Abundant mRNA m1A modification in dinoflagellates: a new layer of gene regulation [m1A-seq-TGIRT]

Dinoflagellates, a class of unicellular eukaryotic phytoplankton, exhibit minimal transcriptional regulation, representing a unique model for exploring gene expression. The biosynthesis, distribution, regulation, and function of mRNA N1-methyladenosine (m1A) remain controversial due to its limited presence in typical eukaryotic mRNA. This study found that m1A, rather than N6-methyladenosine (m6A), is the most prevalent internal mRNA modification in various dinoflagellate species, with an asymmetrically distribution along mature transcripts. In Amphidinium carterae, we identified 6549 m1A sites characterized by a non-tRNA T-loop-like sequence motif within the transcripts of 3196 genes, many involved in regulating carbon and nitrogen metabolism. With enrichment within 3'UTR, dinoflagellate mRNA m1A exhibits negative correlation with translation efficiency. Notably, nitrogen depletion led to a significant decrease in mRNA m1A. Furthermore, our analysis revealed that distinctive patterns of m1A modification influence the expression of metabolism-related genes through translation control. Thus, this study provides a comprehensive map of m1A in dinoflagellate mRNA, highlighting its crucial role as a post-transcriptional regulatory layer and enhancing the understanding of mRNA m1A modification. Overall design: To gain further insight into the roles of m1A, we sought to characterize m1A sites at single-base resolution using m1A-seq-TGIRT. For m1A-seq-TGIRT, 25 µL of prewashed Protein A/G Dynabeads were incubated with 3 µL of anti-m1A antibody (MBL #D345-3) at 4 °C for two hours. Subsequently, 500 ng of fragmented mRNA was used for immunoprecipitation, with 50 ng of purified mRNA taken as input. After digestion with Proteinase K, the RNA was purified using RNA Extraction Reagent (Shanghai Acmec Biochemical Co., Ltd, China) and then subjected to library construction. Briefly, the purified mRNA was treated with Antarctic Phosphatase (NEB) and T4 PNK (NEB) at 37 °C for 30 minutes, followed by purification using the Oligo Clean & Concentrator kit (Zymo Research). The dephosphorylated RNA was then ligated to the 3'-RNA adaptor (/5'Phosphate/AGAUCGGAAGAGCGUCGUG/ddC) using T4 RNA Ligase I (NEB) at 23°C for two hours, purified by Oligo Clean & Concentrator again, and reverse transcribed using TGIRT-III (Haigne, China) with the RT primer (5'-ACACGACGCTCTTCCGA-3') in RT reaction buffer (50 mM Tris–HCl pH 8.3, 75 mM KCl, and 3 mM MgCl2) at 57 °C for two hours. The reaction was terminated with 25 mM EDTA and RNA was degraded with 150 mM NaOH, heated to 70 °C for 12 minutes, and purified with Oligo Clean & Concentrator kit. The 5'-DNA adaptor (/5'Phosphate/AGATCGGAAGAGCACACGTCTG/ddC) was ligated using T4 RNA ligase I (NEB) at 23 °C overnight. The single-stranded cDNA was then amplified by PCR using specific primers (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' and 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3', NNNNNN denotes the 6-bp barcode sequence), and the product was purified with AMPure XP beads (0.9×). Both input and IP samples were sequenced by GenePlus (Shenzhen, China) on DNBSEQ-T7 platform with paired-end 150 bp mode.