Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans

Dimitrios G Zisoulis, Michael T Lovci, Melissa L Wilbert, Kasey R Hutt, Tiffany Y Liang, Amy E Pasquinelli, Gene W Yeo
2010 Nature Structural & Molecular Biology  
nature structural & molecular biology advance online publication a r t i c l e s miRNAs function as ~22-nucleotide (nt) RNAs that target messenger RNAs (mRNAs) for degradation or translational repression 1,2 . A single miRNA can potentially repress hundreds of genes by binding with partial sequence complementarity to mRNAs 3,4 . By combinatorial regulation of thousands of genes, the miRNA pathway critically influences many developmental programs as well as cellular homeostasis, the disruption
more » ... which leads to human disease. Thus, an outstanding challenge has been to distinguish biologically relevant miRNA-target interactions. To date, identification of miRNA target sites has been dependent largely on computational methods that have limited capability for predicting specific and physiologically relevant targets. Addressing this need, several studies have reported biochemical approaches to isolate targets by immunoprecipitation of miRNA effector complexes containing miRNA-mRNA duplexes 5-10 . Despite reduction of the search space for miRNA target sites from within all transcribed genes to a subset of immunoprecipitated RNAs, the identification of miRNA binding sequences is still not directly obtained and usually relies on subsequent computational searches for complementary sites within the precipitated transcripts. Here we have narrowed the regions recognized by miRNA effector complexes to approximately 100-nt sequences. The identification and analysis of sequences directly associated with Argonaute protein in vivo in C. elegans has enabled the discovery of distinct features related to this core component of the miRNA-induced silencing complex (miRISC) as well as its interaction with mRNA target sites. RESULTS ALG- CLIP-seq in C. elegans identifies known miRNA targets As miRNAs guide Argonaute proteins to specific complementary sequences in mRNAs, we applied the CLIP-seq (also referred to as HITS-CLIP) method 11-13 to capture and identify the miRNA and target-site sequences bound by the miRNA complex (miRISC) in developing worms. A recent application of this approach in mouse brain resulted in a map of Argonaute binding sites in this tissue 14 . C. elegans offers several advantages for applying the CLIP-seq procedure to detect global Argonaute protein-RNA interactions. A single Argonaute protein, ALG-1, is largely responsible for miRNA function, and viable alg-1 genetic mutants exist 15 . A short but wellestablished list of miRNA targets expected to be bound by ALG-1 at discrete positions is available 16-31 . Of these targets, extensive studies have confirmed that lin-41 is regulated by let-7 miRNA during the fourth larval (L4) stage via two clustered sequences, let-7 complementary sites 1 and 2 (LCS1 and LCS2) 26-28 . We used this example to optimize the CLIP-seq method to detect bona fide ALG-1 binding sites (Supplementary Fig. 1) . Synchronized L4-stage wild-type (WT) worms and alg-1(gk214) mutants (hereafter referred to as alg-1(-)), which lack the anti-ALG-1 antibody epitope sequence, were treated with UV irradiation to stabilize in vivo protein-RNA interactions (Supplementary Fig. 1a) . A custom antibody specific for the C. elegans ALG-1 protein (Supplementary Fig. 1b ) was used to enrich for ALG-1 complexes expected to include miRNA and target RNA species. Immunoprecipitated complexes were processed for isolation of sequences protected by ALG-1 protein from nuclease digestion. We obtained 3,864,848 and 5,127,241 reads from WT and alg-1(-) CLIP-seq libraries, respectively, out of which 1,651,523 (42.7%) and 695,895 (13.6%) mapped uniquely to the repeat-masked C. elegans genome (Supplementary Fig. 2a) . Using MIResque, a microRNA prediction algorithm designed to analyze small-RNA reads obtained from high-throughput sequencing (S. Aigner and G.W.Y., unpublished data), MicroRNAs (miRNAs) regulate gene expression by guiding Argonaute proteins to specific target mRNA sequences. Identification of bona fide miRNA target sites in animals is challenging because of uncertainties regarding the base-pairing requirements between miRNA and target as well as the location of functional binding sites within mRNAs. Here we present the results of a comprehensive strategy aimed at isolating endogenous mRNA target sequences bound by the Argonaute protein ALG- in C. elegans. Using cross-linking and ALG- immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), we identified extensive ALG- interactions with specific 3′ untranslated region (UTR) and coding exon sequences and discovered features that distinguish miRNA complex binding sites in 3′ UTRs from those in other genic regions. Furthermore, our analyses revealed a striking enrichment of Argonaute binding sites in genes important for miRNA function, suggesting an autoregulatory role that may confer robustness to the miRNA pathway.
doi:10.1038/nsmb.1745 pmid:20062054 pmcid:PMC2834287 fatcat:hohuu3ski5dtbea4ink5i54eha