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.

In yeast and higher eukaryotes, transcription factor TFIIIB is required for accurate initiation of transcription by RNA Polymerase III (Pol III), which synthesizes transfer RNAs (tRNAs), 5S ribosomal RNA (rRNA), and other essential RNA molecules. TFIIIB is composed of three subunits: B double prime 1 (Bdp1), TATA-binding protein (TBP), and TFIIB-related factor 1 (Brf1). Here, we report the molecular characterization of Brf1 in Leishmania major (LmBrf1), a parasitic protozoan that shows distinctive transcription characteristics, including the apparent absence of Pol III general transcription factors TFIIIA and TFIIIC. Although single-knockout parasites of LmBrf1 were obtained, attempts to generate LmBrf1-null mutants were unsuccessful, which suggests that LmBrf1 is essential in promastigotes of L. major. Notably, Northern blot analyses showed that the half-lives of the messenger RNAs (mRNAs) from LmBrf1 and other components of the Pol III transcription machinery (Bdp1 and Pol III subunit RPC1) are very similar (~40 min). Stabilization of these transcripts was observed in stationary-phase parasites. Chromatin immunoprecipitation (ChIP) experiments showed that LmBrf1 binds to tRNA, small nuclear RNA (snRNA), and 5S rRNA genes. Unexpectedly, the results also indicated that LmBrf1 associates to the promoter region of the 18S rRNA genes and to three Pol II-dependent regions here analyzed. Tandem affinity purification and mass spectrometry analyses allowed the identification of a putative TFIIIC subunit. Moreover, several proteins involved in transcription by all three RNA polymerases co-purified with the tagged version of LmBrf1.

BACKGROUND: Deregulation of the RNA polymerase III specific TFIIIB subunit BRF2 occurs in subtypes of human cancers. However, correlations between BRF2 alterations and clinical outcomes in breast cancer are limited. We conducted this review to analyze BRF2 alterations in genomic data sets housed in Oncomine and cBioPortal to identify potential correlations between BRF2 alterations and clinical outcomes.
METHODS: The authors queried both Oncomine and cBioPortal for alterations in BRF2 in human cancers and performed meta-analyses identifying significant correlations between BRF2 and clinical outcomes in invasive breast cancer (IBC).
RESULTS: A meta cancer outlier profile analysis (COPA) of 715 data sets (86,733 samples) in Oncomine identified BRF2 as overexpressed in 60% of breast cancer data sets. COPA scores in IBC data sets (3594 patients) are comparable for HER2 (24.211, median gene rank 60) and BRF2 (29.656, median gene rank 36.5). Overall survival in IBC patients with BRF2 alterations (21%) is significantly decreased (p = 9.332e-3). IBC patients with BRF2 alterations aged 46 to 50 have a significantly poor survival outcome (p = 7.093e-3). Strikingly, in metastatic breast cancer, BRF2 is altered in 33% of women aged 45-50. BRF2 deletions are predominant in this age group.
CONCLUSIONS: This study suggests BRF2 may be an prognostic biomarker in invasive breast carcinoma.

BACKGROUND: Upregulation of RNA polymerase (Pol) III products, including tRNAs and 5S rRNA, in tumor cells leads to enhanced protein synthesis and tumor formation, making it a potential target for cancer treatment. In this study, we evaluated the inhibition of Pol III transcription by triptolide and the anti-cancer effect of this drug in colorectal tumorigenesis.
METHODS: The effect of triptolide on colorectal cancer development was assessed in colorectal cancer mouse models, 3D organoids, and cultured cells. Colorectal cancer cells were treated with triptolide. Pol III transcription was measured by real-time quantitative polymerase chain reaction (PCR). The formation of TFIIIB, a multi-subunit transcription factor for Pol III, was determined by chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP), and fluorescence resonance energy transfer (FRET).
RESULTS: Triptolide reduced both tumor number and tumor size in adenomatous polyposis coli (Apc) mutated (ApcMin/+) mice as well as AOM/DSS-induced mice. Moreover, triptolide effectively inhibited colorectal cancer cell proliferation, colony formation, and organoid growth in vitro, which was associated with decreased Pol III target genes. Mechanistically, triptolide treatment blocked TBP/Brf1interaction, leading to the reduced formation of TFIIIB at the promoters of tRNAs and 5S rRNA.
CONCLUSIONS: Together, our data suggest that inhibition of Pol III transcription with existing drugs such as triptolide provides a new avenue for developing novel therapies for colorectal cancer.

