In Vitro and In Vivo Assays for the Activity of Drosha Complex

Lee, Yoontae; Kim, V. Narry

doi:10.1016/s0076-6879(07)27005-3

Cited by 30 publications

(37 citation statements)

References 34 publications

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“…We evaluated whether Drosha cleaves the NFIB hairpins by in vitro processing assays ( Figure 3E) (Lee and Kim, 2007). Incubation of in vitro transcribed NFIB 3'…”

Section: Drosha Binds and Cleaves The Nfib Mrna Regulating Expressionmentioning

confidence: 99%

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Multipotency of Adult Hippocampal NSCs In Vivo Is Restricted by Drosha/NFIB

et al. 2016

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SUMMARYMulti-lineage neuronal, astrocytic and oligodendrocytic potential is considered a neural stem cell (NSC) trait. However, hippocampal NSCs generate neurons and astrocytes but not oligodendrocytes in vivo and how this is regulated is unknown. Here we show that the RNAseIII Drosha is an intrinsic regulator of stem cell maintenance and differentiation in the adult mouse hippocampus. Inactivation of Drosha results in exhaustion of the NSC pool, premature arrest of neurogenesis, and induction of oligodendrocyte fate commitment. Drosha silences Nuclear Factor IB (NFIB) in hippocampal NSCs by targeting a double-stranded hairpin in the NFIB mRNA, thereby repressing its expression in a Dicer and miRNA-independent manner. We show that NFIB is required and sufficient for oligodendrocyte fate and knockdown of NFIB rescues neurogenesis by Drosha-deficient hippocampal NSCs. Our findings reveal a novel mechanism for stem cell maintenance and oligodendrocyte fate restriction in the adult hippocampus.3

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“…We evaluated whether Drosha cleaves the NFIB hairpins by in vitro processing assays ( Figure 3E) (Lee and Kim, 2007). Incubation of in vitro transcribed NFIB 3'…”

Section: Drosha Binds and Cleaves The Nfib Mrna Regulating Expressionmentioning

confidence: 99%

“…In vitro processing was performed on 5' and 3' UTR NFIB HP RNAs as described previously with minor adaptations (Supplemental Experimental Procedures) (Lee and Kim, 2007).…”

Section: In Vitro Processing Of Nfib Hp Rnasmentioning

confidence: 99%

Multipotency of Adult Hippocampal NSCs In Vivo Is Restricted by Drosha/NFIB

et al. 2016

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“…(D) ADAR1 inhibits pri-miR302 processing. IVT pri-miR302a was incubated with ADAR1 KD HeLa cell lysates in the presence or absence of partially purified Flag-ADAR1 according to the protocol published previously [57]. After incubation, RNAs were extracted for northern blot with denaturing PAGE gels.…”

Section: Adar1 Acts As An Rna-binding Protein That Directly Inhibits mentioning

confidence: 99%

“…In vitro processing assay was carried out as described previously [57]. Briefly, pri-miR302 was IVT with the DIG RNA Labeling Mix (Roche) and AmpliScripe T7/SP6-flash Transcription Kit (Epicentre).…”

Section: In Vitro Processing Assaysmentioning

confidence: 99%

ADAR1 is required for differentiation and neural induction by regulating microRNA processing in a catalytically independent manner

et al. 2015

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Adenosine deaminases acting on RNA (ADARs) are involved in adenosine-to-inosine RNA editing and are implicated in development and diseases. Here we observed that ADAR1 deficiency in human embryonic stem cells (hESCs) significantly affected hESC differentiation and neural induction with widespread changes in mRNA and miRNA expression, including upregulation of self-renewal-related miRNAs, such as miR302s. Global editing analyses revealed that ADAR1 editing activity contributes little to the altered miRNA/mRNA expression in ADAR1-deficient hESCs upon neural induction. Genome-wide iCLIP studies identified that ADAR1 binds directly to pri-miRNAs to interfere with miRNA processing by acting as an RNA-binding protein. Importantly, aberrant expression of miRNAs and phenotypes observed in ADAR1-depleted hESCs upon neural differentiation could be reversed by an enzymatically inactive ADAR1 mutant, but not by the RNA-binding-null ADAR1 mutant. These findings reveal that ADAR1, but not its editing activity, is critical for hESC differentiation and neural induction by regulating miRNA biogenesis via direct RNA interaction.

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“…In vitro processing assay was carried out as using labelled pri-miRNA transcripts incubated with 30 mg of SK-N-BE nuclear extract at 37°C for 90 min in the presence of either 6 nmol of recombinant GST protein or 3 and 6 nmol of recombinant GST-FUS protein 31 . After the incubation, processed RNAs were precipitated and resuspended in 7.5 ml of RNA-loading buffer, heated at 95°for 5 min and then separated on a 12.5% urea-polyacrylamide gel.…”

Section: Ha-fus-g48amentioning

confidence: 99%

An ALS-associated mutation in the FUS 3′-UTR disrupts a microRNA–FUS regulatory circuitry

et al. 2014

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While the physiologic functions of the RNA-binding protein FUS still await thorough characterization, the pathonegetic role of FUS mutations in amyotrophic lateral sclerosis (ALS) is clearly established. Here we find that a human FUS mutation that leads to increased protein expression, and was identified in two ALS patients with severe outcome, maps to the seed sequence recognized by miR-141 and miR-200a in the 3 0 -UTR of FUS. We demonstrate that FUS and these microRNAs are linked by a feed-forward regulatory loop where FUS upregulates miR-141/200a, which in turn impact FUS protein synthesis. We also show that Zeb1, a target of miR-141/200a and transcriptional repressor of these two microRNAs, is part of the circuitry and reinforces it. Our results reveal a possible correlation between deregulation of this regulatory circuit and ALS pathogenesis, and open interesting perspectives in the treatment of these mutations through ad hoc-modified microRNAs.

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In Vitro and In Vivo Assays for the Activity of Drosha Complex

Cited by 30 publications

References 34 publications

Multipotency of Adult Hippocampal NSCs In Vivo Is Restricted by Drosha/NFIB

Multipotency of Adult Hippocampal NSCs In Vivo Is Restricted by Drosha/NFIB

ADAR1 is required for differentiation and neural induction by regulating microRNA processing in a catalytically independent manner

An ALS-associated mutation in the FUS 3′-UTR disrupts a microRNA–FUS regulatory circuitry

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