sRNA/L1 retrotransposition: using siRNAs and miRNAs to expand the applications of the cell culture-based LINE-1 retrotransposition assay

Tristán-Ramos, Pablo; Morell, Santiago; Sánchez, Laura; Toledo, Belen; García‐Pérez, José L.; Heras, Sara R.

doi:10.1098/rstb.2019.0346

Cited by 9 publications

(7 citation statements)

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“…Let-7 regulates human LINE-1 retrotransposition in vitro. To investigate whether there is a causal relationship between the variation in let-7 and miR-34 expression levels and the accumulation of L1 insertions in tumors, we tested the effect of these miRNAs on L1 mobilization using the sRNA/L1 retrotransposition assay, recently developed in our lab 56 . This protocol combines the previously described cell culture-based LINE-1 retrotransposition reporter assay (reviewed in 2 ) with miRNA mimics or inhibitors.…”

Section: Resultsmentioning

confidence: 99%

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The tumor suppressor microRNA let-7 inhibits human LINE-1 retrotransposition

et al. 2020

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Nearly half of the human genome is made of transposable elements (TEs) whose activity continues to impact its structure and function. Among them, Long INterspersed Element class 1 (LINE-1 or L1) elements are the only autonomously active TEs in humans. L1s are expressed and mobilized in different cancers, generating mutagenic insertions that could affect tumor malignancy. Tumor suppressor microRNAs are ∼22nt RNAs that post-transcriptionally regulate oncogene expression and are frequently downregulated in cancer. Here we explore whether they also influence L1 mobilization. We show that downregulation of let-7 correlates with accumulation of L1 insertions in human lung cancer. Furthermore, we demonstrate that let-7 binds to the L1 mRNA and impairs the translation of the second L1-encoded protein, ORF2p, reducing its mobilization. Overall, our data reveals that let-7, one of the most relevant microRNAs, maintains somatic genome integrity by restricting L1 retrotransposition.

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Section: Resultsmentioning

confidence: 99%

“…Retrotransposition assays. Modified versions of previously established L1 retrotransposition assays [80][81][82][83] were performed and are described below 56 . In the Neo/ Blast assays, 2 × 10 5 HeLa JVM cells were plated in 6-well tissue culture plates.…”

Section: Methodsmentioning

confidence: 99%

The tumor suppressor microRNA let-7 inhibits human LINE-1 retrotransposition

et al. 2020

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“…Modified versions of previously established L1 retrotransposition assays [84][85][86][87] were performed and are described below 59 . In the Neo/Blast assays, Luciferase retrotransposition assays were performed as described 87 Binding site mutant rh was generated using an established protocol .…”

