Pol III-Dependent Cabbage BoNR8 Long ncRNA Affects Seed Germination and Growth in Arabidopsis.

Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts longer than 200 nt that are distributed widely in organisms and play many physiological roles. The BoNR8 lncRNA is a 272 nt long transcript yielded by RNA polymerase III in cabbage that was identified as the closest homolog of the AtR8 lncRNA in Arabidopsis. The BoNR8 lncRNA was expressed extensively in the epidermal tissue in the root elongation zone of germinated seeds, and its accumulation was induced by abiotic stresses, auxins and ABA. To investigate the correlation between the BoNR8 lncRNA and germination, BoNR8-overexpressing Arabidopsis plants (BoNR8-AtOX) were prepared. Three independent BoNR8-AtOX lines showed less primary root elongation, incomplete silique development and decreased germination rates. The germination efficiencies were affected strongly by ABA and slightly by salt stress, and ABA-related gene expression was changed in the BoNR8-AtOX lines.

Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts.

RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5' peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential "housekeeping" roles. Many tRNA genes were found to generate long, 3'-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3'-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5'-exonuclease Rat1.

Alteration of BRCA1 expression affects alcohol-induced transcription of RNA Pol III-dependent genes.

Emerging evidence has indicated that alcohol consumption is an established risk factor for breast cancer. Deregulation of RNA polymerase III (Pol III) transcription enhances cellular Pol III gene production, leading to an increase in translational capacity to promote cell transformation and tumor formation. We have reported that alcohol intake increases Pol III gene transcription to promote cell transformation and tumor formation in vitro and in vivo. Studies revealed that tumor suppressors, pRb, p53, PTEN and Maf1 repress the transcription of Pol III genes. BRCA1 is a tumor suppressor and its mutation is tightly related to breast cancer development. However, it is not clear whether BRCA1 expression affects alcohol-induced transcription of Pol III genes. At the present studies, we report that restoring BRCA1 in HCC 1937 cells, which is a BRCA1 deficient cell line, represses Pol III gene transcription. Expressing mutant or truncated BRCA1 in these cells does not affect the ability of repression on Pol III genes. Our analysis has demonstrated that alcohol induces Pol III gene transcription. More importantly, overexpression of BRCA1 in estrogen receptor positive (ER+) breast cancer cells (MCF-7) decreases the induction of tRNA(Leu) and 5S rRNA genes by alcohol, whereas reduction of BRCA1 by its siRNA slightly increases the transcription of the class of genes. This suggests that BRCA1 is associated with alcohol-induced deregulation of Pol III genes. These studies for the first time demonstrate the role of BRCA1 in induction of Pol III genes by alcohol and uncover a novel mechanism of alcohol-associated breast cancer.

Meeting report for "OddPols" 2014: the odds invite an even.

The Ninth International Biennial Conference on RNA Polymerases I and III (the "OddPols") was held on June 19-21, 2014 at the University of Michigan, Ann Arbor, USA. Sponsored by New England Biolabs, the Cayman Chemical Company, the Rackham Graduate School and the University of Michigan Health System, and organized by David Engelke, Craig Pikaard, Lawrence Rothblum, Andrzej Wierzbicki and Astrid Engel. This year at the conference, the "odds" were increased by expanding the usual topics on the advances in RNA polymerases I and III research to include presentations on RNA polymerase IV and V. The keynote speaker, Craig Pikaard, opened the meeting with his presentation entitled "Five nuclear multisubunit RNA polymerases". The meeting drew attendees from fourteen countries that shared their research discoveries through oral and poster presentations. The talks were organized into 11 sessions covering seven distinct topics. Here we present some of the highlights from the meeting using summaries provided by the participants.

Gcn5 and PCAF regulate PPARγ and Prdm16 expression to facilitate brown adipogenesis.

The acetyltransferase Gcn5 is critical for embryogenesis and shows partial functional redundancy with its homolog PCAF. However, the tissue- and cell lineage-specific functions of Gcn5 and PCAF are still not well defined. Here we probe the functions of Gcn5 and PCAF in adipogenesis. We found that the double knockout (DKO) of Gcn5/PCAF inhibits expression of the master adipogenic transcription factor gene PPARγ, thereby preventing adipocyte differentiation. The adipogenesis defects in Gcn5/PCAF DKO cells are rescued by ectopic expression of peroxisome proliferator-activated receptor γ (PPARγ), suggesting Gcn5/PCAF act upstream of PPARγ to facilitate adipogenesis. The requirement of Gcn5/PCAF for PPARγ expression was unexpectedly bypassed by prolonged treatment with an adipogenic inducer, 3-isobutyl-1-methylxanthine (IBMX). However, neither PPARγ ectopic expression nor prolonged IBMX treatment rescued defects in Prdm16 expression in DKO cells, indicating that Gcn5/PCAF are essential for normal Prdm16 expression. Gcn5/PCAF regulate PPARγ and Prdm16 expression at different steps in the transcription process, facilitating RNA polymerase II recruitment to Prdm16 and elongation of PPARγ transcripts. Overall, our study reveals that Gcn5/PCAF facilitate adipogenesis through regulation of PPARγ expression and regulate brown adipogenesis by influencing Prdm16 expression.

