Enhanced ac4C detection in RNA via chemical reduction and cDNA synthesis with modified dNTPs.

The functional analysis of epitranscriptomic modifications in RNA is constrained by a lack of methods that accurately capture their locations and levels. We previously demonstrated that the RNA modification N4-acetylcytidine (ac4C) can be mapped at base resolution through sodium borohydride reduction to tetrahydroacetylcytidine (tetrahydro-ac4C), followed by cDNA synthesis to misincorporate adenosine opposite reduced ac4C sites, culminating in C:T mismatches at acetylated cytidines (RedaC:T). However, this process is relatively inefficient, resulting in <20% C:T mismatches at a fully modified ac4C site in 18S rRNA.

Detection of ac4C in human mRNA is preserved upon data reassessment.

We recently reported the distribution of N4-acetylcytidine (ac4C) in HeLa mRNA at base resolution through chemical reduction and the induction of C:T mismatches in sequencing (RedaC:T-seq). Our results contradicted an earlier report from Schwartz and colleagues utilizing a similar method termed ac4C-seq. Here, we revisit both datasets and reaffirm our findings.

Compartment-specific and ELAVL1-coordinated regulation of intronic polyadenylation isoforms by doxorubicin.

Intronic polyadenylation (IPA) isoforms, which contain alternative last exons, are widely regulated in various biological processes and by many factors. However, little is known about their cytoplasmic regulation and translational status. In this study, we provide the first evidence that the genome-wide patterns of IPA isoform regulation during a biological process can be very distinct between the transcriptome and translatome, and between the nucleus and cytosol.

The multifaceted functions of the Fat mass and Obesity-associated protein (FTO) in normal and cancer cells.

The last decade has seen mRNA modification emerge as a new layer of gene expression regulation. The Fat mass and obesity-associated protein (FTO) was the first identified eraser of N6-methyladenosine (m6A) adducts, the most widespread modification in eukaryotic messenger RNA. This discovery, of a reversible and dynamic RNA modification, aided by recent technological advances in RNA mass spectrometry and sequencing has led to the birth of the field of epitranscriptomics.

FTO-mediated cytoplasmic mA demethylation adjusts stem-like properties in colorectal cancer cell.

Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N,2'-O-dimethyladenosine (mA) demethylase activity.

Antibiotics inhibit sphere-forming ability in suspension culture.

Background: This last decade, a lot of emphasis has been placed on developing new cancer cell culture models, closer to in vivo condition, in order to test new drugs and therapies. In the case of colorectal cancer, the use of patient biopsies to seed 3D primary cultures and mimic tumor initiation necessitates the use of antibiotics to prevent microbial intestinal contamination. However, not only long term use of antibiotics may mask the presence of low levels of microbial contamination, it may also impact cancer cell phenotype.