Photo-Facilitated Detection and Sequencing of 5-Formylcytidine RNA.

5-Formylcytidine (f5 C) is one of the epigenetic nucleotides in tRNA. Despite the evident importance of f5 C in gene expression regulation, little is known about its exact amount and position. To capture this information, we developed a modification-specific functionalization with a semi-stabilized ylide. The chemical labelling exhibited a high selectivity towards f5 C and allowed distinction from similar 5-formyluridine. We realized a detection strategy based on the fluorescence signal of the cyclization product 4,5-pyridin-2-amine-cytidine paC, which exhibited a high quantum yield. The results clearly identified f5 C with a limit of detection at 0.58 nM. This method altered the hydrogen bonding activities of f5 C and modulated its reverse transcription signature in its sequencing profile. We showed that f5 C can be detected from tRNA segments with a single-base resolution. Taken together, this approach is a sensitive, antibody-free, and applicable detection and sequencing method for f5 C-containing RNA.

Alternative splicing (AS) mechanism plays important roles in response to different salinity environments in spotted sea bass.

Salinity represents a critical environmental factor for fishes, and it can directly influence their survival. Transcriptomic analysis at the gene expression level has been extensively conducted to identify functional genes or pathways involved in salinity adaptation in numerous euryhaline fishes. However, the post-transcriptional regulation mechanism in response to salinity changes remains largely unknown. Alternative splicing (AS), the main mechanism accounting for the complexity of the transcriptome and proteome in eukaryotes, plays essential roles in determining organismal responses to environmental changes. In this study, RNA-Seq datasets were used to examine the AS profiles in spotted sea bass (Lateolabrax maculatus), a typical euryhaline fish species. The results showed that 8618 AS events were identified in spotted sea bass. Furthermore, a total of 501 and 162 differential alternative splicing (DAS) events were characterized in the gill and liver under low- and high-salinity environments, respectively. Based on GO enrichment results, DAS genes in both the gill and liver were commonly enriched in 8 GO terms, and their biological functions were implicated in many stages of gene expression regulation, including transcriptional regulation and post-transcriptional regulation. Sanger sequencing and qPCR validations provided additional evidence to ensure the accuracy and reliability of our bioinformatic results. This is the first comprehensive view of AS in response to salinity changes in fish species, providing insights into the post-regulatory molecular mechanisms of euryhaline fishes in salinity adaptation.