Identification of post-transcriptional modifications in honey bees (Apis mellifera) using direct RNA sequencing

Post-transcriptional modifications (PTM) play crucial roles in gene regulation. Among these, the N6-methyladenosine (m⁶A) and Adenosine-to-Inosine (A-to-I) RNA editing are the most prevalent across eukaryotes. In this context, honey bees are the particular interest for their eusociality, since these undergo a division of labor, which is triggered by different gene regulatory mechanisms such as PTM. This study focused on profiling m⁶A modifications and RNA editing sites in honey bees (Apis mellifera) using Oxford Nanopore Sequencing. A novel technology that can sequence and detect PTM by measuring the ionic current disruptions that each nucleotide produces while it is translocated through pores of nanometric scale. Regarding m⁶A, we detected 237 and 184 in the head and thorax tissues, respectively. These were mainly located in the coding sequence of transcripts and occurred at an overall frequency of one per transcript. Gene ontology analysis revealed that m⁶A is placed in genes that modulate crucial biological processes, including protein transport, autophagy, nitrogen metabolism, and many more. Additionally, for its ubiquitous role in the central nervous system, we explored the A-to-I RNA editome of bee heads. Overall, 165 inosines were identified, predominantly located in coding sequences. For its ability to generate mutations, we determined the percentage of non-synonymous mutations (53.9%) triggered by RNA editing. Originally, these sites codified 11 amino acids, which mutated into nine different residues as a product of A-to-I editing. Gene ontology analysis revealed that edited genes play an essential role in brain functions, such as synaptic transmission and related processes. Even though m⁶A and A-to-I have been identified previously using short-read sequencing approaches, this is one of the first studies that has applied direct RNA sequencing to detect post-transcriptional modifications. Through this study, we are pioneering the utilization of direct RNA sequencing to elucidate the functional roles of m⁶A and A-to-I not only in pollinators but also in multiple biological systems.