The eukaryotic biological clock involves a negative transcription-translation feedback loop in which clock genes regulate their own transcription and that of output genes of metabolic significance. While around 10% of the liver transcriptome is rhythmic, only about a fifth is driven by de novo transcription, indicating mRNA processing is a major circadian component. Here, we report that inhibition of transmethylation reactions elongates the circadian period. RNA sequencing then reveals methylation inhibition causes widespread changes in the transcription of the RNA processing machinery, associated with m(6)A-RNA methylation. We identify m(6)A sites on many clock gene transcripts and show that specific inhibition of m(6)A methylation by silencing of the m(6)A methylase Mettl3 is sufficient to elicit circadian period elongation and RNA processing delay. Analysis of the circadian nucleocytoplasmic distribution of clock genes Per2 and Arntl then revealed an uncoupling between steady-state pre-mRNA and cytoplasmic mRNA rhythms when m(6)A methylation is inhibited.

Inhibitors of Na+/H+ exchange proteins block macropinocytosis by lowering the pH near the plasma membrane, which in turn inhibits actin remodeling by Rho family GTPases.

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are the major anions in the large intestinal lumen. They are produced from dietary fiber by bacterial fermentation and are known to have a variety of physiological and pathophysiological effects on the intestine. In the present study, we investigated the expression of the SCFA receptor, GPR43, in the rat distal ileum and colon. Expression of GPR43 was detected by reverse transcriptase/polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. mRNA for GPR43 was detected, by RT-PCR, in extracts of the whole wall and separated mucosa from the ileum and colon and from muscle plus submucosa from the ileum, but not from muscle plus submucosa preparations from the colon. We raised a rabbit antiserum against a synthesized fragment of rat GPR43; this was specific for rat GPR43. GPR43 protein was detected by Western blot analysis in extracts of whole wall and separated mucosa, but not in muscle plus submucosa extracts. By immunohistochemistry, GPR43 immunoreactivity was localized to enteroendocrine cells expressing peptide YY (PYY), whereas 5-hydroxytryptamine (5-HT)-immunoreactive (IR) enteroendocrine cells were not immunoreactive for GPR43. Mast cells of the lamina propria expressing 5-HT were also GPR43-IR. The results of the present study suggest that the PYY-containing enteroendocrine cells and 5-HT-containing mucosal mast cells sense SCFAs via the GPR43 receptor. This is consistent with physiological data showing that SCFAs stimulate the release of PYY and 5-HT from the ileum and colon.

Figure 2. (B) Top: schematic of the structure of membrane-targeted SEpHluorin/mCherry chimaera used to measure pH sm . Bottom: confocal images of SEpHluorin (left) and mCherry fluorescence (right) in A431 cells. Bar, 10 µm.