Structure determination of group II introns

Wiryaman, Timothy; Toor, Navtej

doi:10.1016/j.ymeth.2017.06.020

Cited by 7 publications

(6 citation statements)

References 31 publications

(37 reference statements)

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“…By contrast, X-ray crystallography is, in principle, applicable to large macromolecules. However, X-ray crystallography for RNA is more challenging than for proteins, particularly with respect to construct engineering for obtaining well-diffracting crystals (Wiryaman and Toor 2017;Gomez and Toor 2018) and to phasing (Marcia et al 2013;Marcia 2016). Except for the ribosome, the spliceosome, RNase P, and group I and II introns (Pyle 2014), RNA crystal structures are available only for short molecules (<200 nt long).…”

Section: High-resolution Methodsmentioning

confidence: 99%

The molecular structure of long non-coding RNAs: emerging patterns and functional implications

Chillón

Marcia

2020

Critical Reviews in Biochemistry and Molecular Biology

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Long non-coding RNAs (lncRNAs) are recently-discovered transcripts that regulate vital cellular processes and are crucially connected to diseases. Despite their unprecedented molecular complexity, it is emerging that lncRNAs possess distinct structural motifs. Remarkably, the 3D shape and topology of full-length, native lncRNAs have been visualized for the first time in the last year. These studies reveal that lncRNA structures dictate lncRNA functions. Here, we review experimentally determined lncRNA structures and emphasize that lncRNA structural characterization requires synergistic integration of computational, biochemical and biophysical approaches. Based on these emerging paradigms, we discuss how to overcome the challenges posed by the complex molecular architecture of lncRNAs, with the goal of obtaining a detailed understanding of lncRNA functions and molecular mechanisms in the future.

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Section: High-resolution Methodsmentioning

confidence: 99%

The molecular structure of long non-coding RNAs: emerging patterns and functional implications

Chillón

Marcia

2020

Critical Reviews in Biochemistry and Molecular Biology

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“…A second-step deficient P.li.LSUI2 group II intron was constructed by mutating G to A in EBS3 and U to A in γ′. Lariat-3′ exon and post-catalytic group II intron RNAs were prepared by the non-denaturing purification method 20 , 35 . Lariat-3′ exon and post-catalytic RNA was stored in 10 mM MgCl 2 and 5 mM sodium cacodylate pH 6.5.…”

Section: Methodsmentioning

confidence: 99%

Structural basis for the second step of group II intron splicing

et al. 2018

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The group II intron and the spliceosome share a common active site architecture and are thought to be evolutionarily related. Here we report the 3.7 Å crystal structure of a eukaryotic group II intron in the lariat-3′ exon form, immediately preceding the second step of splicing, analogous to the spliceosomal P complex. This structure reveals the location of the intact 3′ splice site within the catalytic core of the group II intron. The 3′-OH of the 5′ exon is positioned in close proximity to the 3′ splice site for nucleophilic attack and exon ligation. The active site undergoes conformational rearrangements with the catalytic triplex having different configurations before and after the second step of splicing. We describe a complete model for the second step of group II intron splicing that incorporates a dynamic catalytic triplex being responsible for creating the binding pocket for 3′ splice site capture.

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“…To reconstitute group II intron in vitro self-splicing reactions, Ta.it.I1 intron RNA was prepared by in vitro transcription using T7 RNA polymerase (Wiryaman and Toor 2017). Under standard in vitro transcription conditions, with buffers containing 25 mM MgCl 2 , we observed multiple RNA species migrating at lower molecular weights than the expected precursor product (Fig.…”

Section: Resultsmentioning

confidence: 86%

“…In vitro transcription of Ta.it.I1 RNA RNA transcription was performed with sequence confirmed plasmid DNA and T7 RNA polymerase (Wiryaman and Toor 2017). During transcription optimization, the 10× T7 buffer was replaced with a Ta.it.I1 specific 10× T7 buffer containing 5 mM MgCl 2 .…”

Section: Fluorescent Reverse Transcriptase (Rt) Activity Assaysmentioning

confidence: 99%

Transitions between the steps of forward and reverse splicing of group IIC introns

Smathers

Robart

2020

RNA

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Group II introns are mobile genetic elements that perform both self-splicing and intron mobility reactions. These ribozymes are comprised of a catalytic RNA core that binds to an intron-encoded protein (IEP) to form a ribonucleoprotein (RNP) complex. Splicing proceeds through two competing reactions: hydrolysis or branching. Group IIC intron ribozymes have a minimal RNA architecture, and splice almost exclusively through hydrolysis in ribozyme reactions. Addition of the IEP allows the splicing reaction to form branched lariat RNPs capable of intron mobility. Here we examine ribozyme splicing, IEP-dependent splicing, and mobility reactions of a group IIC intron from the thermophilic bacterium Thermoanerobacter italicus (Ta.it.I1). We show that Ta.it.I1 is highly active for ribozyme activity, forming linear hydrolytic intron products. Addition of purified IEP switches activity to the canonical lariat forming splicing reaction. We demonstrate that the Ta.it.I1 group IIC intron coordinates the progression of the forward splicing reaction through a π-π ′ ′ ′ ′ ′ interaction between intron domains II and VI. We further show that branched splicing is supported in the absence of the IEP when the π-π ′ ′ ′ ′ ′ interaction is mutated. We also investigated the regulation of the two steps of reverse splicing during intron mobility into DNA substrates. Using a fluorescent mobility assay that simultaneously visualizes all steps of intron integration into DNA, we show that completion of reverse splicing is tightly coupled to cDNA synthesis regardless of mutation of the π-π ′ ′ ′ ′ ′ interaction.

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Structure determination of group II introns

Cited by 7 publications

References 31 publications

The molecular structure of long non-coding RNAs: emerging patterns and functional implications

The molecular structure of long non-coding RNAs: emerging patterns and functional implications

Structural basis for the second step of group II intron splicing

Transitions between the steps of forward and reverse splicing of group IIC introns

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