A novel monoclonal antibody against human Argonaute proteins reveals unexpected characteristics of miRNAs in human blood cells

Nelson, Peter T.; Planell-Saguer, Mariàngels de; Lamprinaki, Stella; Kiriakidou, Marianthi; Zhang, Paul; O’Doherty, Una; Mourelatos, Zissimos P.

doi:10.1261/rna.646007

Cited by 111 publications

(134 citation statements)

References 30 publications

(31 reference statements)

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“…1A) into Jurkat T cells, which express low levels of endogenous miR-27 (data not shown). The cells were harvested, washed, and lysed 24 h after transfection, and Ago complexes were immunoprecipitated with anti-panAgo antibodies (clone 2A8) (Nelson et al 2007), which 0.4 ± 0.1% FIGURE 1. Ago coimmunoprecipitation of 3 ′ -biotin-tagged miR-27 miRNAs.…”

Section: Resultsmentioning

confidence: 99%

“…Because the Ago epitope is at the C terminus, away from the binding pocket for the miRNA 3 ′ end (Nelson et al 2007), 3 ′ -biotin modifications should not interfere directly with antibody interaction. Levels of miR-27, miR-27 * , and endogenous miR-16 present in the lysate, supernatant, and immunoprecipitated pellets were then analyzed by Northern blot (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The supernatant was subjected to antiAgo immunoprecipitation as follows: Protein G beads (GE Healthcare) were exchanged into lysis buffer, blocked with 200 μg/mL glycogen, 100 μg/mL yeast carrier RNA, and 2 mg/mL BSA (NEB) at 4°C for 30 min and washed twice in lysis buffer. About 30 μL of the blocked beads were incubated with lysate (supernatant after the 16,000g or 10,000g spin) and 12 μg of anti-Ago antibodies (clone 2A8) (Nelson et al 2007) or isotype-matched mouse IgG antibodies as a control (anti-HA; clone 16B12; Covance) for 3 h at 4°C with rotation. The beads were washed 2-3 times with the lysis buffer before RNA extraction.…”

Section: Ago Coimmunoprecipitation Assaymentioning

confidence: 99%

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3′-Biotin-tagged microRNA-27 does not associate with Argonaute proteins in cells

Guo¹,

Steitz

2014

RNA

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Synthetic 3′ -biotin-tagged microRNAs (miRNAs) have often been used to select interacting messenger RNA (mRNA) and noncoding RNA (ncRNA) targets. Here, we examined the extent of association of 3 ′ -end biotinylated miR-27 with Argonaute (Ago) proteins in transfected human cells using a coimmunoprecipitation assay followed by Northern blot analysis. We report that biotinylated miR-27 does not efficiently associate with Ago compared to unmodified miR-27. These results suggest that 3 ′ -end biotin-modified miRNAs are questionable monitors of miRNA function in cells.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Ago Coimmunoprecipitation Assaymentioning

confidence: 99%

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3′-Biotin-tagged microRNA-27 does not associate with Argonaute proteins in cells

Guo¹,

Steitz

2014

RNA

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“…1C; ref. 26). As annotated, the 6 terminal nucleotides of mature miR-451 (23 nt) span the loop region and extend into the complementary strand of the hairpin precursor, which has not been shown to occur in other known miRNAs, so far.…”

Section: Mechanisms Of Mir-451 Biogenesismentioning

confidence: 99%

The Potential Role of miR-451 in Cancer Diagnosis, Prognosis, and Therapy

Pan

Wang

2013

Molecular Cancer Therapeutics

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MicroRNAs (miRNA) are small noncoding RNAs that converge to maintain an intrinsic balance of various processes, including cell proliferation, differentiation, and apoptosis. Recent research efforts have been devoted to translating these basic discoveries into applications that could improve the early diagnosis and therapeutic outcome of patients with cancer. Early studies have shown that miRNA-451 (miR-451) is widely dysregulated in human cancers and plays a critical role in tumorigenesis and tumor progression. In this review, we summarize the potential use of miR-451 for cancer diagnosis, prognosis, and treatment. In addition, we discuss the possible mechanisms of miR-451 dysregulation and future challenges in development of miR-451 as a noninvasive biomarker and a potential therapeutic target in human cancers.

