N6‐methyladenosine (m6A) is the most prevalent modification in mRNA and engages in multiple biological processes. Previous studies indicated that m6A methyltransferase METTL3 (‘writer’) and demethylase FTO (‘eraser’) play critical roles in heart‐related disease. However, in the heart, the function of m6A ‘reader’, such as YTH (YT521‐B homology) domain‐containing proteins remains unclear. Here, we report that the defect in YTHDC1 but not other YTH family members contributes to dilated cardiomyopathy (DCM) in mice. Cardiac‐specific conditional Ythdc1 knockout led to obvious left ventricular chamber enlargement and severe systolic dysfunction. YTHDC1 deficiency also resulted in the decrease of cardiomyocyte contractility and disordered sarcomere arrangement. By means of integrating multiple high‐throughput sequence technologies, including m6A‐MeRIP, RIP‐seq and mRNA‐seq, we identified 42 transcripts as potential downstream targets of YTHDC1. Amongst them, we found that Titin mRNA was decorated with m6A modification and depletion of YTHDC1 resulted in aberrant splicing of Titin. Our study suggests that Ythdc1 plays crucial role in regulating the normal contractile function and the development of DCM. These findings clarify the essential role of m6A reader in cardiac biofunction and provide a novel potential target for the treatment of DCM.
As an aberrant base
in DNA, uracil is generated by either deoxyuridine
(dU) misincorporation or cytosine deamination, and involved in multiple
physiological and pathological processes. Genome-wide profiles of
uracil are important for study of these processes. Current methods
for whole-genome mapping of uracil all rely on uracil-DNA N-glycosylase
(UNG) and are limited in resolution, specificity, and/or sensitivity.
Here, we developed a UdgX cross-linking and polymerase stalling sequencing
(“Ucaps-seq”) method to detect dU at single-nucleotide
resolution. First, the specificity of Ucaps-seq was confirmed on synthetic
DNA. Then the effectiveness of the approach was verified on two genomes
from different sources. Ucaps-seq not only identified the enrichment
of dU at dT sites in pemetrexed-treated cancer cells with globally
elevated uracil but also detected dU at dC sites within the “WRC”
motif in activated B cells which have increased dU in specific regions.
Finally, Ucaps-seq was utilized to detect dU introduced by the cytosine
base editor (nCas9-APOBEC) and identified a novel off-target site
in cellular context. In conclusion, Ucaps-seq is a powerful tool with
many potential applications, especially in evaluation of base editing
fidelity.
Nucleoporins (Nups) are known to be functional in nucleo‐cytoplasmic transport, but the roles of nucleoporins in nonproliferating cells, such as cardiac myocytes, are still poorly understood. In this study, we report that Nup107 regulates cardiac bioelectricity by controlling the nucleo‐cytoplasmic trafficking of Scn5a
mRNA. Overexpression of Nup107 induced the protein expression of Scn5a rather than that of other ion channels, with no effects of their mRNA levels. The analysis for the protein production demonstrated Nup107‐facilitated transport of Scn5a
mRNA. Using RIP‐PCR and luciferase assay, we found that the 5′‐UTR of Scn5a
mRNA was not involved in the interaction, whereas the spatial interaction between Nup107 protein and Scn5a
mRNA was formed when Scn5a
mRNA passing through the nuclear pore. Functionally, Nup107 overexpression in neonatal rat ventricle myocytes significantly increased the currents of Scn5a‐encoded INa channel. Moreover, the close correlation between Nup107 and Nav1.5 protein expression was observed in cardiomycytes and heart tissues subjected to hypoxia and ischaemic insults, suggesting a fast regulation of Nup107 on Nav1.5 channel in cardiac myocytes in a posttranscriptional manner. These findings may provide insights into the emergent control of cardiac electrophysiology through Nup‐mediated modulation of ion channels.
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