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

Abstract

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.

Knockdown long noncoding RNA nuclear paraspeckle assembly transcript 1 suppresses colorectal cancer through modulating miR‐193a‐3p/KRAS

“…Third, the screen process could not be limited to silico tools. Imperfect seed match between RNAs or targeting sites outside of 3’UTR could exist, which leads to false positive predictions . Biotinylated‐RNA pulldown as well as RNA overexpression, combined with RNA sequencing, could be more accurate and effective.…”

“…Imperfect seed match between RNAs or targeting sites outside of 3'UTR could exist, which leads to false positive predictions. 63 Biotinylated-RNA pulldown as well as RNA overexpression, combined with RNA sequencing, could be more accurate and effective. Subsequent experiments are being prepared, as expected to further expound mechanism and provide more exact treatment in CRC.…”

Oligodendrocyte-specific Argonaute profiling identifies microRNAs associated with experimental autoimmune encephalomyelitis

“…In particular, we decided to analyze the putative pathogenic and protective effects mediated respectively by miR-190a-5p and miR-6983-5p, which represent the most significant differentially expressed miRNAs in each category. RNA-seq technology is a reliable approach to capture the genes subjected to the repressing activity of a specific miRNA [36]. Hence, detailed transcriptomic profiling of OPCs over-expressing the two miRNA mimics or the miR-67 control was carried out.…”

Transcriptional and post-transcriptional regulation of the miR171-LaSCL6 module during somatic embryogenesis in Larix kaempferi

“…Many genetic factors, including microRNAs (miRNAs), have been found to control the process of somatic embryogenesis, such as AGAMOUS-LIKE (Gao et al 2020), FUSCA (Liu et al 2018), LEAFY COTYLEDON (Wang et al 2020), miR156 (Long et al 2018), miR166 , and miR171 (Li et al 2017). MiRNAs repress gene expression through mRNA degradation and translational inhibition (Awan et al 2018;Djuranovic et al 2012;Yang et al 2019). Recently, the function of miR171 in somatic embryogenesis of angiosperm and gymnosperm has attracted increasing attention (Li et al 2017(Li et al , 2014Zang et al 2019).…”