An official website of the United States government
Abstract Due to genome segmentation, rotaviruses must co-package eleven distinct genomic RNAs. The packaging is mediated by virus-encoded RNA chaperones, such as the rotavirus NSP2 protein. While the activities of distinct RNA chaperones are well studied on smaller RNAs, little is known about their global effect on the entire viral transcriptome. Here, we used Selective 2′-hydroxyl Acylation Analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP) to examine the secondary structure of the rotavirus transcriptome in the presence of increasing amounts of NSP2. SHAPE-MaP data reveals that despite the well-documented helix-unwinding activity of NSP2 in vitro, its incubation with cognate rotavirus transcripts does not induce a significant change in the SHAPE reactivities. However, a quantitative analysis of mutation rates measured by mutational profiling reveals a global 5-fold rate increase in the presence of NSP2. We demonstrate that the normalization procedure used in deriving SHAPE reactivities from mutation rates can mask an important global effect of an RNA chaperone. Analysis of the mutation rates reveals a larger effect on stems rather than loops. Together, these data provide the first experimentally derived secondary structure model of the rotavirus transcriptome and reveal that NSP2 acts by globally increasing RNA backbone flexibility in a concentration-dependent manner.
more »
« less
This content will become publicly available on December 16, 2025
Template switching enables chemical probing of native RNA structures
RNAs are often studied in nonnative sequence contexts to facilitate structural studies. However, seemingly innocuous changes to an RNA sequence may perturb the native structure and generate inaccurate or ambiguous structural models. To facilitate the investigation of native RNA secondary structure by selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), we engineered an approach that couples minimal enzymatic steps to RNA chemical probing and mutational profiling (MaP) reverse transcription (RT) methods—a process we call template switching and mutational profiling (Switch-MaP). In Switch-MaP, RT templates and additional library sequences are added postprobing through ligation and template switching, capturing reactivities for every nucleotide. For a candidate SAM-I riboswitch, we compared RNA structure models generated by the Switch-MaP approach to those of traditional primer-based MaP, including RNAs with or without appended structure cassettes. Primer-based MaP masked reactivity data in the 5′ and 3′ ends of the RNA, producing ambiguous ensembles inconsistent with the conserved SAM-I riboswitch secondary structure. Structure cassettes enabled unambiguous modeling of an aptamer-only construct but introduced nonnative interactions in the full-length riboswitch. In contrast, Switch-MaP provided reactivity data for all nucleotides in each RNA and enabled unambiguous modeling of secondary structure, consistent with the conserved SAM-I fold. Switch-MaP is a straightforward alternative approach to primer-based and cassette-based chemical probing methods that precludes primer masking and the formation of alternative secondary structures due to nonnative sequence elements.
more »
« less
- Award ID(s):
- 1942398
- PAR ID:
- 10581011
- Publisher / Repository:
- Cold Spring Harbor Laboratory Press
- Date Published:
- Journal Name:
- RNA
- Volume:
- 31
- Issue:
- 1
- ISSN:
- 1355-8382
- Page Range / eLocation ID:
- 113 to 125
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)We report the biological and structural characterization of umbravirus-like associated RNAs (ulaRNAs), a new category of coat-protein dependent subviral RNA replicons that infect plants. These RNAs encode an RNA-dependent RNA polymerase (RdRp) following a −1 ribosomal frameshift event, are 2.7–4.6 kb in length, and are related to umbraviruses, unlike similar RNA replicons that are related to tombusviruses. Three classes of ulaRNAs are proposed, with citrus yellow vein associated virus (CYVaV) placed in Class 2. With the exception of CYVaV, Class 2 and Class 3 ulaRNAs encode an additional open reading frame (ORF) with movement protein-like motifs made possible by additional sequences just past the RdRp termination codon. The full-length secondary structure of CYVaV was determined using Selective 2’ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) structure probing and phylogenic comparisons, which was used as a template for determining the putative structures of the other Class 2 ulaRNAs, revealing a number of distinctive structural features. The ribosome recoding sites of nearly all ulaRNAs, which differ significantly from those of umbraviruses, may exist in two conformations and are highly efficient. The 3′ regions of Class 2 and Class 3 ulaRNAs have structural elements similar to those of nearly all umbraviruses, and all Class 2 ulaRNAs have a unique, conserved 3′ cap-independent translation enhancer. CYVaV replicates independently in protoplasts, demonstrating that the reported sequence is full-length. Additionally, CYVaV contains a sequence in its 3′ UTR that confers protection to nonsense mediated decay (NMD), thus likely obviating the need for umbravirus ORF3, a known suppressor of NMD. This initial characterization lays down a road map for future investigations into these novel virus-like RNAs.more » « less
-
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.more » « less
-
null (Ed.)Abstract The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats.more » « less
-
A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integrationAbstract Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches – like most structured RNAs – are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single-molecule and bulk approaches, we discover how a single Mn2+ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the representativeLactococcus lactisriboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control.more » « less