MYC regulation of cell growth through control of transcription by RNA polymerases I and III.

MYC's tumorigenic potential involves increased ribosome biogenesis and translational capacity, which supply the cell with protein required for enhanced cell growth and subsequent cell division. In addition to activation of protein-encoding genes transcribed by RNA polymerase II, MYC must stimulate transcription by RNA polymerase I and RNA polymerase III to meet this synthetic demand. In the past decade our knowledge of the mechanisms and importance of MYC regulation of RNA polymerases I and III has flourished. Here we discuss MYC's influence on transcription by these "odd" RNA polymerases and the physiological impact of this regulation is evaluated with relevance to cancer development and treatment.

Comparative overview of RNA polymerase II and III transcription cycles, with focus on RNA polymerase III termination and reinitiation.

In eukaryotes, RNA polymerase (RNAP) III transcribes hundreds of genes for tRNAs and 5S rRNA, among others, which share similar promoters and stable transcription initiation complexes (TIC), which support rapid RNAP III recycling. In contrast, RNAP II transcribes a large number of genes with highly variable promoters and interacting factors, which exert fine regulatory control over TIC lability and modifications of RNAP II at different transitional points in the transcription cycle. We review data that illustrate a relatively smooth continuity of RNAP III initiation-elongation-termination and reinitiation toward its function to produce high levels of tRNAs and other RNAs that support growth and development.

Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription.

Human RNA polymerase III transcribes small untranslated RNAs that contribute to the regulation of essential cellular processes, including transcription, RNA processing and translation. Analysis of this transcription system by in vitro transcription techniques has largely contributed to the discovery of its transcription factors and to the understanding of the regulation of human RNA polymerase III transcription. Here we review some of the key steps that led to the identification of transcription factors and to the definition of minimal promoter sequences for human RNA polymerase III transcription.

Investigating transcription reinitiation through in vitro approaches.

By influencing the number of RNA molecules repeatedly synthesized from the same gene, the control of transcription reinitiation has the potential to shape the transcriptome. Transcription reinitiation mechanisms have been mainly addressed in vitro, through approaches based on both crude and reconstituted systems. These studies support the notion that transcription reinitiation and its regulation rely on dedicated networks of molecular interactions within transcription machineries. At the same time, comparison with in vivo transcription rates suggests that additional mechanisms, factors and conditions must exist in the nucleus, whose biochemical elucidation is a fascinating challenge for future in vitro transcription studies.

[Recent progress in adenovirus vectors: focusing on VA-deleted AdV].

First-generation adenovirus vectors (FG-AdVs) are widely used because transduction efficiency of the vectors is very high. However, severe immune responses especially to the liver have been a serious problem of this vector. We succeeded to identify a viral protein that cause the immune responses and reported ''low-inflammatory AdVs'' that mostly solve this problem. However, to develop the ultimate form of this vector, it is necessary to remove virus-associated RNA (VA RNA) genes from the AdV vector genome. VA RNAs are transcribed by polymerase III; they are not essential for viral growth but have important roles to make appropriate circumstances for this virus. Large amount of VA RNAs are required in the late phase to support viral growth. Hence it is difficult to establish 293 cell lines that can support replication of AdVs lacking VA RNA genes (VA-deleted AdVs) supplying sufficient amount of VA RNA in trans. Recently we have developed a method for efficient production of VA-deleted AdVs and succeeded to obtain a high titer of VA-deleted AdVs. Then we construct VA-deleted AdVs expressing shRNA that knockdown the replication of hepatitis C virus (HCV). In fact, VA-deleted AdVs expressing these shRNAs suppressed HCV replication more effectively than conventional FG-AdV. Therefore, we showed that VA RNAs expressed from FG-AdVs probably compete with shRNA in the maturation pathway and reduce the effect of shRNAs. We think that VA-deleted AdV may substitute for current FG-AdVs and become a standard AdV.

