More Like this

1. 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. 

   more » « less
2. Mechanisms and Functions of the RNA Polymerase II General Transcription Machinery during the Transcription Cycle

   https://doi.org/10.3390/biom14020176  

   Archuleta, Stephen R.; Goodrich, James A.; Kugel, Jennifer F. (February 2024, Biomolecules)

   Central to the development and survival of all organisms is the regulation of gene expression, which begins with the process of transcription catalyzed by RNA polymerases. During transcription of protein-coding genes, the general transcription factors (GTFs) work alongside RNA polymerase II (Pol II) to assemble the preinitiation complex at the transcription start site, open the promoter DNA, initiate synthesis of the nascent messenger RNA, transition to productive elongation, and ultimately terminate transcription. Through these different stages of transcription, Pol II is dynamically phosphorylated at the C-terminal tail of its largest subunit, serving as a control mechanism for Pol II elongation and a signaling/binding platform for co-transcriptional factors. The large number of core protein factors participating in the fundamental steps of transcription add dense layers of regulation that contribute to the complexity of temporal and spatial control of gene expression within any given cell type. The Pol II transcription system is highly conserved across different levels of eukaryotes; however, most of the information here will focus on the human Pol II system. This review walks through various stages of transcription, from preinitiation complex assembly to termination, highlighting the functions and mechanisms of the core machinery that participates in each stage. 

   more » « less
3. Nucleolar Stress-inducing Compounds Influence rDNA occupancy of RNA Polymerase I Transcription Machinery

   https://doi.org/10.1101/2025.01.09.632225  

   Yglesias, Matthew V; DeRose, Victoria J (January 2025, bioRxiv)

   Transcription of ribosomal RNA (rRNA) by RNA Polymerase I (Pol I) is often upregulated in cancer to facilitate rapid cell growth and proliferation, and has emerged as a potential target for chemotherapeutic agents. BMH-21 and Pt(II) chemotherapeutic agent oxaliplatin are well documented as inhibitors of Pol I activity, however the underlying mechanisms for this inhibition are not completely understood. Here, we applied chromatin immunoprecipitation sequencing (ChIP-seq) techniques and immunofluorescence imaging to probe the influence of oxaliplatin and BMH-21 on Pol I machinery. We demonstrate oxaliplatin and BMH-21 induce early nucleolar stress leading to the formation of “nucleolar caps” containing Pol I and upstream binding factor (UBF) which corresponds with broad reductions in ribosomal DNA (rDNA) occupancy of Pol I. Distinct occupancy patterns for the two compounds are revealed in ChIP-seq experiments. Taken together, our findings suggest that in vivo, oxaliplatin does not induce Pol I inhibition via interrupting a specific step in Pol I transcription, while treatment with BMH-21 induced unique polymerase stalling at the promoter and terminator regions of the human ribosomal RNA gene. 

   more » « less
4. Shared Transcriptional Machinery at Homologous Alleles Leads to Reduced Transcription in Early Drosophila Embryos

   Deng, Hao; Lim, Bomyi (July 2022, Frontiers in Cell and Developmental Biology)

   The mechanism by which transcriptional machinery is recruited to enhancers and promoters to regulate gene expression is one of the most challenging and extensively studied questions in modern biology. We explored the possibility that interallelic interactions between two homologous alleles might affect gene regulation. Using an MS2- and PP7-based, allele-specific live imaging assay, we visualized de novo transcripts of a reporter gene in hemizygous and homozygous Drosophila embryos. Surprisingly, each homozygous allele produced fewer RNAs than the corresponding hemizygous allele, suggesting the possibility of allelic competition in homozygotes. However, the competition was not observed when the enhancer-promoter interaction was weakened by placing the reporter construct in a different chromosome location or by moving the enhancer further away from the promoter. Moreover, the reporter gene showed reduced transcriptional activity when a partial transcription unit (either an enhancer or reporter gene only) was in the homologous position. We propose that the transcriptional machinery that binds both the enhancer and promoter regions, such as RNA Pol II or preinitiation complexes, may be responsible for the allelic competition. We showed that the degree of allelic interference increased over developmental time as more Pol II was needed to activate zygotic genes. Such allelic competition was observed for an endogenous gene as well. Our study provides new insights into the role of 3D interallelic interactions in gene regulation. 

   more » « less
5. 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. 

   more » « less