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“…While numerous non-canonical miRNA biogenesis pathways have been reported (Ha and Kim 2014), absolute confirmation of REm-1 as a true miRNA will require functional experimentation. RNA co-immunoprecipitation and sequencing of small RNAs associated with members of the Argonaute protein family has been successful in supporting the discovery of authentic miRNAs in both plants and animals (Mourelatos et al 2002, Qi et al 2006, Nelson et al 2007). Non-miRNA small RNAs have also been sequenced from Argonaute co-immunoprecipitations and so while confirmation of REm-1's association with an Argonaute protein would provide support for its designation as a true atypical miRNA, a demonstration a miRNAs capacity to silence targets remains the gold standard.…”

Section: Discussionmentioning

confidence: 99%

RNA interference in early branching metazoans

Calcino¹

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The RNA interference (RNAi) repertoire of metazoans is principally composed of three independent but related systems -endogenous small interfering RNAs (endosiRNAs), micro RNAs (miRNAs) and Piwi-interacting RNAs (piRNAs). Although the endosiRNA pathway has been demonstrated in all five eukaryotic supergroups, the miRNA and piRNA pathways of metazoans likely evolved subsequent to metazoan divergence from other eukaryotes. These innovations evolved in a 100-200 million year period between the emergence of animal multicellularity and the divergence of the sponges. It is not yet known if the organisation and function of these systems, as characterised in bilaterian model species (vertebrates, Drosophila and C. elegans), is shared between early branching phyla (Porifera, Ctenophora, Cnidaria and Placozoa) and bilaterians. Preliminary evidence is mixed with commonalities like the existence of 'ping-pong' piRNA biogenesis in sponges and cnidarians contrasted with the lack of sequence and secondary structure homology of miRNAs between sponges and bilaterians.This thesis focuses on the RNAi systems of three early branching metazoan phyla that represent critical branches in early animal evolution (ie. Porifera, Ctenophora and Cnidaria). As no such tool was available, I developed a bioinformatic pipeline to annotate the RNAi components of mapped small RNA libraries from the demosponge Amphimedon queenslandica, the ctenophore Mnemiopsis leidyi and the cnidarian Nematostella vectensis. Detailed in Chapter 2, this pipeline, named RNAiTool, clusters mapped small RNAs from high-throughput sequencing data and then annotates those clusters based on a simple set of criteria, primarily focused on the read-length of the constituent small RNAs.RNAiTool was also used in the annotation of small RNA datasets from the well-studied bilaterian Drosophila melanogaster, providing a point of comparison for the less wellunderstood non-bilaterian species. As well as interrogating individual datasets, RNAiTool can be used to compare the expression dynamics of endo-siRNA and piRNA cluster between datasets. Although it was used here to investigate the expression dynamics of endo-siRNA and piRNA cluster through development, RNAiTool can be used to assess the expression dynamics of clusters between any test and control datasets.The second major innovation presented in this thesis is the introduction of the uniformity index. This property of small RNA clusters describes the uniformity of small RNA coverage across the length of that cluster. This simple index provides a rapid method 3 for the identification of potential miRNAs and other highly expressed small RNA producing loci from large pools of small RNA clusters. This thesis takes the first steps towards an understanding of the evolution and function of RNAi systems in early branching metazoans, in particular the demosponge A.queenslandica. It is hoped that the framework for RNAi annotation developed here will prove useful for other studies of emerging model RNAi systems. 4

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A novel monoclonal antibody against human Argonaute proteins reveals unexpected characteristics of miRNAs in human blood cells

Cited by 111 publications

References 30 publications

3′-Biotin-tagged microRNA-27 does not associate with Argonaute proteins in cells

3′-Biotin-tagged microRNA-27 does not associate with Argonaute proteins in cells

The Potential Role of miR-451 in Cancer Diagnosis, Prognosis, and Therapy

RNA interference in early branching metazoans

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A novel monoclonal antibody against human Argonaute proteins reveals unexpected characteristics of miRNAs in human blood cells

Cited by 111 publications

References 30 publications

3′-Biotin-tagged microRNA-27 does not associate with Argonaute proteins in cells

3′-Biotin-tagged microRNA-27 does not associate with Argonaute proteins in cells

The Potential Role of miR-451 in Cancer Diagnosis, Prognosis, and Therapy

﻿RNA interference in early branching metazoans

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RNA interference in early branching metazoans