Maf1-mediated repression of RNA polymerase III transcription inhibits tRNA degradation via RTD pathway.

tRNA precursors, which are transcribed by RNA polymerase III, undergo end-maturation, splicing, and base modifications. Hypomodified tRNAs, such as tRNA(Val(AAC)), lacking 7-methylguanosine and 5-methylcytidine modifications, are subject to degradation by a rapid tRNA decay pathway. Here we searched for genes which, when overexpressed, restored stability of tRNA(Val(AAC)) molecules in a modification-deficient trm4Δtrm8Δ mutant. We identified TEF1 and VAS1, encoding elongation factor eEF1A and valyl-tRNA synthetase respectively, which likely protect hypomodified tRNA(Val(AAC)) by direct interactions. We also identified MAF1 whose product is a general negative regulator of RNA polymerase III. Expression of a Maf1-7A mutant that constitutively repressed RNA polymerase III transcription resulted in increased stability of hypomodified tRNA(Val(AAC)). Strikingly, inhibition of tRNA transcription in a Maf1-independent manner, either by point mutation in RNA polymerase III subunit Rpc128 or decreased expression of Rpc17 subunit, also suppressed the turnover of the hypomodified tRNA(Val(AAC)). These results support a model where inhibition of tRNA transcription leads to stabilization of hypomodified tRNA(Val(AAC)) due to more efficient protection by tRNA-interacting proteins.

Conservation between the RNA polymerase I, II, and III transcription initiation machineries.

Recent studies of the three eukaryotic transcription machineries revealed that all initiation complexes share a conserved core. This core consists of the RNA polymerase (I, II, or III), the TATA box-binding protein (TBP), and transcription factors TFIIB, TFIIE, and TFIIF (for Pol II) or proteins structurally and functionally related to parts of these factors (for Pol I and Pol III). The conserved core initiation complex stabilizes the open DNA promoter complex and directs initial RNA synthesis. The periphery of the core initiation complex is decorated by additional polymerase-specific factors that account for functional differences in promoter recognition and opening, and gene class-specific regulation. This review outlines the similarities and differences between these important molecular machines.

Direct regulation of tRNA and 5S rRNA gene transcription by Polo-like kinase 1.

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity.

Transcription by RNA polymerase III: more complex than we thought.

RNA polymerase (Pol) III is highly specialized for the production of short non-coding RNAs. Once considered to be under relatively simple controls, recent studies using chromatin immunoprecipitation followed by sequencing (ChIP-seq) have revealed unexpected levels of complexity for Pol III regulation, including substantial cell-type selectivity and intriguing overlap with Pol II transcription. Here I describe these novel insights and consider their implications and the questions that remain.

17A, a novel non-coding RNA, regulates GABA B alternative splicing and signaling in response to inflammatory stimuli and in Alzheimer disease.

Alternative splicing is a central component of human brain complexity; nonetheless, its regulatory mechanisms are still largely unclear. In this work, we describe a novel non-coding (nc) RNA (named 17A) RNA polymerase (pol) III-dependent embedded in the human G-protein-coupled receptor 51 gene (GPR51, GABA B2 receptor). The stable expression of 17A in SHSY5Y neuroblastoma cells induces the synthesis of an alternative splicing isoform that abolish GABA B2 intracellular signaling (i.e., inhibition of cAMP accumulation and activation of K(+) channels). Indeed, 17A is expressed in human brain, and we report that it is upregulated in cerebral tissues derived from Alzheimer disease patients. We demonstrate that 17A expression in neuroblastoma cells enhances the secretion of amyloid ß peptide (Aß) and the Aß x-42/Αß x-40 peptide ratio and that its synthesis is induced in response to inflammatory stimuli. These data correlate, for the first time, the activity of a novel pol III-dependent ncRNA to alternative splicing events and, possibly, to neurodegeneration induced by abnormal GABA B function. We anticipate that further analysis of pol III-dependent regulation of alternative splicing will disclose novel regulatory pathways associated to brain physiology and/or pathology.

A mammalian herpesvirus uses noncanonical expression and processing mechanisms to generate viral MicroRNAs.

Canonical primary microRNA (pri-miRNA) precursors are transcribed by RNA polymerase II and then processed by the Drosha endonuclease to generate approximately 60 nt pre-miRNA hairpins. Pre-miRNAs in turn are cleaved by Dicer to generate mature miRNAs. Previously, some short introns, called miRtrons, were reported to fold into pre-miRNA hairpins after splicing and debranching, and miRNAs can also be excised by Dicer cleavage of rare endogenous short hairpin RNAs. Here we report that the miRNAs encoded by murine gamma-herpesvirus 68 (MHV68) are also generated via atypical mechanisms. Specifically, MHV68 miRNAs are transcribed from RNA polymerase III promoters located within adjacent viral tRNA-like sequences. The resultant pri-miRNAs, which bear a 5' tRNA moiety, are not processed by Drosha but instead by cellular tRNase Z, which cleaves 3' to the tRNA to liberate pre-miRNA hairpins that are then processed by Dicer to yield the mature viral miRNAs.

Mature and functional viral miRNAs transcribed from novel RNA polymerase III promoters.

