N6,2′-O-dimethyladenosine (m⁶Am) is a post-transcriptional modification that may be associated with regulatory roles in the control of cellular functions. Therefore, it is crucial to accurately identify transcriptome-wide m⁶Am sites to understand underlying m⁶Am-dependent mRNA regulation mechanisms and biological functions. Here, we used three sequence-based feature-encoding schemes, including one-hot, nucleotide chemical property (NCP), and nucleotide density (ND), to represent RNA sequence samples. Additionally, we proposed an ensemble deep learning framework, named DLm6Am, to identify m⁶Am sites. DLm6Am consists of three similar base classifiers, each of which contains a multi-head attention module, an embedding module with two parallel deep learning sub-modules, a convolutional neural network (CNN) and a Bi-directional long short-term memory (BiLSTM), and a prediction module. To demonstrate the superior performance of our model’s architecture, we compared multiple model frameworks with our method by analyzing the training data and independent testing data. Additionally, we compared our model with the existing state-of-the-art computational methods, m6AmPred and MultiRM. The accuracy (ACC) for the DLm6Am model was improved by 6.45% and 8.42% compared to that of m6AmPred and MultiRM on independent testing data, respectively, while the area under receiver operating characteristic curve (AUROC) for the DLm6Am model was increased by 4.28% and 5.75%, respectively. All the results indicate that DLm6Am achieved the best prediction performance in terms of ACC, Matthews correlation coefficient (MCC), AUROC, and the area under precision and recall curves (AUPR). To further assess the generalization performance of our proposed model, we implemented chromosome-level leave-out cross-validation, and found that the obtained AUROC values were greater than 0.83, indicating that our proposed method is robust and can accurately predict m⁶Am sites.

N6-methyladenosine (m⁶A) is the most abundant within eukaryotic messenger RNA modification, which plays an essential regulatory role in the control of cellular functions and gene expression. However, it remains an outstanding challenge to detect mRNA m⁶A transcriptome-wide at base resolution via experimental approaches, which are generally time-consuming and expensive. Developing computational methods is a good strategy for accurate in silico detection of m⁶A modification sites from the large amount of RNA sequence data. Unfortunately, the existing computational models are usually only for m⁶A site prediction in a single species, without considering the tissue level of species, while most of them are constructed based on low-confidence level data generated by an m⁶A antibody immunoprecipitation (IP)-based sequencing method, thereby restricting reliability and generalizability of proposed models. Here, we review recent advances in computational prediction of m⁶A sites and construct a new computational approach named im6APred using ensemble deep learning to accurately identify m⁶A sites based on high-confidence level data in multiple tissues of mammals. Our model im6APred builds upon a comprehensive evaluation of multiple classification methods, including four traditional classification algorithms and three deep learning methods and their ensembles. The optimal base–classifier combinations are then chosen by five-fold cross-validation test to achieve an effective stacked model. Our model im6APred can produce the area under the receiver operating characteristic curve (AUROC) in the range of 0.82–0.91 on independent tests, indicating that our model has the ability to learn general methylation rules on RNA bases and generalize to m⁶A transcriptome-wide identification. Moreover, AUROCs in the range of 0.77–0.96 were achieved using cross-species/tissues validation on the benchmark dataset, demonstrating differences in predictive performance at the tissue level and the need for constructing tissue-specific models for m⁶A site prediction.