High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down

Li, Xiaohua; Pritykin, Yuri; Concepcion, Carla P.; Lu, Yuheng; Rocca, Gaspare La; Zhang, Minsi; King, Bryan; Cook, Peter J.; Au, Yu Wah; Popow, Olesja; Paulo, João A.; Otis, Hannah G.; Mastroleo, Chiara; Ogrodowski, Paul; Schreiner, Ryan; Haigis, Kevin M.; Betel, Doron; Leslie, Christina; Ventura, Andrea

doi:10.1016/j.molcel.2020.05.009

Cited by 37 publications

(64 citation statements)

References 89 publications

(111 reference statements)

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“… AGenome browser view of the region encompassing miR‐290 and miR‐295/4 MREs (red) within lncRNA‐c1 . Halo‐enhanced AGO2 pull‐down (Li et al , 2020) read density for two independent replicates (between 0–127 is represented in the y ‐axis). BPairwise alignment between miR‐290‐3p (top) and miR‐294‐3p (bottom) and respective predicted miRNA response elements (MRE) within lncRNA‐c1 . Seed‐complementary MRE start position within annotated lncRNA‐c1 transcript is indicated inside parentheses. C GFP‐lncRNA‐c1 expression 24 h following transfection of mESCs with 5, 15 or 30 mM mmu‐miR294‐3p inhibitors (Inhibitor, triangles) or small RNA negative control (NC, squares).…”

Section: Resultsmentioning

confidence: 99%

“…Central band of boxplot represents median, box depicts 25–75 quantiles of distribution, and whiskers represent the 5 th and 95 th quantiles of the distribution. C, DSmall RNA and Poly(A)‐selected RNA sequencing based estimates of the fold difference ( y ‐axis) in (C) miRNA and (D) mRNA expression, respectively, relative to day 0, during a 12 days’ time course ( x ‐axis) following treatment of DTCM23/49XY mESCs with 4‐OHT and consequent loss of DICER function. Points represent the average miRNA or mRNA expression and error bars the standard deviation based on three independent biological replicates. ECumulative distribution plot of the fold difference in expression after 8 days of 4‐OHT treatment for mRNAs, relative to day 0 of treatment (tpm ≥ 1) with ( n = 6,034) and without ( n = 7,887) evidence of AGO2‐binding by HEAP (Li et al , 2020). FDistribution of the relative fold change after 8 days of 4‐OHT treatment in steady‐state abundance, relative to day 0 of treatment, for mESC‐expressed (tpm ≥ 1) mRNAs ( n = 19,306, red), cytosolic ( n = 445, blue) and nuclear ( n = 529, grey) lncRNAs (two‐tailed Mann–Whitney U ‐test, mRNAs vs cytosolic lncRNAs P ‐value < 2 × 10 −16 and cytosolic vs nuclear lncRNAs P = 0.875).…”

Section: Resultsmentioning

confidence: 99%

“…Ribosome profiling data in mESCs (Ingolia et al , 2011) support that mRNAs (50.4%) are more frequently associated with translating ribosomes than cytosolic or nuclear lncRNAs (6.6% and 4.0%, respectively, two‐tailed chi‐square test, P ‐value < 2 × 10 −16 , Fig 1A). We took advantage of publicly available Halo‐enhanced Ago2 pull‐down (HEAP) data in mESCs (Li et al , 2020) to assess whether cytosolic lncRNAs are associated with miRISC. We found that the fraction of mESC‐expressed mRNAs with experimental evidence for AGO2 binding is significantly higher than for cytosolic lncRNAs (25.7% and 4.7%, respectively, two‐tailed chi‐square test, P ‐value < 2 × 10 −26 ).…”

Section: Resultsmentioning

confidence: 99%

“…Points represent the average miRNA or mRNA expression and error bars the standard deviation based on three independent biological replicates. E Cumulative distribution plot of the fold difference in expression after 8 days of 4-OHT treatment for mRNAs, relative to day 0 of treatment (tpm ≥ 1) with (n = 6,034) and without (n = 7,887) evidence of AGO2-binding by HEAP (Li et al, 2020). F Distribution of the relative fold change after 8 days of 4-OHT treatment in steady-state abundance, relative to day 0 of treatment, for mESC-expressed (tpm ≥ 1) mRNAs (n = 19,306, red), cytosolic (n = 445, blue) and nuclear (n = 529, grey) lncRNAs (two-tailed Mann-Whitney U-test, mRNAs vs cytosolic lncRNAs Pvalue < 2 × 10 À16 and cytosolic vs nuclear lncRNAs P = 0.875).…”

