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1. Thiolutin has complex effects in vivo but is a direct inhibitor of RNA polymerase II in vitro

   https://doi.org/10.1093/nar/gkad1258  

   Qiu, Chenxi ; Arora, Payal ; Malik, Indranil ; Laperuta, Amber J. ; Pavlovic, Emily M. ; Ugochukwu, Scott ; Naik, Mandar ; Kaplan, Craig D. ( January 2024 , Nucleic Acids Research)

   Thiolutin is a natural product transcription inhibitor with an unresolved mode of action. Thiolutin and the related dithiolopyrrolone holomycin chelate Zn2+ and previous studies have concluded that RNA Polymerase II (Pol II) inhibition in vivo is indirect. Here, we present chemicogenetic and biochemical approaches to investigate thiolutin's mode of action in Saccharomyces cerevisiae. We identify mutants that alter sensitivity to thiolutin. We provide genetic evidence that thiolutin causes oxidation of thioredoxins in vivo and that thiolutin both induces oxidative stress and interacts functionally with multiple metals including Mn2+ and Cu2+, and not just Zn2+. Finally, we show direct inhibition of RNA polymerase II (Pol II) transcription initiation by thiolutin in vitro in support of classical studies that thiolutin can directly inhibit transcription in vitro. Inhibition requires both Mn2+ and appropriate reduction of thiolutin as excess DTT abrogates its effects. Pause prone, defective elongation can be observed in vitro if inhibition is bypassed. Thiolutin effects on Pol II occupancy in vivo are widespread but major effects are consistent with prior observations for Tor pathway inhibition and stress induction, suggesting that thiolutin use in vivo should be restricted to studies on its modes of action and not as an experimental tool.

    

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2. RNA Polymerase I Is Uniquely Vulnerable to the Small-Molecule Inhibitor BMH-21

   https://doi.org/10.3390/cancers14225544  

   Jacobs, Ruth Q. ; Fuller, Kaila B. ; Cooper, Stephanie L. ; Carter, Zachariah I. ; Laiho, Marikki ; Lucius, Aaron L. ; Schneider, David A. ( November 2022 , Cancers)

   Cancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and represses cancer cell growth. Several recent studies have uncovered key mechanisms by which BMH-21 inhibits ribosome biosynthesis but the selectivity of BMH-21 for Pol I has not been directly measured. Here, we quantify the effects of BMH-21 on Pol I, RNA polymerase II (Pol II), and RNA polymerase III (Pol III) in vitro using purified components. We found that BMH-21 directly impairs nucleotide addition by Pol I, with no or modest effect on Pols II and III, respectively. Additionally, we found that BMH-21 does not affect the stability of any of the Pols’ elongation complexes. These data demonstrate that BMH-21 directly exploits unique vulnerabilities of Pol I. 

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3. The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans

   https://doi.org/10.1073/pnas.1915312116  

   Song, Jiarui ; Logeswaran, Dhenugen ; Castillo-González, Claudia ; Li, Yang ; Bose, Sreyashree ; Aklilu, Behailu Birhanu ; Ma, Zeyang ; Polkhovskiy, Alexander ; Chen, Julian J.-L. ; Shippen, Dorothy E. ( December 2019 , Proceedings of the National Academy of Sciences)

   null (Ed.)

   Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana , called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3′ long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates. 

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4. AID–RNA polymerase II transcription-dependent deamination of IgV DNA

   https://doi.org/10.1093/nar/gkz821  

   Pham, Phuong ; Malik, Sohail ; Mak, Chiho ; Calabrese, Peter C ; Roeder, Robert G ; Goodman, Myron F ( September 2019 , Nucleic Acids Research)

   Abstract Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes to produce high-affinity antibodies. SHM requires IgV transcription by RNA polymerase II (Pol II). A eukaryotic transcription system including AID has not been reported previously. Here, we reconstitute AID-catalyzed deamination during Pol II transcription elongation in conjunction with DSIF transcription factor. C→T mutations occur at similar frequencies on non-transcribed strand (NTS) and transcribed strand (TS) DNA. In contrast, bacteriophage T7 Pol generates NTS mutations predominantly. AID-Pol II mutations are strongly favored in WRC and WGCW overlapping hot motifs (W = A or T, R = A or G) on both DNA strands. Single mutations occur on 70% of transcribed DNA clones. Mutations are correlated over a 15 nt distance in multiply mutated clones, suggesting that deaminations are catalyzed processively within a stalled or backtracked transcription bubble. Site-by-site comparisons for biochemical and human memory B-cell mutational spectra in an IGHV3-23*01 target show strongly favored deaminations occurring in the antigen-binding complementarity determining regions (CDR) compared to the framework regions (FW). By exhibiting consistency with B-cell SHM, our in vitro data suggest that biochemically defined reconstituted Pol II transcription systems can be used to investigate how, when and where AID is targeted. 

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5. A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription

   https://doi.org/10.1371/journal.ppat.1010850  

   Dissanayaka Mudiyanselage, Shachinthaka D. ; Ma, Junfei ; Pechan, Tibor ; Pechanova, Olga ; Liu, Bin ; Wang, Ying ( September 2022 , PLOS Pathogens)

   Daròs, José-Antonio (Ed.)

   Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription. 

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