miR‐TRAP: A Benchtop Chemical Biology Strategy to Identify microRNA Targets

Baigude, Huricha; Ahsanullah,; Li, Zhonghan; Zhou, Ying; Rana, Tariq M.

doi:10.1002/anie.201201512

Cited by 50 publications

(50 citation statements)

References 32 publications

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“…To demonstrate direct interactions between miR-1298 and its target mRNAs in vivo, we turned to a recently reported method miR-TRAP (miRNA target RNA affinity purification) (25). For this, psoralen, a highly photoreactive probe, was conjugated to the 2′ position of the sugar ring of an amine-modified uridine residue 3′ of the miR-1298 seed sequence, and biotin was conjugated to the 3′ end for affinity purification (Figure 3B).…”

Section: Resultsmentioning

confidence: 99%

“…miRNA mimics were chemically modified at 2 distinct positions in the antisense/guide strand of miR-1298, as previously described (25). Briefly, psoralen (Pso) was covalently attached to the 2′ position of the sugar ring of a uridine within the miRNA-1298 seed sequence (position 5 from the 5′ end), and a biotin was conjugated to the 3′ end for affinity purification.…”

Section: Methodsmentioning

confidence: 99%

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miR-1298 Inhibits Mutant KRAS-Driven Tumor Growth by Repressing FAK and LAMB3

et al. 2016

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Global microRNA functional screens can offer a strategy to identify synthetic lethal interactions in cancer cells that might be exploited therapeutically. In this study, we applied this strategy to identify novel gene interactions in KRAS mutant cancer cells. In this manner, we discovered miR-1298, a novel miRNA that inhibited the growth of KRAS-driven cells both in vitro and in vivo. Using miR-TRAP affinity purification technology, we identified the tyrosine kinase FAK and the laminin subunit LAMB3 as functional targets of miR-1298. Silencing of FAK or LAMB3 recapitulated the synthetic lethal effects of miR-1298 expression in KRAS-driven cancer cells, whereas co-expression of both proteins was critical to rescue miR-1298-induced cell death. Expression of LAMB3 but not FAK was upregulated by mutant KRAS. In clinical specimens, elevated LAMB3 expression correlated with poorer survival in lung cancer patients with an oncogenic KRAS gene signature, suggesting a novel candidate biomarker in this disease setting. Our results define a novel regulatory pathway in KRAS-driven cancers which offers a potential therapeutic target for their eradication

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

miR-1298 Inhibits Mutant KRAS-Driven Tumor Growth by Repressing FAK and LAMB3

et al. 2016

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“…PAR-CLIP (Hafner et al, 2010) and miR-TRAP (Baigude et al, 2012) both include photoactivatable ribonucleosides in transfected miRNA mimics to allow specific cross-linking sites and higher wavelength cross-linking, which is less harmful to cells and improves RNA recovery. The PAR-CLIP method has been used to achieve single nucleotide resolution of the binding site due to the specificity of the cross-linking.…”

Section: Immunoprecipitation-based Target Identification Techniquesmentioning

confidence: 99%

“…Both errors will introduce false positives. The miR-TRAP method seeks to avoid this issue by inclusion of a biotin tag on transfected miRNA in an effort to select only for complexes containing specific miRNAs (Baigude et al, 2012). Perhaps most promising of new technologies, crosslinking, ligation, and sequencing of hybrids (CLASH) of RNA pulled down with AGO complexes, may provide the ability to simultaneously discover mRNAs being downregulated by RISC and the specific miRNA(s) which target them, as a miRNA sequence and a fragment of its targeted RNA sequence will be ligated together and sequenced as a single chimeric sequence (Helwak et al, 2013).…”

Section: Immunoprecipitation-based Target Identification Techniquesmentioning

confidence: 99%

Developing microRNA screening as a functional genomics tool for disease research

Lemons¹,

Maurya²,

Subramaniam³

et al. 2013

Front. Physiol.

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Originally discovered as regulators of developmental timing in C. elegans, microRNAs (miRNAs) have emerged as modulators of nearly every cellular process, from normal development to pathogenesis. With the advent of whole genome libraries of miRNA mimics suitable for high throughput screening, it is possible to comprehensively evaluate the function of each member of the miRNAome in cell-based assays. Since the relatively few microRNAs in the genome are thought to directly regulate a large portion of the proteome, miRNAome screening, coupled with the identification of the regulated proteins, might be a powerful new approach to gaining insight into complex biological processes.

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“…23,24 In addition to the aforementioned techniques for identifying the native targets of endogenous microRNAs, target discovery based on affinity pulldown after cellular delivery of exogenous microRNA mimetics has also been reported. [25][26][27][28] A potential caveat of these methods is that exogenously delivered miRNAs do not necessarily engage with the same set of targets as the endogenous miRNAs.…”

Section: Introductionmentioning

confidence: 99%

TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells

Chen

et al. 2017

RNA Biology

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MicroRNAs are small non-coding RNAs acting as posttranscriptional repressors of gene expression. Identifying mRNA targets of a given miRNA remains an outstanding challenge in the field. We have developed a new experimental approach, TargetLink, that applied locked nucleic acid (LNA) as the affinity probe to enrich target genes of a specific microRNA in intact cells. TargetLink also consists a rigorous and systematic data analysis pipeline to identify target genes by comparing LNA-enriched sequences between experimental and control samples. Using miR-21 as a test microRNA, we identified 12 target genes of miR-21 in a human colorectal cancer cell by this approach. The majority of the identified targets interacted with miR-21 via imperfect seed pairing. Target validation confirmed that miR-21 repressed the expression of the identified targets. The cellular abundance of the identified miR-21 target transcripts varied over a wide range, with some targets expressed at a rather low level, confirming that both abundant and rare transcripts are susceptible to regulation by microRNAs, and that TargetLink is an efficient approach for identifying the target set of a specific microRNA in intact cells. C20orf111, one of the novel targets identified by TargetLink, was found to reside in the nuclear speckle and to be reliably repressed by miR-21 through the interaction at its coding sequence.

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miR‐TRAP: A Benchtop Chemical Biology Strategy to Identify microRNA Targets

Cited by 50 publications

References 32 publications

miR-1298 Inhibits Mutant KRAS-Driven Tumor Growth by Repressing FAK and LAMB3

miR-1298 Inhibits Mutant KRAS-Driven Tumor Growth by Repressing FAK and LAMB3

Developing microRNA screening as a functional genomics tool for disease research

TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells

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