skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Disease-associated inosine misincorporation into RNA hinders translation
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.  more » « less
Award ID(s):
2047629
PAR ID:
10371075
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ;
Publisher / Repository:
Oxford University Press
Date Published:
Journal Name:
Nucleic Acids Research
Volume:
50
Issue:
16
ISSN:
0305-1048
Page Range / eLocation ID:
p. 9306-9318
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; (, Journal of Experimental Botany)
    Bozhkov, Peter (Ed.)
    Abstract The Arabidopsis thaliana T2 family endoribonuclease RNS2 localizes to the vacuole and functions in rRNA degradation. Loss of RNS2 activity impairs rRNA turnover and leads to constitutive autophagy, a process for degradation of cellular components. Autophagy is normally activated during environmental stress and is important for stress tolerance and homeostasis. Here we show that restoration of cytosolic purine nucleotide levels rescues the constitutive autophagy phenotype of rns2-2 seedlings, whereas inhibition of purine synthesis induces autophagy in wild-type seedlings. rns2-2 seedlings have reduced activity of the target of rapamycin (TOR) kinase complex, a negative regulator of autophagy, and this phenotype is rescued by addition of inosine to increase purine levels. Activation of TOR in rns2-2 by exogenous auxin blocks the enhanced autophagy, indicating a possible involvement of the TOR signaling pathway in the activation of autophagy in the rns2-2 mutant. Our data suggest a model in which loss of rRNA degradation in rns2-2 leads to a reduction in cytoplasmic nucleotide concentrations, which in turn inhibits TOR activity, leading to activation of autophagy to restore homeostasis. 
    more » « less
  2. ; ; ; ; ; ; ; ; ; (, PLOS ONE)
    Ginsberg, Stephen D. (Ed.)
    Lysosomes play important roles in catabolism, nutrient sensing, metabolic signaling, and homeostasis. NPC1 deficiency disrupts lysosomal function by inducing cholesterol accumulation that leads to early neurodegeneration in Niemann-Pick type C (NPC) disease. Mitochondria pathology and deficits in NPC1 deficient cells are associated with impaired lysosomal proteolysis and metabolic signaling. It is thought that activation of the transcription factor TFEB, an inducer of lysosome biogenesis, restores lysosomal-autophagy activity in lysosomal storage disorders. Here, we investigated the effect of trehalose, a TFEB activator, in the mitochondria pathology of NPC1 mutant fibroblastsin vitroand in mouse developmental Purkinje cellsex vivo. We found that in NPC1 mutant fibroblasts, serum starvation or/and trehalose treatment, both activators of TFEB, reversed mitochondria fragmentation to a more tubular mitochondrion. Trehalose treatment also decreased the accumulation of Filipin+cholesterol in NPC1 mutant fibroblasts. However, trehalose treatment in cerebellar organotypic slices (COSCs) from wild-type andNpc1nmf164mice caused mitochondria fragmentation and lack of dendritic growth and degeneration in developmental Purkinje cells. Our data suggest, that although trehalose successfully restores mitochondria length and decreases cholesterol accumulation in NPC1 mutant fibroblasts, in COSCs, Purkinje cells mitochondria and dendritic growth are negatively affected possibly through the overactivation of the TFEB-lysosomal-autophagy pathway. 
    more » « less
  3. ; ; ; ; (, Nucleic Acids Research)
    Abstract Circular RNAs (circRNAs) are covalently closed RNAs that are present in all eukaryotes tested. Recent RNA sequencing (RNA-seq) analyses indicate that although generally less abundant than messenger RNAs (mRNAs), over 1.8 million circRNA isoforms exist in humans, much more than the number of currently known mRNA isoforms. Most circRNAs are generated through backsplicing that depends on pre-mRNA structures, which are influenced by intronic elements, for example, primate-specific Alu elements, leading to species-specific circRNAs. CircRNAs are mostly cytosolic, stable and some were shown to influence cells by sequestering miRNAs and RNA-binding proteins. We review the increasing evidence that circRNAs are translated into proteins using several cap-independent translational mechanisms, that include internal ribosomal entry sites, N6-methyladenosine RNA modification, adenosine to inosine RNA editing and interaction with the eIF4A3 component of the exon junction complex. CircRNAs are translated under conditions that favor cap-independent translation, notably in cancer and generate proteins that are shorter than mRNA-encoded proteins, which can acquire new functions relevant in diseases. 
    more » « less
  4. ; ; ; ; ; (, Science Advances)
    In animal models,Nipbldeficiency phenocopies gene expression changes and birth defects seen in Cornelia de Lange syndrome, the most common cause of which isNipblhaploinsufficiency. Previous studies inNipbl+/−mice suggested that heart development is abnormal as soon as cardiogenic tissue is formed. To investigate this, we performed single-cell RNA sequencing on wild-type andNipbl+/−mouse embryos at gastrulation and early cardiac crescent stages.Nipbl+/−embryos had fewer mesoderm cells than wild-type and altered proportions of mesodermal cell subpopulations. These findings were associated with underexpression of genes implicated in driving specific mesodermal lineages. In addition,Nanogwas found to be overexpressed in all germ layers, and many gene expression changes observed inNipbl+/−embryos could be attributed toNanogoverexpression. These findings establish a link betweenNipbldeficiency,Nanogoverexpression, and gene expression dysregulation/lineage misallocation, which ultimately manifest as birth defects inNipbl+/−animals and Cornelia de Lange syndrome. 
    more » « less
  5. (, Stem cell and regenerative medicine)
    null (Ed.)
    The present study explores an RNA we have discovered in human heart that induces differentiation of mouse embryonic stem cells and human induced pluripotent stem cells into cardiomyocytes in vitro. We have designated this RNA as Cardiac Inducing RNA or CIR. We now find that CIR also induces mouse embryonic fibroblasts (MEF) to form cardiomyocytes in vitro. For these studies, human-derived CIR is transfected into MEF using lipofectamine. The CIR-transfected mouse fibroblasts exhibit spindle-shaped cells, characteristic of myocardial cells in culture, and express cardiac-specific troponin-T and cardiac tropomyosin. As such, the CIR-induced conversion of the fibroblasts into cardiomyocytes in vitro appears to take place without initial dedifferentiation into pluripotent stem cells. Instead, after CIR transfection using a lipofectamine transfection system, over the next 8 days there appears to be a direct transdifferentiation of ˃80% of the cultured fibroblasts into definitive cardiomyocytes. Fewer than ˂7% of the untreated controls using non-active RNA or lipofectamine by itself show cardiomyocyte characteristics. Thus, discovery of CIR may hold significant potential for future use in repair/regeneration of damaged myocardial tissue in humans after myocardial infarction or other disease processes such that affected patients may be able to return to pre-heart-disease activity levels. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email