Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase

Pimentel, Sydney C.; Upton, Heather; Collins, Kathleen

doi:10.1016/j.jbc.2022.101624

Cited by 8 publications

(9 citation statements)

References 53 publications

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“…Furthermore, the best fit is obtained if the bmRT catalytic site nucleotide (labeled as -1 position in Figure 3B) and the next nucleotide (labeled as -2 position in Figure 3B) are assumed to be always unpaired, indicating that the helicase site of bmRT is two nucleotides from the catalytic site at position -3 (indicated in Figure 3B). Indeed, structure prediction based on homology modeling of bmRT suggests that position -2 cannot accommodate dsRNA 11 , in agreement with this conclusion.…”

Section: Resultssupporting

confidence: 73%

“…In other words, we need to establish the offset between the constriction site of the pore and the catalytic site of bmRT 15,16 . To this end, we exploited a particular feature of this enzyme when it reaches the 5’ end of an RNA template: it extends its cDNA product via non-templated addition generating up to five 3’ overhang nucleotides 10,11 . Based on the range of positions at which the enzyme stops threading RNA into the nanopore, we estimated that the distance between the enzyme’s catalytic site and the constriction site of the nanopore is 17 nt ( Supplementary Figure 6 ).…”

Section: Resultsmentioning

confidence: 99%

“…Its RT architecture shares homology with viral and bacterial RTs with the fingers, palm, and thumb subdomains arranged in a right-handed fashion. In vivo full-length bmRT binds to its mRNA, creates a nick in the genome at its insertion site in a segment of the ribosomal RNA precursor gene, and uses the nick and mRNA as a template to synthetize cDNA into the genome 10,11 . In humans, non-LTR retroelements, such as the long interspersed nuclear element 1, can constitute about 20% of the genome and contribute to gene regulation and genome stability and evolution 12 .…”

Section: Introductionmentioning

confidence: 99%

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Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism

Shaw

Craig

Amiri

et al. 2023

Preprint

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Techniques that can directly sequence RNAs and determine secondary structures are essential to establish how an RNA molecule folds and how folding affects its function. We describe the development of a direct RNA nanopore sequencing technique using an engineered Bombyx mori retroelement reverse transcriptase (RT) to thread RNA through the Mycobacterium smegmatis porin A (MspA) nanopore in single-nucleotide steps. First, we establish the map that correlates RNA sequence and MspA ion current. We find that during sequencing the RT can sense the strength of RNA secondary structures 11-12 nt downstream of its front boundary, modifying its stepping dynamics accordingly. Using this feature, we achieve simultaneous RNA nanopore sequencing and structure detection, without the need of prior conversion to complementary DNA (cDNA) or RNA modifications. The sequence-dependent ion currents open the way to utilize the MspA nanopore to investigate the single-molecule activity of other processive RNA translocases such as the ribosome.

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Section: Resultssupporting

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism

Shaw

Craig

Amiri

et al. 2023

Preprint

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“…1G). This target heteroduplex is surrounded by residues important for RT activity ( 18 ), and the cryo-EM density shows incorporation of the ddT chain terminator nucleotide into the bottom strand (Fig. 1H).…”

Section: Reconstitution and Cryo-em Structure Of An R2 Tprt Complexmentioning

confidence: 99%

Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription

Frangieh

Macrae

Zhang

2023

Science

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Non-LTR retrotransposons, or Long Interspersed Nuclear Elements (LINEs), are an abundant class of eukaryotic transposons that insert into genomes by target-primed reverse transcription (TPRT). During TPRT, a target DNA sequence is nicked and primes reverse transcription of the retrotransposon RNA. Here, we report the cryo-electron microscopy structure of the Bombyx mori R2 non-LTR retrotransposon initiating TPRT at its ribosomal DNA target. The target DNA sequence is unwound at the insertion site and recognized by an upstream motif. An extension of the reverse transcriptase (RT) domain recognizes the retrotransposon RNA and guides the 3′ end into the RT active site to template reverse transcription. We used Cas9 to retarget R2 in vitro to non-native sequences, suggesting future use as a reprogrammable RNA-based gene-insertion tool.

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“…The template next interacts with the R0 loop, which forms a ‘lid’ over the template RNA. This loop is a portion of the R0 region, also called the N-terminal extension (NTE)-0, which is found in non-LTR retrotransposons, the group IIC intron and HCV RdRp, but not in viral RTs 30 , and has been demonstrated to be important for template jumping and/or switching activity 35 , 36 (‘Domain comparison of ORF2p and other RTs’). The downstream template makes extensive interactions continuing until the n +8 position with fingers, palm, wrist and thumb (Fig.…”

Section: Five Orf2p Core Domains All Bind Nucleic Acidmentioning

confidence: 99%

Structures, functions and adaptations of the human LINE-1 ORF2 protein

Baldwin,

van Eeuwen,

Hoyos

et al. 2023

Nature

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The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a ‘copy and paste’ mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p ‘core’ (residues 238–1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6–8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

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Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase

Cited by 8 publications

References 53 publications

Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism

Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism

Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription

Structures, functions and adaptations of the human LINE-1 ORF2 protein

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