CLIP-tag is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. Data in b– e represent results from two independent experiments. This package contains 50 nmol of CLIP-Biotin substrate, sufficient to make 10 ml of a 5 M solution for the labeling of CLIP-tag fusion proteins in cells. e, PTBP1 LACE-seq detected cDNA ends were accumulated at CU-rich motifs. d, WebLogo showing the base frequency at and around the PTBP1–RNA crosslinking sites. c, PTBP1 LACE-seq reads were terminated before the CUUCCU motif in the EIF4G1/SNORD66 transcript. The yellow oval represents PTBP1 protein. In the schematic, the black arrow denotes a CLIP read, while the purple arrow stands for a LACE-seq read. b, LACE-seq peaks showed a 5-nt shift relative to bulk CLIP-seq and iCLIP.
Among them, LC-MS/MS and colorimetry can detect the. The current technical methods used to detect m6A include high-throughput sequencing, colorimetry, and LC-MS. The internal modification of mRNA is used to maintain the stability of mRNA. A circled B represents biotin modification. N6-methyladenosine, also called m6A, is a base modification behavior widely found on mRNA. 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.Ī, Schematic of the LACE-seq method. Furthermore, the Ago2 and endo-siRNA complexes fine-tune the transcriptome by slicing long terminal repeat retrotransposon-derived chimeric transcripts. Unexpectedly, transcriptomics and proteomics analysis of Ago2 −/− oocytes revealed that Ago2 interacts with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally.
The researchers determined the binding sites and regulatory mechanisms for several RBPs, including Argonaute 2 (Ago2), Mili, Ddx4 and Ptbp1, in mature mouse oocytes. 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. However, current methods are unable to identify the in vivo targets of a RBP in these low-abundance cells. RNA-binding proteins (RBPs) have essential functions during germline and early embryo development.
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