Dissecting the sequence and structural determinants guiding m6A deposition and evolution via inter- and intra-species hybrids.

BACKGROUND: N6-methyladenosine (m6A) is the most abundant mRNA modification, and controls mRNA stability. m6A distribution varies considerably between and within species. Yet, it is unclear to what extent this variability is driven by changes in genetic sequences ('cis') or cellular environments ('trans') and via which mechanisms. RESULTS: Here we dissect the determinants governing RNA methylation via interspecies and intraspecies hybrids in yeast and mammalian systems, coupled with massively parallel reporter assays and m6A-QTL reanalysis. We find that m6A evolution and variability is driven primarily in 'cis', via two mechanisms: (1) variations altering m6A consensus motifs, and (2) variation impacting mRNA secondary structure. We establish that mutations impacting RNA structure - even when distant from an m6A consensus motif - causally dictate methylation propensity. Finally, we demonstrate that allele-specific differences in m6A levels lead to allele-specific changes in gene expression. CONCLUSIONS: Our findings define the determinants governing m6A evolution and diversity and characterize the consequences thereof on gene expression regulation.

Dissecting the basis for differential substrate specificity of ADAR1 and ADAR2.

Millions of adenosines are deaminated throughout the transcriptome by ADAR1 and/or ADAR2 at varying levels, raising the question of what are the determinants guiding substrate specificity and how these differ between the two enzymes. We monitor how secondary structure modulates ADAR2 vs ADAR1 substrate selectivity, on the basis of systematic probing of thousands of synthetic sequences transfected into cell lines expressing exclusively ADAR1 or ADAR2. Both enzymes induce symmetric, strand-specific editing, yet with distinct offsets with respect to structural disruptions: -26 nt for ADAR2 and -35 nt for ADAR1. We unravel the basis for these differences in offsets through mutants, domain-swaps, and ADAR homologs, and find it to be encoded by the differential RNA binding domain (RBD) architecture. Finally, we demonstrate that this offset-enhanced editing can allow an improved design of ADAR2-recruiting therapeutics, with proof-of-concept experiments demonstrating increased on-target and potentially decreased off-target editing.

Exclusion of m6A from splice-site proximal regions by the exon junction complex dictates m6A topologies and mRNA stability.

N6-methyladenosine (m6A), a widespread destabilizing mark on mRNA, is non-uniformly distributed across the transcriptome, yet the basis for its selective deposition is unknown. Here, we propose that m6A deposition is not selective. Instead, it is exclusion based: m6A consensus motifs are methylated by default, unless they are within a window of ∼100 nt from a splice junction. A simple model which we extensively validate, relying exclusively on presence of m6A motifs and exon-intron architecture, allows in silico recapitulation of experimentally measured m6A profiles. We provide evidence that exclusion from splice junctions is mediated by the exon junction complex (EJC), potentially via physical occlusion, and that previously observed associations between exon-intron architecture and mRNA decay are mechanistically mediated via m6A. Our findings establish a mechanism coupling nuclear mRNA splicing and packaging with the covalent installation of m6A, in turn controlling cytoplasmic decay.

Cloning of DNA oligo pools for in vitro expression.

Oligo library pools are powerful tools for systematic investigation of genetic and transcriptomic machinery such as promoter function and gene regulation, non-coding RNAs, or RNA modifications. Here, we provide a detailed protocol for cloning DNA oligo pools made up of tens of thousands of different constructs, aiming to preserve the complexity of the pools. This system would be suitable for expression in cell lines and can be followed up by next-generation sequencing analysis. For complete details on the use and execution of this profile, please refer to Uzonyi et al. (2021).

Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution.

