the presence of antibodies by applying the commercial test - EUROLINE SSc Profile. Due to the presence of a-RNA Pol III, patients were divided into two groups: the a-RNA Pol III (+) SSc group of 19 patients and the a-RNA Pol III (-) SSc group of 107 patients. RESULTS: A-RNA Pol III were present in 19/126 patients with SSc (15%), 13/19 (68.4%) patients had no other SSc marker antibodies. A-RNA Pol III were more common in patients with diffuse cutaneous SSc (p = 0.049). We showed a significant positive association between a-RNA Pol III and occurrence of malignancy (p = 0.007), scleroderma renal crisis (p = 0.001) and decreased DLCO (p = 0.007). CONCLUSIONS: Anti-a-RNA Pol III antibodies are common in patients with SSc, particularly with a diffuse subtype. In more than 50% of patients with a-RNA Pol III antibodies, they may be present as the sole marker of antibodies. In SSc, a-RNA Pol III antibodies are frequently associated with malignancy occurrence, kidney and lung involvement.

miRNA deregulation targets specific pathways in leiomyosarcoma development: an in silico analysis.

BACKGROUND: MicroRNA (miRNA) mediate post-transcriptional gene repression and are involved in a variety of human diseases, including cancer. Soft tissue sarcomas are rare malignancies with a variety of histological subtypes which may occur virtually anywhere in the human body. Leiomyosarcoma is one of the most common subtypes, shows a smooth muscle phenotype and its cancerogenesis is still unclear. The aim of our study was to investigate the potential role of miRNA differential expression in leiomyosarcoma development. METHODS: We first employed the Sarcoma microRNA Expression Database, a repository that describes the patterns of over 1000 miRNA expression in various human sarcoma types, to identify differentially expressed miRNA comparing leiomyosarcoma and smooth muscle samples. Subsequently, we identified putative target genes of those miRNAs with the TargetScan prediction tool. Finally, we evaluated whether the retrieved pool of putative targets was enriched in genes belonging to specific molecular pathways by means of the Enrichr analysis tool. Protein-protein network analysis was analyzed by means of the STRING web tool. RESULTS: Out of 1120 miRNAs tested, the expression of 301 miRNAs was statistically significantly different between leiomyosarcoma and smooth muscle samples. The hypothetical targets could be predicted for 172 miRNAs. 438 genes were predicted to be the targets with high confidence (cumulative weighted context score cut-off level less than - 1.0) and analyzed for belonging to specific molecular pathways. Pathway analysis suggested that RNA Polymerase III, tRNA functions and synaptic neurotransmission (with special regard to dopamine mediated signaling) could be involved in leiomyosarcoma development. CONCLUSIONS: Our results demonstrate that data mining of publicly available repositories can be useful to suggest molecular pathways underlying the pathogenesis of rare tumors such as leiomyosarcoma.

Transcriptional regulation of microRNA-100, -146a, and -150 genes by p53 and NFκB p65/RelA in mouse striatal STHdh(Q7)/ Hdh(Q7) cells and human cervical carcinoma HeLa cells.

MicroRNA (miRNA) genes generally share many features common to those of protein coding genes. Various transcription factors (TFs) and co-regulators are also known to regulate miRNA genes. Here we identify novel p53 and NFκB p65/RelA responsive miRNAs and demonstrate that these 2 TFs bind to the regulatory sequences of miR-100, -146a and -150 in both mouse striatal and human cervical carcinoma cells and regulate their expression. p53 represses the miRNAs while NFκB p65/RelA induces them. Further, we provide evidence that exogenous p53 inhibits NFκB p65/RelA activity by reducing its nuclear content and competing with it for CBP binding. This suggests for the existence of a functional cross-talk between the 2 TFs in regulating miRNA expression. Moreover, promoter occupancy assay reveals that exogenous p53 excludes NFκB p65/RelA from its binding site in the upstream sequence of miR-100 gene thereby causing its repression. Thus, our work identifies novel p53 and NFκB p65/RelA responsive miRNAs in human and mouse and uncovers possible mechanisms of co-regulation of miR-100. It is to be mentioned here that cross-talks between p53 and NFκB p65/RelA have been observed to define the outcome of several biological processes and that the pro-apoptotic effect of p53 and the pro-survival functions of NFκB can be largely mediated via the biological roles of the miRNAs these TFs regulate. Our observation with cell lines thus provides an important platform upon which further work is to be done to establish the biological significance of such co-regulation of miRNAs by p53 and NFκB p65/RelA.