Murid herpesvirus 4 (MuHV-4) microRNAs were previously cloned from latently infected tumor cells and predicted to be processed from a series of RNA polymerase III primary transcripts. We detected maturely processed MuHV-4 miRNAs within total RNA from lytically infected cells in vitro and infected tissues ex vivo, using a highly sensitive reverse ligation meditated RT-PCR strategy. We determined that the MuHV-4 microRNAs are biologically active during infection by a luciferase reporter system. We experimentally demonstrated that transcription of the MuHV-4 microRNAs is by RNA polymerase III by alpha-amanitin insensitivity and by specific deletion of the RNA polymerase III type 2-like promoter elements of MuHV-4, resulting in the complete loss of miRNA detection and function. Finally, we demonstrate that these 10 viral miRNAs, each transcribed from highly conserved and novel polymerase III promoter elements, vary markedly in their relative abundance and activity.

The JNKs differentially regulate RNA polymerase III transcription by coordinately modulating the expression of all TFIIIB subunits.

RNA polymerase (pol) III-dependent transcription is subject to stringent regulation by tumor suppressors and oncogenic proteins and enhanced RNA pol III transcription is essential for cellular transformation and tumorigenesis. Since the c-Jun N-terminal kinases (JNKs) display both oncogenic and tumor suppressor properties, the roles of these proteins in regulating RNA pol III transcription were examined. In both mouse and human cells, loss or reduction in JNK1 expression represses RNA pol III transcription. In contrast, loss or reduction in JNK2 expression induces transcription. The JNKs coordinately regulate expression of all 3 TFIIIB subunits. While JNK1 positively regulates TBP expression, the RNA pol III-specific factors, Brf1 and Bdp1, JNK2 negatively regulates their expression. Brf1 is coregulated with TBP through the JNK target, Elk-1. Reducing Elk-1 expression decreases Brf1 expression. Decreasing JNK1 expression reduces Elk-1 occupancy at the Brf1 promoter, while decreasing JNK2 expression enhances recruitment of Elk-1 to the Brf1 promoter. In contrast, regulation of Bdp1 occurs through JNK-mediated alterations in TBP expression. Altered TBP expression mimics the effect of reduced JNK1 or JNK2 levels on Bdp1 expression. Decreasing JNK1 expression reduces the occupancy of TBP at the Bdp1 promoter, while decreasing JNK2 expression enhances recruitment of TBP to the Bdp1 promoter. Together, these results provide a molecular mechanism for regulating RNA pol III transcription through the coordinate control of TFIIIB subunit expression and elucidate opposing functions for the JNKs in regulating a large class of genes that dictate the biosynthetic capacity of cells.

InvAluable junk: the cellular impact and function of Alu and B2 RNAs.

The short interspersed elements (SINEs) Alu and B2 are retrotransposons that litter the human and mouse genomes, respectively. Given their abundance, the manner in which these elements impact the host genome and what their biological functions might be is of significant interest. Finding that Alu and B2 SINEs are transcribed, both as distinct RNA polymerase III transcripts and as part of RNA polymerase II transcripts, and that these SINE encoded RNAs indeed have biological functions has refuted the historical notion that SINEs are merely "junk DNA." This article reviews currently known cellular functions of both RNA polymerase II and RNA polymerase III transcribed Alu and B2 RNAs. These RNAs, in different forms, control gene expression by participating in processes as diverse as mRNA transcriptional control, A-to-I editing, nuclear retention, and alternative splicing. Future studies will likely reveal additional contributions of Alu and B2 RNAs as regulators of gene expression.

Sub1 functions in osmoregulation and in transcription by both RNA polymerases II and III.

Sub1 is implicated in transcriptional activation, elongation, and mRNA 3'-end formation in budding yeast. To gain more insight into its function, we performed a synthetic genetic array screen with SUB1 that uncovered genetic interactions with genes involved in the high-osmolarity glycerol (HOG) osmoresponse pathway. We find that Sub1 and the HOG pathway are redundant for survival in moderate osmolarity. Chromatin immunoprecipitation analysis shows that Sub1 is recruited to osmoresponse gene promoters during osmotic shock and is required for full recruitment of TBP, TFIIB, and RNA polymerase II (RNAP II) at a subset of these genes. Furthermore, we detect Sub1 at the promoter of every constitutively transcribed RNAP II and, unexpectedly, at every RNAP III gene tested, but not at the RNAP I-transcribed ribosomal DNA promoter. Significantly, deletion of SUB1 reduced levels of promoter-associated RNAP II or III at these genes, but not TBP levels. Together these data suggest that, in addition to a general role in polymerase recruitment at constitutive RNAP II and RNAP III genes, during osmotic shock, Sub1 facilitates osmoresponse gene transcription by enhancing preinitiation complex formation.