The N6-methyladenosine (m6A) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous m6A-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, Mettl3-KO severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers’ expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers’ role during early development that is Ythdf1/2/3 gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.

RNA-binding proteins (RBPs) are critical regulators of post-transcriptional gene expression, and aberrant RBP-RNA interactions can promote cancer progression. Here, we interrogate the function of RBPs in cancer using pooled CRISPR-Cas9 screening and identify 57 RBP candidates with distinct roles in supporting MYCdriven oncogenic pathways. We find that disrupting YTHDF2-dependent mRNA degradation triggers apoptosis in triple-negative breast cancer (TNBC) cells and tumors. eCLIP and m 6 A sequencing reveal that YTHDF2 interacts with mRNAs encoding proteins in the MAPK pathway that, when stabilized, induce epithelial-to-mesenchymal transition and increase global translation rates. scRibo-STAMP profiling of translating mRNAs reveals unique alterations in the translatome of single cells within YTHDF2-depleted solid tumors, which selectively contribute to endoplasmic reticulum stress-induced apoptosis in TNBC cells. Thus, our work highlights the therapeutic potential of RBPs by uncovering a critical role for YTHDF2 in counteracting the global increase of mRNA synthesis in MYC-driven breast cancers.

Background Circular RNAs (circRNAs) are stable, often highly expressed RNA transcripts with potential to modulate other regulatory RNAs. A few circRNAs have been shown to bind RNA-binding proteins (RBPs); however, little is known about the prevalence and distribution of these interactions in different biological contexts. Methods We conduct an extensive screen of circRNA-RBP interactions in the ENCODE cell lines HepG2 and K562. We profile circRNAs in deep-sequenced total RNA samples and analyze circRNA-RBP interactions using a large set of eCLIP data with binding sites of 150 RBPs. We validate interactions for select circRNAs and RBPs by performing RNA immunoprecipitation and functionally characterize our most interesting candidates by conducting knockdown studies followed by RNA-Seq. Results We generate a comprehensive catalog of circRNA-RBP interactions in HepG2 and K562 cells. We show that KHSRP binding sites are enriched in flanking introns of circRNAs and that KHSRP depletion affects circRNA biogenesis. We identify circRNAs that are highly covered by RBP binding sites and experimentally validate individual circRNA-RBP interactions. We show that circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, is almost completely covered with GRWD1 binding sites in HepG2 cells, and that circCDYL depletion counteracts the effect of GRWD1 depletion. Furthermore, we confirm interactions between circCDYL and RBPs in bladder cancer cells and demonstrate that circCDYL depletion affects hallmarks of cancer and perturbs the expression of key cancer genes, e.g., TP53. Finally, we show that elevated levels of circCDYL are associated with overall survival of bladder cancer patients. Conclusions Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

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15The N6-methyladenosine (m 6 A) modification is the most prevalent post-transcriptional mRNA 16 modification, regulating mRNA decay, translation and splicing. It plays a major role during normal 17 development, differentiation, and disease progression. The modification is dynamically regulated 18 by a set of writer, eraser and reader proteins. The YTH-domain family of proteins: Ythdf1, Ythdf2, 19and Ythdf3, are three homologous m 6 A binding proteins, which have different cellular functions. 20However, their sequence similarity and their tendency to bind the same targets suggest that they 21 may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer for each of 22 E7.5, and to embryonic lethality. By using systematic genotyping of viable offspring, we found 81 that in early development there is compensation between the readers, which is dosage-82 dependent, i.e. Ythdf2-hetrozygouse mice need to have at least one functional copy of another 83Ythdf reader to escape mortality. Furthermore, we used mESCs to analyze the function of each 84Ythdf reader separately, and together. We found that only triple-KO mESCs are not able to 85 differentiate properly, and present a prolonged mRNA degradation rate, similar to the effect 86shown in Mettl3-KO, while no significant effect is seen in the single-KOs. This suggests that just 87 like in early development, in mouse ESCs, a system in which all the readers are expressed in the 88 same cells and compartment, there is a redundancy between Ythdf readers, which enables 89 compensation in the absence of the other. 90 91Results 92 93Mettl3 writer plays an essential role in oogenesis and spermatogenesis 94 95We started by systematically testing the three readers in a specific system in-vivo, focusing on 96 spermatogenesis and oogenesis. m 6 A writers Mettl3 and Mettl14 and m 6 A erasers FTO and 97ALKBH5 were found to be essential for proper gametogenesis in mouse. Their KO typically leads 98to defective maturation of sperm or ova, and hypofertility (Xu et al.