NC: non-canonical; C: canonical

NC: non-canonical; C: canonical. targets identified by TargetLink, was found to reside in the nuclear speckle and to be reliably repressed by miR-21 through the interaction at its coding sequence. KEYWORDS: Colorectal cancer, LNA, locked nucleic acid, microRNA, microRNA target, miR-21, targetlink, target identification == Abbreviations == locked nucleic acid microRNA Argonaute protein size exclusion chromatography high pressure liquid chromatography guanidinium thiocyanate buffer knockout wild type ultraviolet wild type cells UV-crosslinked wild type cells not UV-crosslinked miR-21 knockout cells UV crosslinked 3-untranslated region == Introduction == microRNAs are small noncoding RNAs that play important roles in regulating gene expression. A specific microRNA can target multiple genes, and can perform diverse roles in development, physiology and/or disease of different cell types. 1Because of their complex involvement in numerous pathways and in different cell types, it is important to identify targets of a given microRNA in specific cell types and under specific conditions. Despite extensive efforts to develop both computational and experimental approaches to predict or identify miRNA targets, target identification of a given miRNA remains an outstanding challenge in the field. 2A number of bioinformatics programs predict miRNA targets on a genomic scale exploiting the rule of seed matching conserved Watson-Crick pairing to the 5 region of miRNA centered on nucleotides 27, which is called the miRNA seed. 3, 4The various algorithms developed thus far, however , often yield different sets of predictions that do not overlap. 3, 5(Fig. S1). Further, computational methods rarely take into the consideration of how the variation in biologic contexts may affect RNA conformation or RNA-protein interaction, both of which can influence the accessibility of RNAs and hence their engagement with the microRNA/Argonaute silencing complex6-11Established genome-wide experimental methods for identifying targets of endogenous microRNAs include high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (CLIP). 12-14In this method, the isolated Argonaute protein (Ago) binding sites are analyzed computationally SR 3576 Rtn4r to infer the possible microRNA(s) that could have been responsible for tethering Ago to those binding sites. However , at the miRNome level, it is not always straightforward to pair the isolated Ago-binding sequences with a SR 3576 specific microRNA based on seed matching, especially when considering that non-canonical microRNA:: target interactions represent a rather frequent feature of microRNA targeting. 2, 15-22More recently, a crosslinking and ligation approach termed CLASH was reported in which crosslinked miRNA-target sequence are ligated to generate chimeras for sequencing. 22This method should overcome the ambiguity in assigning the Ago-bound sequence to the microRNA responsible for recruiting the target gene to the RISC complex. 21A drawback of the method, however , is its relatively low efficiency of chimera generation and capture. 23, 24In addition to the aforementioned techniques for identifying the native targets of endogenous microRNAs, target discovery based on affinity pulldown after cellular delivery of exogenous microRNA mimetics has also been reported. 25-28A potential caveat of these methods is that exogenously delivered miRNAs do not necessarily engage with the same set of targets as the endogenous miRNAs. Despite these important advances in target identification on a miRNome level, there are still pressing needs to develop new experimental methods to improve the efficiency and accuracy of target identification. Moreover, since the majority of studies on microRNA focus on functional analysis of a single microRNA of interest, methods designed to recover the sequences directly bound by a specific endogenous microRNA in intact living cells should greatly facilitate such investigations. We have developed a new approach, TargetLink, for identifying target genes directly bound by a specific microRNA. TargetLink involves crosslinking microRNA and its target genes in intact live cells by UV illumination. Cell lysates containing the crosslinked microRNA-Argonaute-mRNA ternary complexes were prepared and subjected to affinity purification using locked nucleic acid (LNA) as the capture probe to pull down complementary microRNA and its crosslinked target genes. The affinity-purified RNAs were then deproteinized, converted to cDNAs, and sequenced. Using miR-21 as a test microRNA, we have also developed a data analysis pipeline by comparing the sequenced reads of the crosslinked sample with the control sample without UV illumination, as well as with the crosslinked sample from an SR 3576 isogenic cell line lacking miR-21. Our analysis identified 12 target genes of miR-21 in a human colorectal cancer cell. Remarkably, the expression level of these 12 target genes varied over a wide range, confirming that both abundant and rare transcripts are susceptible to SR 3576 regulation by microRNAs. Among several.