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Comparing two approaches of miR-34a target identification, biotinylated-miRNA pulldown vs miRNA overexpression

ABSTRACT: Comparing two approaches of miR-34a target identification, biotinylated-miRNA pulldown vs miRNA overexpression

PROVIDER: PRJNA524346 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8634190_1.fastq.gz	Fastqsanger.gz
	SRR8634190_2.fastq.gz	Fastqsanger.gz
	SRR8634187_1.fastq.gz	Fastqsanger.gz
	SRR8634187_2.fastq.gz	Fastqsanger.gz
	SRR8634188_1.fastq.gz	Fastqsanger.gz

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Comparing two approaches of miR-34a target identification, biotinylated-miRNA pulldown vs miRNA overexpression

Project description:Here we show that biotin-labelled miR-34a can be loaded to AGO2, and AGO2 immunoprecipitation can pulldown biotinylated miR-34a (Bio-miR pulldown). RNA-sequencing (RNA-seq) of the Bio-miR pulldown RNAs efficiently identified miR-34a mRNA targets, which could be verified with luciferase assays. In contrast to the approach of Bio-miR pulldown, RNA-seq of miR-34a overexpression samples had limited value in identifying direct targets of miR-34a. It seems that pulldown of 30 -Biotin-tagged miRNA can identify bona fide microRNA targets at least for miR34a.

2019-02-27 | GSE127211 | GEO

Comparing two approaches of miR-34a target identification, biotinylated-miRNA pulldown vs miRNA overexpression.

Project description:microRNAs (miRNAs) are critical regulators of gene expression. For elucidating functional roles of miRNAs, it is critical to identify their direct targets. There are debates about whether pulldown of biotinylated miRNA mimics can be used to identify miRNA targets or not. Here we show that biotin-labelled miR-34a can be loaded to AGO2, and AGO2 immunoprecipitation can pulldown biotinylated miR-34a (Bio-miR pulldown). RNA-sequencing (RNA-seq) of the Bio-miR pulldown RNAs efficiently identified miR-34a mRNA targets, which could be verified with luciferase assays. In contrast to the approach of Bio-miR pulldown, RNA-seq of miR-34a overexpression samples had limited value in identifying direct targets of miR-34a. It seems that pulldown of 3'-Biotin-tagged miRNA can identify bona fide microRNA targets at least for miR-34a.

| S-EPMC5786020 | biostudies-literature

Identification of SART3 as a pre-miR-34a–binding protein

Project description:MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of nearly all biological processes. Global miRNA biogenesis is altered in many cancers and RNA-binding proteins (RBPs) have been shown to play a role in this process, presenting a promising avenue for targeting miRNA dysregulation in disease. miR-34a exhibits tumor-suppressive functions by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor Bcl-2, among other regulatory pathways such as Wnt, TGF-, and Notch signaling. Many cancers show downregulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tumor growth in vivo; however, the post-transcriptional mechanisms by which miR-34a is lost in cancer are not entirely understood. Here, we have used a proteomics-mediated approach to identify Squamous cell carcinoma antigen recognized by T-cells 3 (SART3) as a putative pre-miR-34a-binding protein. SART3 is a spliceosome recycling factor and nuclear RBP with no previously reported role in miRNA regulation. We demonstrate that SART3 binds pre-miR-34a with specificity over pre-let-7d and begin to elucidate a new functional role for this protein in non-small lung cancer cells. Overexpression of SART3 led to increased miR-34a levels, downregulation of the miR-34a target genes CDK4 and CDK6, and cell cycle arrest in the G1 phase. In vitro binding studies showed that the RNA-recognition motifs within the SART3 sequence are responsible for selective pre-miR-34a binding. Collectively, our results present evidence for an influential role of SART3 in miR-34a biogenesis and cell cycle progression.

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Gas5 Pulldown Total and small RNAseq

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Project description:miR-34a is strongly induced upon TPA-induced megakaryocyte differentiation of K562 cells. To investigate the gene networks regulated by this miRNA during the process of differentiation we performed gene microarray analysis in K562 cells overexpressing miR-34a or a control sequence.

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Halo-enhanced Ago2 pulldown (HEAP) to identify miRNA targets in vivo

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miR-150 Pulldown-sequencing data

Project description:Human CD4+ Total memory cells (CD4+CD25-CD45RA-CCR7+/-) were activated for 5d with anti-CD3/anti-CD28 antibodies and transfected with biotinylated miR-150 mimic or biotinylated mimic negative control by Exiqon. After 24h cells were lysed and miRNA:mRNA complexes pulled-down with streptavidin agarose O/N at 4°C. RNA was then purified using TRI-reagent and RNA zymospin columns. Three independent donors were used for this experiment. Inputs were also sequenced. Total RNA was sequenced in single end mode. This experiment is assessing direct targets bound by miR-150-5p in human T cells.

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