Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers

Abstract

m6A methylation provides an essential layer of regulation in organismal development, and is aberrant in a range of cancers and neuro-pathologies. The information encoded by m6A methylation is integrated into existing RNA regulatory networks by RNA binding proteins that recognise methylated sites, the m6A readers. m6A readers include a well-characterised class of dedicated proteins, the YTH proteins, as well as a broader group of multi-functional regulators where recognition of m6A is only partially understood. Molecular insight in this recognition is essential to build a mechanistic understanding of global m6A regulation. In this study, we show that the reader IMP1 recognises the m6A using a dedicated hydrophobic platform that assembles on the methyl moiety, creating a stable high-affinity interaction. This recognition is conserved across evolution and independent from the underlying sequence context but is layered upon the strong sequence specificity of IMP1 for GGAC RNA. This leads us to propose a concept for m6A regulation where methylation plays a context-dependent role in the recognition of selected IMP1 targets that is dependent on the cellular concentration of available IMP1, differing from that observed for the YTH proteins.

RNA m6A modification, signals for degradation or stabilisation?

“…A recent structural study has further revealed that the hydrophobic groove within KH4 of IGF2BPs contains a dedicated and evolutionarily conserved structural element that conveys m 6 A specificity. While this m 6 A recognition is independent of the underlying RNA sequence context, there is a preference for GGAC, with double mutations of V523I/P524S shown to significantly reduce cognate binding [ 55 ]. Interaction between IGF2BPs and m 6 A is observed only at certain m 6 A sites, as revealed by cross-linking and immunoprecipitation followed by high-throughput sequencing (CLIP-seq) analysis [ 15 , 55 ], in contrast with YTH-domain-containing proteins.…”

“…While this m 6 A recognition is independent of the underlying RNA sequence context, there is a preference for GGAC, with double mutations of V523I/P524S shown to significantly reduce cognate binding [ 55 ]. Interaction between IGF2BPs and m 6 A is observed only at certain m 6 A sites, as revealed by cross-linking and immunoprecipitation followed by high-throughput sequencing (CLIP-seq) analysis [ 15 , 55 ], in contrast with YTH-domain-containing proteins. This suggests that m 6 A does not represent a universal layer of regulation in IGF2BPs target selection.…”

“…This suggests that m 6 A does not represent a universal layer of regulation in IGF2BPs target selection. Additionally, it is important to note that the binding affinity of IGF2BPs for m 6 A-containing RNAs is considerably weaker compared with that of YTH domains [ 47 , 55 ]. Moreover, RNA structural data suggest that IGF2BPs may recognise the structural changes induced by the so-called ‘m 6 A-switch [ 56 ]'.…”

Unraveling the RNA Tapestry: A Symphony of Innovations in m6A Research Technology

“…[30] Insulinlike growth factor 2 mRNA-binding proteins 1, 2, and 3 (IGF2BP1-3), and Fragile X mental retardation 1 (FMR1) bind m 6 A with high-affinity utilizing their K homolog (KH) domain, which undergoes a local structural rearrangement and creates an open hydrophobic platform to accommodate the methyl group upon binding to m 6 A. [31][32][33] m 6 A modification of mRNA has been shown to play a significant role at multiple levels of regulation to help maintain homeostasis of host organism. At molecular level, it influences the metabolism of mRNA by mediating its splicing, export, translation, and degradation.…”

Interconnections between m6A RNA modification, RNA structure, and protein–RNA complex assembly

“…IMP1, a recently described m 6 A reader, interacts with the modification via a dedicated hydrophobic platform, enabling a high-affinity interaction ( Table 1 ). This interaction is sequence-independent but is embedded into the methylation-independent sequence preference of IMP1, GGAC, which has significant similarity with the METTL3/METTL14 DRACH motif ( Nicastro et al, 2023 ). The methyl group within the binding sequence results only in a small change in the on-rate from k on = 1.3 × 10 5 ± 0.1 × 10 5 M −1 s −1 to k on = 8.7 × 10 4 ± 0.1 × 10 4 M −1 s −1 but a more significant decrease in the off-rate from k off = 2.7 × 10 −3 ± 0.4 × 10 −3 s −1 to k off = 3.2 × 10 −4 ± 0.7 × 10 −4 s −1 , highlighting how the modification can specifically increase the lifetime of a protein–RNA complex.…”