Eukaryotic transcription is a highly regulated fundamental life process. A large number of regulatory proteins and complexes, many of them with sequence-specific DNA-binding activity are known to influence transcription by RNA polymerase (pol) II with a fine precision. In comparison, only a few regulatory proteins are known for pol III, which transcribes genes encoding small, stable, non-translated RNAs. The pol III transcription is precisely regulated under various stress conditions. We used pol III transcription complex (TC) components TFIIIC (Tfc6), pol III (Rpc128) and TFIIIB (Brf1) as baits and mass spectrometry to identify their potential interactors in vivo. A large interactome constituting chromatin modifiers, regulators and factors of transcription by pol I and pol II supports the possibility of a crosstalk between the three transcription machineries. The association of proteins and complexes involved in various basic life processes like ribogenesis, RNA processing, protein folding and degradation, DNA damage response, replication and transcription underscores the possibility of the pol III TC serving as a signaling hub for communication between the transcription and other cellular physiological activities under normal growth conditions. We also found an equally large number of proteins and complexes interacting with the TC under nutrient starvation condition, of which at least 25% were non-identical under the two conditions. The data reveal the possibility of a large number of signaling cues for pol III transcription against adverse conditions, necessary for an efficient co-ordination of various cellular functions.

The role of transcription factors (TFs) on nucleosome positioning, RNA polymerase recruitment, and transcription initiation has been extensively characterized. Here, we propose that a subset of TFs such as Reb1, Abf1, Rap1, and TFIIIB also serve a major function in partitioning transcription units by assisting the Nrd1p-Nab3p-Sen1p Pol II termination pathway.

BACKGROUND: RNA polymerase (pol) III transcribes a variety of untranslated RNAs responsible for regulating cellular growth and is deregulated in a variety of cancers. In this study, we examined gender differences in RNA pol III transcription in vitro and in vivo.
METHODS: Expression levels of U6 snRNA, tMet, and known modulators of RNA pol III transcription were assayed in male and female derived adenocarcinoma (AC) lung cancer cell lines and male and female C57BL/6J mice using real time quantitative PCR. Methylation status of the U6 snRNA promoter was determined for lung and liver tissue isolated from male and female C57BL/6J mice by digesting genomic DNA with methylation sensitive restriction enzymes and digestion profiles were analyzed by qPCR using primers spanning the U6 promoter.
RESULTS: Here, we demonstrate that RNA pol III transcription is differentially regulated by EGCG in male and female derived AC lung cancer cell lines. Basal RNA pol III transcript levels are significantly different in male and female derived AC lung cancer cell lines. These data prompted an investigation of gender specific differences in RNA pol III transcription in vivo in lung and liver tissue. Herein, we report that U6 snRNA RNA pol III transcription is significantly stimulated in the liver tissue of male C57BL/6J mice. Further, the increase in U6 transcription correlates with a significant inhibition in the expression of p53, a negative regulator of RNA pol III transcription, and demethylation of the U6 promoter in the liver tissue of male C57BL/6J mice.
CONCLUSIONS: To the best of our knowledge, this is the first study demonstrating gender specific differences in RNA pol III transcription both in vivo and in vitro and further highlights the need to include both male and female cell lines and animals in experimental design.

RNA polymerase III-transcribed U6 snRNA genes have gene-external promoters that contain TATA boxes. U6 TATA sequences are bound by TFIIIB that in Drosophila contains the three subunits TBP, Brf1, and Bdp1. The overall structure of TFIIIB is still not well understood. We have therefore studied the mode of TFIIIB binding to DNA by site-specific protein-DNA photo-cross-linking. The results indicate that a portion of Brf1 is sandwiched between Bdp1 and TBP upstream of the TATA box. Furthermore, Bdp1 traverses the DNA under the N-terminal stirrup of TBP to interact with the DNA (and very likely Brf1) downstream of the TATA sequence.

RNA polymerase III (RNAPIII) synthesizes diverse, small, non-coding RNAs with many important roles in the cellular metabolism. One of the open questions of RNAPIII transcription is whether and how additional factors are involved. Recently, Nab2 was identified as the first messenger ribonucleoprotein particle (mRNP) biogenesis factor with a function in RNAPIII transcription.

RNA polymerase III (RNAPIII) synthesizes most small RNAs, the most prominent being tRNAs. Although the basic mechanism of RNAPIII transcription is well understood, recent evidence suggests that additional proteins play a role in RNAPIII transcription. Here, we discovered by a genome-wide approach that Nab2, a poly(A)-binding protein important for correct poly(A) tail length and nuclear mRNA export, is present at all RNAPIII transcribed genes. The occupancy of Nab2 at RNAPIII transcribed genes is dependent on transcription. Using a novel temperature-sensitive allele of NAB2, nab2-34, we show that Nab2 is required for the occupancy of RNAPIII and TFIIIB at target genes. Furthermore, Nab2 interacts with RNAPIII, TFIIIB, and RNAPIII transcripts. Importantly, impairment of Nab2 function causes an RNAPIII transcription defect in vivo and in vitro. Taken together, we establish Nab2, an important mRNA biogenesis factor, as a novel player required for RNAPIII transcription by stabilizing TFIIIB and RNAPIII at promoters.