Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading

Gagnon, Keith T.; Li, Liande; Janowski, Bethany A.; Corey, David R.

doi:10.1038/nprot.2014.135

Cited by 194 publications

(172 citation statements)

References 34 publications

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“…AGO2 is found in the endoplasmic reticulum (ER), which is contiguous with the nuclear envelope (Stalder et al 2013). We have previously developed protocols for separating nuclei from ER (Gagnon et al 2014b). Western blot analysis demonstrated that nuclear preparations lacked ER protein or cytoplasmic contaminants (Fig.…”

Section: Purification Of Nuclear or Cytoplasmic Ago2mentioning

confidence: 99%

“…Purification of cytoplasmic and nuclear extract from T47D or Flag-AGO2 cells was performed as described previously (Gagnon et al 2014b) with a few modifications. Rather than using sonication to lyse nuclei, a 27-gauge needle was used.…”

Section: Extract Preparationmentioning

confidence: 99%

“…Western blots to determine the purity of nuclear and cytoplasmic fractions from TurboNuclease or DNase-treated T47D or Flag-AGO2 cells were performed as before (Gagnon et al 2014b). For comparing nuclear and cytoplasmic fractions by Western blot, the same cell equivalents of extract were separated by electrophoresis (one-half the volume of nuclear extract per one volume of cytoplasmic).…”

Section: Extract Analysismentioning

confidence: 99%

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Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells

Kalantari

Hicks

et al. 2016

RNA

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Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with Flag-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners.

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Section: Purification Of Nuclear or Cytoplasmic Ago2mentioning

confidence: 99%

Section: Extract Preparationmentioning

confidence: 99%

Section: Extract Analysismentioning

confidence: 99%

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Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells

Kalantari

Hicks

et al. 2016

RNA

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“…This study highlighted the importance of ensuring that isolated nuclei are free from endoplasmic reticulum (ER) to avoid contamination with cytoplasmic AGO-containing complexes. Recently, a comprehensive protocol was developed to ensure that purified nuclei are free from ER contamination [134] .…”

Section: Tgsmentioning

confidence: 99%

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

Méndez

2015

WJV

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“…To recapitulate this effect in vitro, siRNAs were designed against the two lncRNAs using the Dharmacon siDesign tool (http://dharmacon.gelifesciences.com/design-center/). SiRNAs have previously been used to reduce the expression of nuclear lncRNAs and it is now known that the factors required for RNAi are indeed present in the nuclear compartment [254,[355][356][357]. SiRNA C1-2 and siRNA C1-3 were chosen for further experiments to silence CUPID1, with siRNA C1-3 being less efficient but still producing a consistent 50-70% reduction in CUPID1 levels (Figure 4.18A).…”

Section: Chaptermentioning

confidence: 99%

The long range regulation of breast cancer associated genes

Betts¹

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PrefacePage iBreast cancer is the most common non-cutaneous cancer of women and a major cause of mortality [1]. Genetic factors are the single biggest risk factor and 75% of the risk derives from common but low penetrance single nucleotide polymorphisms (SNPs) [2]. These are found using genome-wide association studies (GWAS) and the majority localize to non-coding regions of the genome [3] The identification of CCND1 as an interacting partner of PRE1 and PRE2 was done using a candidate gene approach with 3C (chromosome conformation capture) targeted confirmation of interaction. To detect other potential targets of PRE1 and PRE2 at the 11q13 locus using an agnostic approach, the 4Cseq and 5C techniques were used. These revealed a number of local interaction regions covering six gene promoters. A novel strategy using knockdown of enhancer RNA transcribed from PRE1 then identified the IGHMBP2 and CPT1A genes as likely additional targets of PRE1. Genome editing with transcription activator-like effector nucleases (TALENS) was also used, creating isogenic cell lines to clarify the effects of the SNPs in their native genomic context. Further functional work is required, however the range of techniques employed has substantially expanded our understanding of the 11q13 breast cancer risk locus and forms a template for future investigations of GWAS risk loci.To identify noncoding RNAs expressed at the 11q13 locus that may be interacting with PRE1 or PRE2 required the use of RNA Capture-seq. RNA Capture-seq involves a targeted enrichment step that greatly increases the sequencing depth at regions of interest and can reveal lowly expressed transcripts that may have not been found by traditional RNA-seq [7]. This identified one novel long non-coding RNA expressed from the (+) strand that was named CUPID1 and a second arising from the same locus on the (-) strand named CUPID2. They were located in the nucleus, oestrogen induced and both expressed relatively highly in ERα positive breast cancer cell lines but not in ERα The 11q13 locus is amplified in around 20% of breast cancers and is thought to contain a number of driver genes including CCND1 [8]. Given that CUPID1 and CUPID2 are widely over-expressed inERα positive breast cancer cell lines, it was hypothesized that they may also have a role in driving tumour growth. Publically available RNAseq data showed CUPID2 to be highly expressed in breast and renal cancer but not in normal tissue. Stable cell lines over-expressing the lncRNAs were then generated and subsequent oncogenic assays revealed that CUPID2 increased cellular proliferation and the efficiency of colony formation in breast cancer cells. A murine xenograft model confirmed these findings in vivo by demonstrating a marked increase in tumour size for the mice injected with cells over-expressing CUPID2. There is thus evidence that CUPID2 behaves as an oncogene and may be an additional driver of the 11q13 amplicon in ERα positive breast cancer.In summary, CPT1A and IGHMBP2 were identified as potential mediat...

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Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading

Cited by 194 publications

References 34 publications

Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells

Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

The long range regulation of breast cancer associated genes

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