Context-dependent functional compensation between Ythdf m⁶A reader proteins

Abstract

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.

The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation

“…YTHDF proteins consist of an N-terminal intrinsically disordered region (IDR) followed by the YTH domain ( Patil et al, 2018 ). Early reports seemed to indicate functional specialization of vertebrate YTHDF proteins for either translational activation or mRNA decay ( Wang et al, 2014 ; Wang et al, 2015 ; Li et al, 2017 ; Shi et al, 2017 ), whereas recent studies support functional redundancy among the three YTHDFs in mammals and zebrafish ( Kontur et al, 2020 ; Lasman et al, 2020 ; Zaccara and Jaffrey, 2020 ), similar to the functional overlap described earlier for a subgroup of plant YTHDFs ( Arribas-Hernández et al, 2018 ).…”

“…For those ECTs with overlapping expression patterns, competition for targets leading to different molecular outcomes is an interesting possibility. Further genetic and biochemical studies on additional ECTs are necessary to understand redundant and specific functions of YTHDF proteins, a question that is currently under debate also in animal systems ( Kontur et al, 2020 ; Lasman et al, 2020 ; Zaccara and Jaffrey, 2020 ).…”

Evolution of the Neocortex Through RNA-Binding Proteins and Post-transcriptional Regulation

“…The functional redundancy was contributed to the RBPs' similar binding affinities and tendencies toward all m 6 A sites (Zaccara and Jaffrey, 2020). Another recent study corroborated these findings in vivo after systematically knocking out each of the Ythdf paralogs separately, or together (Lasman et al, 2020). As a result, the authors proposed context-dependent redundancy since a complete lack of Ythdf2 cannot be compensated by other two paralogs, probably due to differences in their spatial cytoplasmic expression, while lack of either Ythdf1 or Ythdf3 can be functionally compensated for by the two other paralogs.…”

“…Ythdf2 KOs showed delayed cortical development, as reflected in the dramatically decreased thickness of the CP and SVZ at earlier stages (E12–E14), followed by increased mortality rates at later stages of neurogenesis (E14–E18) ( Li et al, 2018 ). Contrarily, CRISPR/Cas9-generated single Ythdf1 or Ythdf3 KOs did not share the same lethal destiny ( Lasman et al, 2020 ). In Ythdf2 KOs, impaired Ythdf2-mediated decay of neuronal related m 6 A-modified transcripts was found to be the underlying cause of retarded early cortical development, which is the stage when Ythdf2 is normally highly expressed.…”

“…The functional redundancy is also dose-dependent since a partial lack of Ythdf2 requires the presence of at least one other reader to enable embryonic vitality. To confirm the redundancy hypothesis, the authors measured the m 6 A mRNA half-life in single or triple Ythdf KO mouse embryonic stem cells (ESC), and reported that their half-life is longer only in triple KO mouse ESC ( Lasman et al, 2020 ; Figure 10B ). The Ythdf gene thus evolved from having one copy in Drosophila , which directly cooperates with the RBP Fmr1 (FMRP homolog) to translationally suppress mRNAs involved in axonal growth ( Worpenberg et al, 2021 ), to having three mammalian paralogs at high sequence identities, probably made by gene duplication events ( Pervaiz et al, 2019 ).…”