Selection of the AUG start codon is a key step in translation initiation requiring hydrolysis of GTP in the eIF2•GTP•Met-tRNA_i^Met ternary complex (TC) and subsequent P_i release from eIF2•GDP•P_i. It is thought that eIF1 prevents recognition of non-AUGs by promoting scanning and blocking P_i release at non-AUG codons. We show that Sui⁻ mutations in Saccharomyces cerevisiae eIF1, which increase initiation at UUG codons, reduce interaction of eIF1 with 40S subunits in vitro and in vivo, and both defects are diminished in cells by overexpressing the mutant proteins. Remarkably, Sui⁻ mutation ISQLG_93–97ASQAA (abbreviated 93–97) accelerates eIF1 dissociation and P_i release from reconstituted preinitiation complexes (PICs), whereas a hyperaccuracy mutation in eIF1A (that suppresses Sui⁻ mutations) decreases the eIF1 off-rate. These findings demonstrate that eIF1 dissociation is a critical step in start codon selection, which is modulated by eIF1A. We also describe Gcd⁻ mutations in eIF1 that impair TC loading on 40S subunits or destabilize the multifactor complex containing eIF1, eIF3, eIF5, and TC, showing that eIF1 promotes PIC assembly in vivo beyond its important functions in AUG selection.

Translation initiation factor 1A stimulates 40S‐binding of the eukaryotic initiation factor 2 (eIF2)/GTP/Met‐tRNAiMet ternary complex (TC) and promotes scanning in vitro . eIF1A contains an OB‐fold present in bacterial IF1 plus N‐ and C‐terminal extensions. Truncating the C‐terminus (Δ C ) or mutating OB‐fold residues ( 66–70 ) of eIF1A reduced general translation in vivo but increased GCN4 translation (Gcd− phenotype) in a manner suppressed by overexpressing TC. Consistent with this, both mutations diminished 40S‐bound TC, eIF5 and eIF3 in vivo , and Δ C impaired TC recruitment in vitro . The assembly defects of the OB‐fold mutation can be attributed to reduced 40S‐binding of eIF1A, whereas Δ C impairs eIF1A function on the ribosome. A substitution in the C‐terminal helix ( 98 – 101 ) also reduced 43S assembly in vivo . Rather than producing a Gcd− phenotype, however, 98 – 101 impairs GCN4 derepression in a manner consistent with defective scanning by reinitiating ribosomes. Indeed, 98 – 101 allows formation of aberrant 48S complexes in vitro and increases utilization of non‐AUG codons in vivo . Thus, the OB‐fold is crucial for ribosome‐binding and the C‐terminal domain of eIF1A has eukaryotic‐specific functions in TC recruitment and scanning.

The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytosine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI’s capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome.

Circular RNAs (circRNAs) exhibit unique properties due to their covalently closed nature. Models of circRNAs synthesis and function are emerging but much remains undefined about this surprisingly prevalent class of RNA. Here, we identified exonic circRNAs from human and mouse RNA-sequencing datasets, documenting multiple new examples. Addressing function, we found that many circRNAs co-sediment with ribosomes, indicative of their translation potential. By contrast, circRNAs with potential to act as microRNA sponges were scarce, with some support for a collective sponge function by groups of circRNAs. Addressing circRNA biogenesis, we delineated several features commonly associated with circRNA occurrence. CircRNA-producing genes tend to be longer and to contain more exons than average. Back-splice acceptor exons are strongly enriched at ordinal position 2 within genes, and circRNAs typically have a short exon span with two exons being the most prevalent. The flanking introns either side of circRNA loci are exceptionally long. Of note also, single-exon circRNAs derive from unusually long exons while multi-exon circRNAs are mostly generated from exons of regular length. These findings independently validate and extend similar observations made in a number of prior studies. Furthermore, we analysed high-resolution RNA polymerase II occupancy data from two separate human cell lines to reveal distinctive transcription dynamics at circRNA-producing genes. Specifically, RNA polymerase II traverses the introns of these genes at above average speed concomitant with an accentuated slow-down at exons. Collectively, these features indicate how a perturbed balance between transcription and linear splicing creates important preconditions for circRNA production. We speculate that these preconditions need to be in place so that looping interactions between flanking introns can promote back-splicing to raise circRNA production to appreciable levels.