Subcellular localization shapes the fate of RNA polymerase III.

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.

Development of a gRNA Expression and Processing Platform for Efficient CRISPR-Cas9-Based Gene Editing and Gene Silencing in Candida tropicalis.

Candida tropicalis, a nonmodel diploid microbe, has been applied in industry as a chassis cell. Metabolic engineering of C. tropicalis is challenging due to a lack of gene editing and regulation tools. Here, we report a tRNA:guide RNA (gRNA) platform for boosting gene editing and silencing efficiency in C. tropicalis. As the endogenous tRNA-processing system enables autocleavage for producing a large number of mature gRNAs, a tRNAGly sequence from the genome of C. tropicalis ATCC 20336 was selected for constructing the tRNA:gRNA platform. In the CRISPR-Cas9 system, the tRNA:gRNA platform proved to be efficient in single-gene and multi-gene editing. Furthermore, based on the tRNA:gRNA platform, a CRISPR interference (CRISPRi) system was developed to construct an efficient dCas9-mediated gene expression regulation system for C. tropicalis. The CRISPRi system was employed to regulate the expression of the exogenous gene GFP3 (green fluorescent protein) and the endogenous gene ADE2 (phosphoribosylaminoimidazole carboxylase). Different regions of GFP3 and ADE2 were targeted with the gRNAs processed by the tRNAGly, and the transcription levels of GFP3 and ADE2 were successfully downregulated to 23.9% ± 4.1% and 38.0% ± 7.4%, respectively. The effects of the target regions on gene regulation were also investigated. Additionally, the regulation system was applied to silence ERG9 (squalene synthase) to enhance ß-carotene biosynthesis in a metabolically modified C. tropicalis strain. The results suggest that the endogenous tRNAGly and the CRISPRi system have great potential for metabolic engineering of C. tropicalis. IMPORTANCE In the nonmodel yeast Candida tropicalis, a lack of available RNA polymerase type III (Pol III) promoters hindered the development of guide RNA (gRNA) expression platforms for the establishment of CRISPR-Cas-mediated genome editing and silencing strategies. Here, a tRNA:gRNA platform was constructed. We show that this platform allows efficient and precise expression and processing of different gRNAs from a single polycistronic gene capable of mediating multi-gene editing in combination with CRISPR-Cas9. Furthermore, in combination with dCas9, the tRNA:gRNA platform was efficiently used for silencing of exogenous and endogenous genes, representing the first CRISPR interference tool (CRISPRi) in C. tropicalis. Importantly, the established CRISPRi-tRNA:gRNA tool was also used for metabolic engineering by regulating ß-carotene biosynthesis in C. tropicalis. The results suggest that the tRNA:gRNA platform and the CRISPRi system will further advance the application of the CRISPR-Cas-based editing and CRISPRi systems for metabolic engineering in C. tropicalis.

RNA Pol III promoters-key players in precisely targeted plant genome editing.

The clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein Cas) system is a powerful and highly precise gene-editing tool in basic and applied research for crop improvement programs. CRISPR/Cas tool is being extensively used in plants to improve crop yield, quality, and nutritional value and make them tolerant to environmental stresses. CRISPR/Cas system consists of a Cas protein with DNA endonuclease activity and one CRISPR RNA transcript that is processed to form one or several short guide RNAs that direct Cas9 to the target DNA sequence. The expression levels of Cas proteins and gRNAs significantly influence the editing efficiency of CRISPR/Cas-mediated genome editing. This review focuses on insights into RNA Pol III promoters and their types that govern the expression levels of sgRNA in the CRISPR/Cas system. We discussed Pol III promoters structural and functional characteristics and their comparison with Pol II promoters. Further, the use of synthetic promoters to increase the targeting efficiency and overcome the structural, functional, and expressional limitations of RNA Pol III promoters has been discussed. Our review reports various studies that illustrate the use of endogenous U6/U3 promoters for improving editing efficiency in plants and the applicative approach of species-specific RNA pol III promoters for genome editing in model crops like Arabidopsis and tobacco, cereals, legumes, oilseed, and horticultural crops. We further highlight the significance of optimizing these species-specific promoters' systematic identification and validation for crop improvement and biotic and abiotic stress tolerance through CRISPR/Cas mediated genome editing.

Eukaryotic RNA Polymerases: The Many Ways to Transcribe a Gene.

In eukaryotic cells, three nuclear RNA polymerases (RNA pols) carry out the transcription from DNA to RNA, and they all seem to have evolved from a single enzyme present in the common ancestor with archaea. The multiplicity of eukaryotic RNA pols allows each one to remain specialized in the synthesis of a subset of transcripts, which are different in the function, length, cell abundance, diversity, and promoter organization of the corresponding genes. We hypothesize that this specialization of RNA pols has conditioned the evolution of the regulatory mechanisms used to transcribe each gene subset to cope with environmental changes. We herein present the example of the homeostatic regulation of transcript levels versus changes in cell volume. We propose that the diversity and instability of messenger RNAs, transcribed by RNA polymerase II, have conditioned the appearance of regulatory mechanisms based on different gene promoter strength and mRNA stability. However, for the regulation of ribosomal RNA levels, which are very stable and transcribed mainly by RNA polymerase I from only one promoter, different mechanisms act based on gene copy variation, and a much simpler regulation of the synthesis rate.

Host-Informed Expression of CRISPR Guide RNA for Genomic Engineering in Komagataella phaffii.

There is growing interest in the use of nonmodel microorganisms as hosts for biopharmaceutical manufacturing. These hosts require genomic engineering to meet clinically relevant product qualities and titers, but the adaptation of tools for editing genomes, such as CRISPR-Cas9, has been slow for poorly characterized hosts. Specifically, a lack of biochemical characterization of RNA polymerase III transcription has hindered reliable expression of guide RNAs in new hosts. Here, we present a sequencing-based strategy for the design of host-specific cassettes for modular, reliable, expression of guide RNAs. Using this strategy, we achieved up to 95% gene editing efficiency in the methylotrophic yeast Komagataella phaffii. We applied this approach for the rapid, multiplexed engineering of a complex phenotype, achieving humanized product glycosylation in two sequential steps of engineering. Reliable extension of simple gene editing tools to nonmodel manufacturing hosts will enable rapid engineering of manufacturing strains tuned for specific product profiles and potentially decrease the costs and timelines for process development.

Growth signals employ CGGBP1 to suppress transcription of Alu-SINEs.

CGGBP1 (CGG triplet repeat-binding protein 1) regulates cell proliferation, stress response, cytokinesis, telomeric integrity and transcription. It could affect these processes by modulating target gene expression under different conditions. Identification of CGGBP1-target genes and their regulation could reveal how a transcription regulator affects such diverse cellular processes. Here we describe the mechanisms of differential gene expression regulation by CGGBP1 in quiescent or growing cells. By studying global gene expression patterns and genome-wide DNA-binding patterns of CGGBP1, we show that a possible mechanism through which it affects the expression of RNA Pol II-transcribed genes in trans depends on Alu RNA. We also show that it regulates Alu transcription in cis by binding to Alu promoter. Our results also indicate that potential phosphorylation of CGGBP1 upon growth stimulation facilitates its nuclear retention, Alu-binding and dislodging of RNA Pol III therefrom. These findings provide insights into how Alu transcription is regulated in response to growth signals.