The identification of endogenous targets remains an important challenge in understanding

The identification of endogenous targets remains an important challenge in understanding microRNA (miRNA) function. approaches are often based on common motifs such as the “seed” sequence found in nucleotides 2-7 of the miRNA and the propensity for miRNAs to bind to the 3’UTR of messenger RNAs. However these programs often miss non-canonical target sites and have high rates of false positives as the presence of a binding site does not make sure regulation. Furthermore there is little consensus among miRNA target prediction programs. To address these problems several labs have utilized high-throughput sequencing following ultraviolet (UV) crosslinking and immunoprecipitation (CLIP-seq or HITS-CLIP) and other similar methods to identify Argonaute/miRNA binding sites [4-6 9 In HITS-CLIP target RNAs are identified by first crosslinking with UV light which generates a covalent bond between the protein of interest and the RNA. These protein-RNA complexes are then immunoprecipitated. By subsequently adding 5′ and 3′ adapters and removing the protein through Proteinase K digestion the isolated target RNAs are reverse-transcribed amplified and identified through high-throughput sequencing. These studies have revealed the prevalence of non-3’UTR binding sites in target mRNAs [4-6] new binding motifs [12] and have the potential to improve target prediction approaches. Since its development HITS-CLIP has become increasingly used for determining target sites for a range of RNA-binding proteins particularly in the fields of splicing and small RNA research. At first miRNA target site identification was limited to the size of the RNA fragment isolated after RNA trimming and immunoprecipitation. However analysis of sequencing data revealed that mutations and deletions occur during generation of the cDNA library at the crosslinking site due to the presence of an amino Myrislignan acid or short peptide that remains attached after Proteinase K treatment. From these data it is possible to determine RNA-protein interaction locations with increased resolution [13 14 An adaptation on HITS-CLIP known as photoactivatable-ribonucleoside-enhanced (PAR)-CLIP uses photoreactive ribonucleoside analogs and UV-A light to increase crosslink efficiency and the incidence of point mutations at the crosslinking site [6]. However PAR-CLIP requires tissues and cells to be pre-treated with the photoreactive ribonucleoside analogs before crosslinking. This may result in toxic effects [15] that may alter endogenous conditions. Furthermore primer extension studies and analysis of HITS-CLIP data have demonstrated that a significant portion of reverse transcription products terminate at the site of crosslinking [16 17 To address these problems the Ule Lab developed a new method known as individual-nucleotide resolution CLIP (iCLIP) [18 19 In brief iCLIP is designed to isolate all cDNA products generated during reverse transcription including cDNAs that truncate at the crosslinking site. Additionally iCLIP uses barcoded primers during generation of the cDNA library to control for PCR artifacts that may occur with library amplification [20]. A recent analysis of HITS-CLIP and iCLIP datasets has Serpine1 revealed that 80% of cDNAs truncate at the crosslink site and are absent in HITS-CLIP data [17]. This analysis also showed that the use of RNase I which can cleave between any ribonucleotide pairs in iCLIP alleviates the bias in target site identification Myrislignan that results from the use of other enzymes during RNA trimming. These advantages allow iCLIP to recover additional reads and provide increased confidence in the identification of bona fide target sites. As a complement to both HITS-CLIP and PAR-CLIP which have been previously adapted for use in [5 21 we have developed a procedure to bring the advantages of the iCLIP protocol [19] to for the identification of Argonaute/miRNA regulated RNAs (Figure 1). For this application offers several advantages: (1) Argonaute-like-gene 1 (ALG-1) is largely responsible for miRNA function (2) crosslinking in a living animal preserves ALG-1:RNA binding context and (3) viable genetic mutants exist to use as specificity controls. These advantages along with those of iCLIP allow us to Myrislignan identify miRNA:mRNA interaction sites in an entire organism with increased sensitivity and resolution. Fig. 1 Outline of the ALG-1 iCLIP method: i) crosslinking.