Section: Retrotransposition Assaysmentioning

confidence: 99%

The tumor suppressor microRNA let-7 inhibits human LINE-1 retrotransposition

Tristán-Ramos

Rubio-Roldán

Peris

et al. 2020

Preprint

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19Nearly half of the human genome is made of transposable elements (TEs) 20 whose activity continues to impact its structure and function. Among them, 21 Long INterspersed Element class 1 (LINE-1 or L1) elements are the only 22 autonomously active TEs in humans. L1s are expressed and mobilized in 23 different cancers, generating mutagenic insertions that could affect 24 malignancy. Tumor suppressor microRNAs are 22nt RNAs that post-25 transcriptionally regulate oncogene expression and are frequently 26 downregulated in cancer. Here we explore whether they also influence L1 27 mobilization. We found that downregulation of let-7 correlates with 28 accumulation of L1 insertions in human lung cancer. Furthermore, we 29 demonstrate that let-7 binds to the L1 mRNA and impairs the translation of the 30 second L1-encoded protein, ORF2p, reducing its mobilization. Overall, our 31 data uncover a new role for let-7, one of the most relevant microRNAs, which 32 is to maintain somatic genome integrity by restricting L1 retrotransposition. 33 34 35 Transposable elements (TEs) account for nearly half of the human genome 1 . 36 However, the only TE that remains autonomously active nowadays is a non-37 Long Terminal Repeat (LTR) retrotransposon known as Long INterspersed 38 Element class 1 (LINE-1 or L1), whose mobilization continues to impact our 39 genome 2 . LINE-1s comprise >20% of our DNA 3 but only about 80-100 of the 40 ~500,000 L1 copies present in the average human genome are full-length 41 elements that retain the ability to mobilize and are thus called 42 Retrotransposition-Competent L1s (RC-L1s) 4 . RC-L1s belong to the human-43 2 specific L1Hs subfamily, are 6kb long and encode two proteins (L1-ORF1p and 44 L1-ORF2p) that are indispensable for retrotransposition 5 . However, ORF2p is 45 expressed at a significantly lower level than ORF1p 6,7 , and these differences are 46 thought to be controlled at the level of translation 8 . L1-ORF1p is a 40kDa RNA 47 binding protein with nucleic acid chaperone activity 9,10 , whereas L1-ORF2p is a 48 150 kDa protein with Endonuclease (EN) and Reverse Transcriptase (RT) 49 activities 11,12 . RC--and-ism, involving 50 reverse transcription of an RNA intermediate and insertion of its cDNA copy at 51 a new site in the genome (reviewed in 2 ). Briefly, retrotransposition starts with 52 the transcription of a full-length RC-L1 bicistronic mRNA, which is exported to 53 the cytoplasm and translated, giving rise to L1-ORF1p and L1-ORF2p that bind 54 preferentially to the same L1 mRNA to form a ribonucleoparticle (RNP) 13 . The 55 RNP gains access to the nucleus where retrotransposition occurs by a 56 mechanism known as Target Primed Reverse Transcription (TPRT) 14,15 . During 57 TPRT, the endonuclease activity of L1-ORF2p nicks the genomic DNA, and its 58 reverse transcriptase activity uses the L1 mRNA as a template to generate a new 59 copy of the element in a different genomic location. L1 can target all regions of 60 the genome, but integration is locally dictated by the presence of...

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“…Burns and colleagues [28] report one such method to profile new LINE-1 insertions in single cells. Retrotransposition assays in cell culture are useful tools to study transposon replication, and García-Pérez and colleagues [29] now combine this technology with the use of small RNAs to gain new insights into the regulation of retrotransposition. Finally, mutant models with disrupted transposon silencing pathways can be valuable experimental tools for discovering new transposons, and Panaud and colleagues [30] report one such line in rice.…”

Section: Specialized Tools For Te Researchmentioning

confidence: 99%

Crossroads between transposons and gene regulation

Branco

Chuong

2020

Phil. Trans. R. Soc. B

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Transposons are mobile genetic elements that have made a large contribution to genome evolution in a largely species-specific manner. A wide variety of different transposons have invaded genomes throughout evolution, acting in a first instance as ‘selfish’ elements, whose success was determined by their ability to self-replicate and expand within the host genome. However, their coevolution with the host has created many crossroads between transposons and the regulation of host gene expression. Transposons are an abundant source of transcriptional modulatory elements, such as gene promoters and enhancers, splicing and termination sites, and regulatory non-coding RNAs. Moreover, transposons have driven the evolution of host defence mechanisms that have been repurposed for gene regulation. However, dissecting the potential functional roles of transposons remains challenging owing to their evolutionary path, as well as their repetitive nature, which requires the development of specialized analytical tools. In this special issue, we present a collection of articles that lay out current paradigms in the field and discuss a vision for future research. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.

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sRNA/L1 retrotransposition: using siRNAs and miRNAs to expand the applications of the cell culture-based LINE-1 retrotransposition assay

Cited by 9 publications

References 86 publications

The tumor suppressor microRNA let-7 inhibits human LINE-1 retrotransposition

The tumor suppressor microRNA let-7 inhibits human LINE-1 retrotransposition

The tumor suppressor microRNA let-7 inhibits human LINE-1 retrotransposition

Crossroads between transposons and gene regulation

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