RNA-binding proteins (RBPs) have essential functions during germline and early embryo development. However, current methods are unable to identify the in vivo targets of a RBP in these low-abundance cells. Here, by coupling RBP-mediated reverse transcription termination with linear amplification of complementary DNA ends and sequencing, researchers from the Chinese Academy of Sciences have developed the LACE-seq method for identifying RBP-regulated RNA networks at or near the single-oocyte level. The researchers determined the binding sites and regulatory mechanisms for several RBPs, including Argonaute 2 (Ago2), Mili, Ddx4 and Ptbp1, in mature mouse oocytes. Unexpectedly, transcriptomics and proteomics analysis of Ago2−/− oocytes revealed that Ago2 interacts with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally. Furthermore, the Ago2 and endo-siRNA complexes fine-tune the transcriptome by slicing long terminal repeat retrotransposon-derived chimeric transcripts. The precise mapping of RBP-binding sites in low-input cells opens the door to studying the roles of RBPs in embryonic development and reproductive diseases.
Overview of the LACE-seq method
a, Schematic of the LACE-seq method. A circled B represents biotin modification. N, random nucleotide; V can be A, G or C. b, LACE-seq peaks showed a 5-nt shift relative to bulk CLIP-seq and iCLIP. In the schematic, the black arrow denotes a CLIP read, while the purple arrow stands for a LACE-seq read. The yellow oval represents PTBP1 protein. c, PTBP1 LACE-seq reads were terminated before the CUUCCU motif in the EIF4G1/SNORD66 transcript. d, WebLogo showing the base frequency at and around the PTBP1–RNA crosslinking sites. e, PTBP1 LACE-seq detected cDNA ends were accumulated at CU-rich motifs. Dashed lines denote randomized controls. Data in b–e represent results from two independent experiments.