RNA Matchmaking: Finding Cellular Pairing Partners

Graveley, Brenton R.

doi:10.1016/j.molcel.2016.07.001

Cited by 23 publications

(18 citation statements)

References 14 publications

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“…To address the limitations of chemical probing experiments, new strategies have been developed that use scanning mutagenesis and chemical probing (mutate-and-map) to identify interacting nucleotides (10) or detect RNA duplexes by crosslinking and proximity ligation (11,12).…”

Section: Introductionmentioning

confidence: 99%

RNA base pairing complexity in living cells visualized by correlated chemical probing

Mustoe¹,

Lama²,

Irving³

et al. 2019

Preprint

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2 ABSTRACT RNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having notable deficiencies. Here we convert the common reagent dimethyl sulfate (DMS) into a useful probe of all four RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base pairing interactions in cells. PAIR-MaP has superior resolution and accuracy compared to alternative experiments, can resolve alternative pairing interactions of structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and two bacterial mRNA 5'-UTRs reveals new functionally important and complex structures undetectable by conventional analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells. !

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Section: Introductionmentioning

confidence: 99%

RNA base pairing complexity in living cells visualized by correlated chemical probing

Mustoe¹,

Lama²,

Irving³

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…In eukaryotes, RNA structure affects gene expression through modulating all steps of pre-mRNA processing including splicing [7], cleavage and polyadenylation [8], and RNA editing [9]. The loss of functional RNA structure has been increasingly reported as implicated in hereditary disease and cancer [10,11,12,13].Recent progress in high-throughput sequencing techniques enabled several experimental strategies to determine RNA structure in vivo [14,15,16]. Chemical RNA structure probing can reveal which bases are single-or double-stranded, but it cannot determine which nucleotides form base pairs [17,18,19,20,21].In order to identify the interacting partners, RNA structure probing has to be combined with de novo RNA structure prediction [22], but due to a number of technical limitations such methods cannot account for base pairings that are far apart [23].…”

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confidence: 99%

Conserved long-range base pairings are associated with pre-mRNA processing of human genes

Kalmykova

Kalinina

Denisov

et al. 2020

Preprint

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The ability of nucleic acids to form double-stranded structures is essential for all living systems on Earth. While DNA employs it for genome replication, RNA molecules fold into complicated secondary and tertiary structures. Current knowledge on functional RNA structures in human protein-coding genes is focused on locally-occurring base pairs. However, chemical crosslinking and proximity ligation experiments have demonstrated that long-range RNA structures are highly abundant. Here, we present the most complete to-date catalog of conserved long-range RNA structures in the human transcriptome, which consists of 1.1 million pairs of conserved complementary regions (PCCRs). PCCRs tend to occur within introns proximally to splice sites, suppress intervening exons, circumscribe circular RNAs, and exert an obstructive effect on cryptic and inactive splice sites. The double-stranded structure of PCCRs is supported by a significant decrease of icSHAPE nucleotide accessibility, high abundance of A-to-I RNA editing sites, and frequent nearby occurrence of forked eCLIP peaks. Introns with PCCRs show a distinct splicing pattern in response to RNA Pol II slowdown suggesting that splicing is widely affected by co-transcriptional RNA folding. Additionally, transcript starts and ends are strongly enriched in regions between complementary parts of PCCRs, leading to an intriguing hypothesis that RNA folding coupled with splicing could mediate co-transcriptional suppression of premature cleavage and polyadenylation events. PCCR detection procedure is highly sensitive with respect to bona fide validated RNA structures at the expense of having a high false positive rate, which cannot be reduced without loss of sensitivity.The catalog of PCCRs is visualized through a UCSC Genome Browser track hub to facilitate further genome research on long-range RNA structures.Double-stranded structure is a key feature of nucleic acids that enables replicating the genomic information and underlies fundamental cellular processes [1,2]. Many RNAs adopt functional secondary structures, and mRNAs are no exception although their main role is to encode proteins [3,4,5,6]. In eukaryotes, RNA structure affects gene expression through modulating all steps of pre-mRNA processing including splicing [7], cleavage and polyadenylation [8], and RNA editing [9]. The loss of functional RNA structure has been increasingly reported as implicated in hereditary disease and cancer [10,11,12,13].Recent progress in high-throughput sequencing techniques enabled several experimental strategies to determine RNA structure in vivo [14,15,16]. Chemical RNA structure probing can reveal which bases are single-or double-stranded, but it cannot determine which nucleotides form base pairs [17,18,19,20,21].In order to identify the interacting partners, RNA structure probing has to be combined with de novo RNA structure prediction [22], but due to a number of technical limitations such methods cannot account for base pairings that are far apart [23]. Photo-inducible RNA crosslinking a...

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“…Chemical RNA structure probing methods are insufficient to determine long-range base pairings since they can only reveal which bases are single-stranded, but cannot identify the interacting partners (63)(64)(65)(66)(67). Other rapidly emerging technologies such as RNA in situ conformation sequencing (RIC-seq) can be used instead to profile long-range RNA structures (68)(69)(70)(71). The published RIC-seq data for HeLa cells confirm R1R3 and R3R4 base pairings as well as over 50 other base paired regions in exon 7a/7b MXE cluster with a potential impact on splicing (68) ( Figure S6).…”

Section: Discussionmentioning

confidence: 99%

Multiple competing RNA structures dynamically control alternative splicing in human ATE1 gene

Kalinina

Skvortsov

Kalmykova

et al. 2020

Preprint

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The mammalian Ate1 gene encodes an arginyl transferase enzyme, which is essential for embryogenesis, male meiosis, and regulation of the cytoskeleton. Reduced levels of Ate1 are associated with malignant transformations and serve as a prognostic indicator of prostate cancer metastasis. The tumor suppressor function of Ate1 depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. Here, we report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1-R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore the RNA structure also revert splicing to that of the wild type. Blocking the competing base pairings by locked nucleic acid (LNA)/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of the ultra-longrange R2R5 interaction changes the ratio of mutually exclusive isoforms in the endogenous Ate1 pre-mRNA. The upstream exon 7a becomes more included than the downstream exon 7b in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted. In sum, we demonstrated that mutually exclusive splicing in Ate1 is controlled by two independent, dynamically interacting and functionally distinct RNA structure modules. The molecular mechanism proposed here opens new horizons for the development of therapeutic solutions, including antisense correction of splicing.

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RNA Matchmaking: Finding Cellular Pairing Partners

Cited by 23 publications

References 14 publications

RNA base pairing complexity in living cells visualized by correlated chemical probing

RNA base pairing complexity in living cells visualized by correlated chemical probing

Conserved long-range base pairings are associated with pre-mRNA processing of human genes

Multiple competing RNA structures dynamically control alternative splicing in human ATE1 gene

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