skip to main content


Title: A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription
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
Award ID(s):
2145967
NSF-PAR ID:
10403228
Author(s) / Creator(s):
; ; ; ; ;
Editor(s):
Daròs, José-Antonio
Date Published:
Journal Name:
PLOS Pathogens
Volume:
18
Issue:
9
ISSN:
1553-7374
Page Range / eLocation ID:
e1010850
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. 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
  2. Abstract

    The largest subunit of RNA polymerase II (Pol II) has an unusual carboxyl‐terminal domain (CTD). This domain is composed of a tandemly repeating heptapeptide, Y1S2P3T4S5P6S7, that has multiple roles in regulating Pol II function and processing newly synthesized RNA. Transient phosphorylation of Ser2 and Ser5 of the YS2PTS5PS repeat have well‐defined roles in recruiting different protein complexes and coordinating sequential steps in gene transcription. As such, these phospho‐marks encipher a molecular recognition code, colloquially termed the CTD code. In contrast, the contribution of phospho‐Threonine 4 (pThr4/pT4) to the CTD code remains opaque and contentious. Fuelling the debate on the relevance of this mark to gene expression are the findings that replacing Thr4 with a valine or alanine has varied impact on cellular function in different species and independent proteomic analyses disagree on the relative abundance of pThr4 marks. Yet, substitution with negatively charged residues is lethal and even benign mutations selectively disrupt synthesis and 3′ processing of distinct sets of coding and non‐coding transcripts. Suggestive of non‐canonical roles, pThr4 marked Pol II regulates distinct gene classes in a species‐ and signal‐responsive manner. Hinting at undiscovered roles of this elusive mark, multiple signal‐responsive kinases phosphorylate Thr4 at target genes. Here, we focus on this under‐explored residue and postulate that the pThr4 mark is superimposed on the canonical CTD code to selectively regulate expression of targeted genes without perturbing genome‐wide transcriptional processes.

    This article is categorized under:

    RNA Processing > 3′ End Processing

    RNA Processing > Processing of Small RNAs

    RNA Processing > Splicing Regulation/Alternative Splicing

     
    more » « less
  3. Abstract

    RNA polymerase III (Pol III) transcription initiation requires the action of the transcription factor IIIB (TFIIIB) and is highly regulated. Here, we determine the structures of Pol III pre-initiation complexes (PICs) using single particle cryo-electron microscopy (cryo-EM). We observe stable Pol III–TFIIIB complexes using nucleic acid scaffolds mimicking various functional states, in which TFIIIB tightly encircles the upstream promoter DNA. There is an intricate interaction between TFIIIB and Pol III, which stabilizes the winged-helix domains of the C34 subunit of Pol III over the active site cleft. The architecture of Pol III PIC more resembles that of the Pol II PIC than the Pol I PIC. In addition, we also obtain a 3D reconstruction of Pol III in complex with TFIIIB using the elongation complex (EC) scaffold, shedding light on the mechanism of facilitated recycling of Pol III prior to transcription re-initiation.

     
    more » « less
  4. 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. 
    more » « less
  5. Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as the information carrier. Two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rolling-circle synthesis), and describe emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could be propagated as hereditary material in cellular contexts.

     
    more » « less