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Abstract Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during in vitro transcription results in T7 polymerase-mediated inosine misincorporation in luciferase RNA. In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis. 

Internal ribosomal entry sites (IRESs) are RNA secondary structures that mediate translation independent from the m7G RNA cap. The dicistronic luciferase assay is the most frequently used method to measure IRES-mediated translation. While this assay is quantitative, it requires numerous controls and can be time-consuming. Circular RNAs generated by splinted ligation have been shown to also accurately report on IRES-mediated translation, however suffer from low yield and other challenges. More recently, cellular sequences were shown to facilitate RNA circle formation through backsplicing. Here, we used a previously published backsplicing circular RNA split GFP reporter to create a highly sensitive and quantitative split nanoluciferase (NanoLuc) reporter. We show that NanoLuc expression requires backsplicing and correct orientation of a bona fide IRES. In response to cell stress, IRES-directed NanoLuc expression remained stable or increased while a capped control reporter decreased in translation. In addition, we detected NanoLuc expression from putative cellular IRESs and the Zika virus 5′ untranslated region that is proposed to harbor IRES function. These data together show that our IRES reporter construct can be used to verify, identify and quantify the ability of sequences to mediate IRES-translation within a circular RNA. 

Protein synthesis by the ribosome is coordinated by an intricate series of large-scale conformational rearrangements. Structural studies can provide information about long-lived states, however biological kinetics are controlled by the intervening free-energy barriers. While there has been progress describing the energy landscapes of bacterial ribosomes, very little is known about the energetics of large-scale rearrangements in eukaryotic systems. To address this topic, we constructed an all-atom model with simplified energetics and performed simulations of subunit rotation in the yeast ribosome. In these simulations, the small subunit (SSU; ∼1 MDa) undergoes spontaneous and reversible rotation events (∼8∘). By enabling the simulation of this rearrangement under equilibrium conditions, these calculations provide initial insights into the molecular factors that control dynamics in eukaryotic ribosomes. Through this, we are able to identify specific inter-subunit interactions that have a pronounced influence on the rate-limiting free-energy barrier. We also show that, as a result of changes in molecular flexibility, the thermodynamic balance between the rotated and unrotated states is temperature-dependent. This effect may be interpreted in terms of differential molecular flexibility within the rotated and unrotated states. Together, these calculations provide a foundation, upon which the field may begin to dissect the energetics of these complex molecular machines.