Inhibition of cytoplasmic cap methylation identifies 5’ TOP mRNAs as recapping targets and reveals recapping sites downstream of native 5’ ends

Cap homeostasis is the cyclical process of decapping and recapping that maintains the translation and stability of a subset of the transcriptome. Previous work showed levels of some recapping targets decline following transient expression of an inactive form of RNMT (ΔN-RNMT), likely due to degradation of mRNAs with improperly methylated caps. The current study examined transcriptome-wide changes following inhibition of cytoplasmic cap methylation. This identified mRNAs with 5’-terminal oligopyrimidine (TOP) sequences as the largest single class of recapping targets. Cap end mapping of several TOP mRNAs identified recapping events at native 5’ ends and downstream of the TOP sequence of EIF3K and EIF3D. This provides the first direct evidence for downstream recapping. Inhibition of cytoplasmic cap methylation was also associated with mRNA abundance increases for a number of transcription, splicing, and 3’ processing factors. Previous work suggested a role for alternative polyadenylation in target selection, but this proved not to be the case. However, inhibition of cytoplasmic cap methylation resulted in a shift of upstream polyadenylation sites to annotated 3’ ends. Together, these results solidify cap homeostasis as a fundamental process of gene expression control and show cytoplasmic recapping can impact regulatory elements present at the ends of mRNA molecules. Human U2OS osteosarcoma cells stably expressing the tetracycline repressor (U2OS-TR) were described previously in (Otsuka et al., 2009). To generate cells with tetracycline-inducible ΔN-RNMT stably integrated, U2OS-TR cells were transfected with pcDNA3/TO-ΔN-RNMT. U2OS-TR/ΔN-RNMT (n=10) and parental U2OS-TR (n=6) were grown in 10cm dishes. After 48 hr half of the samples were induced with doxycycline. 24 hours later the cells were lysed, RNA extracted , and QuantSeq REV 3' mRNA-Seq libraries prepared. Induced (+doxycycline) vs uninduced (-doxycycline) gene expression was separately compared for the U2OS-TR/ΔN-RNMT and parental U2OS-TR cell lines. See also BioProject PRJNA547607 and SRA project SRP201120.