Group II Intron RNPs and Reverse Transcriptases: From Retroelements to Research Tools

Belfort, Marlene; Lambowitz, Alan M.

doi:10.1101/cshperspect.a032375

Cited by 27 publications

(27 citation statements)

References 88 publications

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“…However, based on secondary structure determination for nine different lncRNAs, it is evident that these lncRNAs do contain modular structures. In fact, regarding RNA systems in general, there are other RNAs of similar sizes (e.g., group I and II intron 26,27,47 ) with well-defined high-resolution crystallographic and cryo-EM structures. Large RNA-protein complexes, such as the ribosome and spliceosome have also yielded high-resolution cryo-EM structures 26,48 .…”

Section: Discussionmentioning

confidence: 99%

Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution

et al. 2020

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Long non-coding RNAs (lncRNAs) constitute a significant fraction of the transcriptome, playing important roles in development and disease. However, our understanding of structure-function relationships for this emerging class of RNAs has been limited to secondary structures. Here, we report the 3-D atomistic structural study of epigenetic lncRNA, Braveheart (Bvht), and its complex with CNBP (Cellular Nucleic acid Binding Protein). Using small angle X-ray scattering (SAXS), we elucidate the ensemble of Bvht RNA conformations in solution, revealing that Bvht lncRNA has a well-defined, albeit flexible 3-D structure that is remodeled upon CNBP binding. Our study suggests that CNBP binding requires multiple domains of Bvht and the RHT/AGIL RNA motif. We show that RHT/AGIL, previously shown to interact with CNBP, contains a highly flexible loop surrounded by more ordered helices. As one of the largest RNA-only 3-D studies, the work lays the foundation for future structural studies of lncRNA-protein complexes.

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Section: Discussionmentioning

confidence: 99%

Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution

et al. 2020

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“…To address this weakness, we have been developing RNA-seq methods using the RTs encoded by mobile group II introns, bacterial retrotransposons that are evolutionary ancestors of introns and retroelements in eukaryotes 6–9 . Unlike retroviral RTs, which evolved to help retroviruses evade host defenses by introducing and propagating mutational variations 5 , group II intron RTs evolved to function in retrohoming, a retrotranposition mechanism that requires faithful synthesis of a full-length cDNA of a long, highly structured group II intron RNA 10 .…”

Section: Introductionmentioning

confidence: 99%

Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

Yao

et al. 2019

Sci Rep

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Thermostable group II intron reverse transcriptases (TGIRTs) with high fidelity and processivity have been used for a variety of RNA sequencing (RNA-seq) applications, including comprehensive profiling of whole-cell, exosomal, and human plasma RNAs; quantitative tRNA-seq based on the ability of TGIRT enzymes to give full-length reads of tRNAs and other structured small ncRNAs; high-throughput mapping of post-transcriptional modifications; and RNA structure mapping. Here, we improved TGIRT-seq methods for comprehensive transcriptome profiling by rationally designing RNA-seq adapters that minimize adapter dimer formation. Additionally, we developed biochemical and computational methods for remediating 5′- and 3′-end biases, the latter based on a random forest regression model that provides insight into the contribution of different factors to these biases. These improvements, some of which may be applicable to other RNA-seq methods, increase the efficiency of TGIRT-seq library construction and improve coverage of very small RNAs, such as miRNAs. Our findings provide insight into the biochemical basis of 5′- and 3′-end biases in RNA-seq and suggest general approaches for remediating biases and decreasing adapter dimer formation.

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“…To address this issue, we have been developing RNA-seq applications using the RTs encoded by mobile group II introns, bacterial retrotransposons that are evolutionary ancestors of introns and retroelements in eukaryotes (Mohr et al 2013;Nottingham et al 2016;Qin et al 2016;Belfort and Lambowitz 2019). Unlike retroviral RTs, which evolved to help retroviruses evade host defenses by introducing and propagating mutational variations (Hu and Hughes 2012), group II intron RTs evolved to function in retrohoming, a retrotranposition mechanism that requires faithful synthesis of a full-length cDNA of a long, highly structured group II intron RNA (Lambowitz and Belfort 2015).…”

Section: Introductionmentioning

confidence: 99%

Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

Yao

et al. 2018

Preprint

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Thermostable group II intron reverse transcriptases (TGIRTs) with high fidelity and processivity have been used for a variety of RNA sequencing (RNA-seq) applications, including comprehensive profiling of whole-cell, exosomal, and human plasma RNAs; quantitative tRNAseq based on the ability of TGIRT enzymes to give full-length reads of tRNAs and other structured small ncRNAs; high-throughput mapping of post-transcriptional modifications; and RNA structure mapping. Here, we improved TGIRT-seq methods for comprehensive transcriptome profiling by (i) rationally designing RNA-seq adapters that minimize adapter dimer formation, and (ii) developing biochemical and computational methods that remediate 5'and 3'-end biases. These improvements, some of which may be applicable to other RNA-seq methods, increase the efficiency of TGIRT-seq library construction and improve coverage of very small RNAs, such as miRNAs. Our findings provide insight into the biochemical basis of 5'-and 3'-end biases in RNA-seq and suggest general approaches for remediating biases and decreasing adapter dimer formation.

show abstract

Group II Intron RNPs and Reverse Transcriptases: From Retroelements to Research Tools

Cited by 27 publications

References 88 publications

Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution

Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution

Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

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