BioProject

The translational reactivation of maternal mRNAs encoding the drivers of vertebrate meiosis is accomplished mainly by cytoplasmic polyadenylation. The Cytoplasmic Polyadenylation Elements (CPEs) present in the 3’ UTR of these transcripts, together with their cognate CPE-binding proteins (CPEBs), define a combinatorial code that determines the timing and extent of translational activation upon meiosis resumption. In addition, the RNA-binding protein Musashi1 (Msi1) regulates the polyadenylation of CPE-containing mRNAs by an as yet undefined CPEB-dependent or -independent mechanism. Here we show that Msi1 alone does not support cytoplasmic polyadenylation, but its binding triggers the remodeling of RNA structure, thereby exposing adjacent CPEs and stimulating polyadenylation. Thus, Msi1 directs the preferential use of specific CPEs, which in turn affects the timing and extent of polyadenylation during meiotic progression. Genome-wide analysis of CPEB1- and Msi-associated mRNAs identified 491 common targets, thus revealing a new layer of CPE-mediated translational control. Overall design: To obtain a global perspective of the CPEB1- and Msi1-regulated transcripts, and their degree of overlap, we performed CPEB1 and Msi1 RNA immunoprecipitation (RIP) from immature oocytes, followed by microarray hybridization. Stage VI oocytes were lysed and immunoprecipitations were performed with the indicated antibody. Co-Immunoprecipitated RNAs were eluted in proteinase K buffer (200 mM Tris pH7.5, 100M NaCl, 10 mM EDTA, 1%SDS) with 100 µg of proteinase K (30 min at 37ºC).. For each IP, ¼ of the IP was kept to verify the efficiency of the IP by Western blot. GeneChip Xenopus laevis Genome 2.0 Array on GeneChip Xenopus laevis Genome 2.0 Array were processed with Bioconductor 34, using RMA background correction, quantile normalization and RMA summarization 35. Significantly enriched target genes in CPEB1 vs. preserum and HA-MSI vs. HA samples were detected with the phenoTest package36, using a fold-change threshold of 1.5 times or more and a Benjamini-Hogberg adjusted p-value < 0.05