Section: No Evidence For Mirna-dependent Destabilization Of Noncodingmentioning

confidence: 99%

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Translation is required for miRNA‐dependent decay of endogenous transcripts

et al. 2020

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Post-transcriptional repression of gene expression by miRNAs occurs through transcript destabilization or translation inhibition. mRNA decay is known to account for most miRNA-dependent repression. However, because transcript decay occurs co-translationally, whether target translation is a requirement for miRNA-dependent transcript destabilization remains unknown. To decouple these two molecular processes, we used cytosolic long noncoding RNAs (lncRNAs) as models for endogenous transcripts that are not translated. We show that, despite interacting with the miRNA-loaded RNA-induced silencing complex, the steady-state abundance and decay rates of these transcripts are minimally affected by miRNA loss. To further validate the apparent requirement of translation for miRNA-dependent decay, we fused two lncRNA candidates to the 3'-end of a proteincoding gene reporter and found this results in their miRNA-dependent destabilization. Further analysis revealed that the few natural lncRNAs whose levels are regulated by miRNAs in mESCs tend to associate with translating ribosomes, and possibly represent misannotated micropeptides, further substantiating the necessity of target translation for miRNA-dependent transcript decay. In summary, our analyses suggest that translation is required for miRNA-dependent transcript destabilization, and demonstrate that the levels of coding and noncoding transcripts are differently affected by miRNAs.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: No Evidence For Mirna-dependent Destabilization Of Noncodingmentioning

confidence: 99%

See 2 more Smart Citations

Translation is required for miRNA‐dependent decay of endogenous transcripts

et al. 2020

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“…Finally, owing to advances in sequencing technologies, the field is rapidly progressing from studying a single miRNA and a few of its targets to identifying miRNA-target networks associated with distinct cell types and developmental stages ( Nowakowski et al, 2018 ). This effort is complemented by new technologies that allow precise mapping of miRNA-target interactions (mTI) in specific cell types ( Tan et al, 2013 ; Li et al, 2020 ). Integrating miRNA and target expression profile with mTI mapping will deliver, for the first time, the landscape of miRNA repression in specific cell types.…”

Section: Discussionmentioning

confidence: 99%

MicroRNAs Instruct and Maintain Cell Type Diversity in the Nervous System

Zolboot

Zampa

et al. 2021

Front. Mol. Neurosci.

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Characterizing the diverse cell types that make up the nervous system is essential for understanding how the nervous system is structured and ultimately how it functions. The astonishing range of cellular diversity found in the nervous system emerges from a small pool of neural progenitor cells. These progenitors and their neuronal progeny proceed through sequential gene expression programs to produce different cell lineages and acquire distinct cell fates. These gene expression programs must be tightly regulated in order for the cells to achieve and maintain the proper differentiated state, remain functional throughout life, and avoid cell death. Disruption of developmental programs is associated with a wide range of abnormalities in brain structure and function, further indicating that elucidating their contribution to cellular diversity will be key to understanding brain health. A growing body of evidence suggests that tight regulation of developmental genes requires post-transcriptional regulation of the transcriptome by microRNAs (miRNAs). miRNAs are small non-coding RNAs that function by binding to mRNA targets containing complementary sequences and repressing their translation into protein, thereby providing a layer of precise spatial and temporal control over gene expression. Moreover, the expression profiles and targets of miRNAs show great specificity for distinct cell types, brain regions and developmental stages, suggesting that they are an important parameter of cell type identity. Here, we provide an overview of miRNAs that are critically involved in establishing neural cell identities, focusing on how miRNA-mediated regulation of gene expression modulates neural progenitor expansion, cell fate determination, cell migration, neuronal and glial subtype specification, and finally cell maintenance and survival.

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HaloTag‐Based Reporters for Fluorescence Imaging and Biosensing

2023

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Visualizing the structure and dynamics of biomolecules is critical to understand biological function, and requires methods to fluorescently label targets of interest in their cellular context. Self‐labelling proteins, which combine a genetically encoded tag with a small‐molecule fluorophore, have attracted considerable attention for this purpose, as they can overcome limitations of fluorescent proteins. Among them, the HaloTag protein is the most broadly used, showing fast specific labelling with a small, easy to functionalize and cell‐permeant ligand. Synthetic chemistry and protein engineering have provided a portfolio of powerful imaging tools exploiting HaloTag, along with general methods to optimize and adapt them to specific applications. Here, we provide an overview of fluorescent reporters based on the HaloTag protein for imaging and biosensing, highlighting engineering strategies and general applications.

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High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down

Cited by 37 publications

References 89 publications

Translation is required for miRNA‐dependent decay of endogenous transcripts

Translation is required for miRNA‐dependent decay of endogenous transcripts

MicroRNAs Instruct and Maintain Cell Type Diversity in the Nervous System

HaloTag‐Based Reporters for Fluorescence Imaging and Biosensing

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