N6-methyladenosine (m6A) is the most prevalent modification of messenger RNA in mammals. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels: site, gene and sample. Current approaches address these needs in a limited manner. Here we develop m6A-seq2, relying on multiplexed m6A-immunoprecipitation of barcoded and pooled samples. m6A-seq2 allows a big increase in throughput while reducing technical variability, requirements of input material and cost. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an orthogonal alternative to mass spectrometry-based approaches. Finally, we develop a computational approach for gene-level quantitation of m6A. We demonstrate that using this metric, roughly 30% of the variability in RNA half life in mouse embryonic stem cells can be explained, establishing m6A as a main driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-mediated regulation at three different levels.

A single-embryo, single-cell time-resolved model for mouse gastrulation.

Mouse embryonic development is a canonical model system for studying mammalian cell fate acquisition. Recently, single-cell atlases comprehensively charted embryonic transcriptional landscapes, yet inference of the coordinated dynamics of cells over such atlases remains challenging. Here, we introduce a temporal model for mouse gastrulation, consisting of data from 153 individually sampled embryos spanning 36 h of molecular diversification. Using algorithms and precise timing, we infer differentiation flows and lineage specification dynamics over the embryonic transcriptional manifold. Rapid transcriptional bifurcations characterize the commitment of early specialized node and blood cells. However, for most lineages, we observe combinatorial multi-furcation dynamics rather than hierarchical transcriptional transitions. In the mesoderm, dozens of transcription factors combinatorially regulate multifurcations, as we exemplify using time-matched chimeric embryos of Foxc1/Foxc2 mutants. Our study rejects the notion of differentiation being governed by a series of binary choices, providing an alternative quantitative model for cell fate acquisition.

Deciphering the principles of the RNA editing code via large-scale systematic probing.

Adenosine-to-inosine editing is catalyzed by ADAR1 at thousands of sites transcriptome-wide. Despite intense interest in ADAR1 from physiological, bioengineering, and therapeutic perspectives, the rules of ADAR1 substrate selection are poorly understood. Here, we used large-scale systematic probing of ∼2,000 synthetic constructs to explore the structure and sequence context determining editability. We uncover two structural layers determining the formation and propagation of A-to-I editing, independent of sequence. First, editing is robustly induced at fixed intervals of 35 bp upstream and 30 bp downstream of structural disruptions. Second, editing is symmetrically introduced on opposite sites on a double-stranded structure. Our findings suggest a recursive model for RNA editing, whereby the structural alteration induced by the editing at one site iteratively gives rise to the formation of an additional editing site at a fixed periodicity, serving as a basis for the propagation of editing along and across both strands of double-stranded RNA structures.

Dynamic Interaction of Eukaryotic Initiation Factor 4G1 (eIF4G1) with eIF4E and eIF1 Underlies Scanning-Dependent and -Independent Translation.

Translation initiation of most mRNAs involves m7G-cap binding, ribosomal scanning, and AUG selection. Initiation from an m7G-cap-proximal AUG can be bypassed resulting in leaky scanning, except for mRNAs bearing the translation initiator of short 5' untranslated region (TISU) element. m7G-cap binding is mediated by the eukaryotic initiation factor 4E (eIF4E)-eIF4G1 complex. eIF4G1 also associates with eIF1, and both promote scanning and AUG selection. Understanding of the dynamics and significance of these interactions is lacking. We report that eIF4G1 exists in two complexes, either with eIF4E or with eIF1. Using an eIF1 mutant impaired in eIF4G1 binding, we demonstrate that eIF1-eIF4G1 interaction is important for leaky scanning and for avoiding m7G-cap-proximal initiation. Intriguingly, eIF4E-eIF4G1 antagonizes the scanning promoted by eIF1-eIF4G1 and is required for TISU. In mapping the eIF1-binding site on eIF4G1, we unexpectedly found that eIF4E also binds it indirectly. These findings uncover the RNA features underlying regulation by eIF4E-eIF4G1 and eIF1-eIF4G1 and suggest that 43S ribosome transition from the m7G-cap to scanning involves relocation of eIF4G1 from eIF4E to eIF1.