Main

RNA is a critical molecule in the central dogma of molecular biology, which describes the flow of genetic information from DNA to RNA to protein.

RNA molecules play a crucial role in various cellular processes, including gene expression, regulation and catalysis. Given the importance of RNA in biological systems, there is a growing demand for efficient and accurate methods to analyse RNA sequences. The analysis of RNA sequences has traditionally been performed using experimental techniques such as RNA sequencing and microarrays1,2. However, these methods are often expensive and time-consuming and require large amounts of input RNA. In recent years, there has been increasing interest in using computational methods based on machine learning models to analyse RNA sequences.

Pretrained language models, on the other hand, have shown great success in various natural language processing tasks, including text classification3, question answering4 and language translation5. Advancements in the field of natural language processing have led to the successful adoption of pretrained language models like BERT6 to model and analyse nucleotides (nts) and ribonucleotides from trillions of DNA/RNA sequences. For example, preMLI7 employs rna2vec to produce RNA word vector representations. The RNA sequence features are then mined independently, and the two feature vectors are concatenated as the input for the prediction task. DNABERT8 has been proposed to extract features from DNA sequences via the pretrained language model BERT-alike, and its derivatives9,10 with task-agnostic extensions have been studied to solve DNA analytical tasks in an ad hoc manner11. Moreover, based on T5 (ref. 12), Rm-LR13 integrates two large-scale RNA language pretrained models to learn local key features and collect discriminative sequential information. A bilinear attention network is then used to integrate the learned features. However, there is still some work focusing on generic models that performs well on varying downstream tasks derived from one set of pretrained weights. RNA-FM14 trains a foundation model for the community to fit all the ncRNA sequences, although it only uses naive token masking as a pretraining strategy, which may lose high-density information hidden in continuous RNA subsequences. This problem is further compounded by the fact that RNA is a more complex molecule than DNA15, due to the presence of additional modifications and higher-order structures, and existing pretrained models are not optimized for RNA analysis.

In response to this challenge, we have developed a pretrained RNA language model: RNAErnie. As shown in Fig. 1, this model is built upon the Enhanced Representation through Knowledge Integration (ERNIE) framework and incorporates multilayer and multihead transformer blocks, each having a hidden state dimension of 768. Pretraining is conducted using an extensive corpus consisting of approximately 23 million RNA sequences meticulously curated from RNAcentral16. The proposed motif-aware pretraining strategy involves base-level masking, subsequence-level masking and motif-level random masking, which effectively captures both subsequence and motif-level knowledge17,18,19, enriching the representation of RNA sequences as illustrated in Fig. 2a. Additionally, RNAErnie tokenizes coarse-grained RNA types as special vocabularies and appends the tokens of coarse-grained RNA types at the end of every RNA sequence during pretraining. By doing so, the model gains the potential to discern the distinct characteristics of various RNA types, facilitating domain adaption to various downstream tasks.

Fig. 1: Overview of the design of the proposed model and its applications.
figure 1

The RNAErnie model consists of 12 transformer layers. In the motif-aware pretraining phase, RNAErnie is trained on a dataset of approximately 23 million sequences extracted from the RNAcentral database using self-supervised learning with motif-aware multilevel random masking. In the type-guided fine-tuning phase, RNAErnie first predicts the possible coarse-grained RNA types using output embeddings and then leverages the predicted types as auxiliary information for fine-tuning the model with task-specific heads. w/, with; w/o, without; MLM, masked language modelling; norm., normalization.

Source data

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Fig. 2: Motif-aware pretraining and type-guided fine-tuning strategies.
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a, Motif-aware multilevel masking strategy for RNAErnie pretraining. Built upon ERNIE transformer blocks, the design incorporates three levels of masking: base, subsequence and motif. All masking levels are applied during the pretraining phase. b, Type-guided fine-tuning for downstream tasks. The RNAErnie approach first leverages an RNAErnie basic block to predict the top-K most possible coarse-grained RNA types. Then, it stacks an additional layer of K downstream modules with shared parameters for fine-tuning and outputs the ensemble result for tasks.

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Specifically, a type-guided fine-tuning strategy is employed, incorporating the predicted RNA types as ‘auxiliary information’ within a stacking architecture, as shown in Fig. 2b. Upon receiving an RNA sequence as input, the model first employs a pretrained RNAErnie block to generate output embeddings. Subsequently, it predicts the potential coarse-grained RNA types based on these embeddings. The sequence and the predicted RNA types are then fed into a downstream network, which consists of RNAErnie blocks and task-specific heads. This approach enables the model to accommodate a diverse range of RNA types and enhances its utility in a broad spectrum of RNA analytical tasks. More specifically, to adapt the distribution shifts between pretraining datasets and target domains, RNAErnie leverages domain adaptation20 that composites the pretrained backbone with downstream modules in three neural architectures: frozen backbone with trainable head (FBTH), trainable backbone with trainable head (TBTH) and stacking for type-guided fine-tuning (STACK). In this way, the proposed method can either end-to-end optimize the backbone and task-specific heads or fine-tune task-specific heads with embeddings extracted from the frozen backbone, subject to the downstream applications.

The conducted experiments highlight the immense potential of RNAErnie in advancing RNA analysis. The model demonstrates strong performance across diverse downstream tasks, showcasing its versatility and effectiveness as a generic solution. Additionally, the innovative strategies employed in RNAErnie show promise in enhancing the performance of other pretrained models in RNA analysis. These findings position RNAErnie as a valuable asset, empowering researchers with a powerful tool to unravel the complexities of RNA-related investigations.

Results

In this section, we present the experiment results for RNAErnie evaluation on both unsupervised learning (RNA grouping) and supervised learning (RNA sequence classification, RNA–RNA interaction prediction and RNA secondary structure prediction) tasks. For additional experiment settings and results (such as long-sequence classification, SARS-CoV-2 variant evolutionary path visualization and so on), please refer to Supplementary Information Section C.

Unsupervised clustering of RNAErnie-extracted features

Various types of RNA exhibit distinct functions and structures, and it is expected that these characteristics are captured within the embeddings generated by our proposed model (RNAErnie) using raw RNA sequences. To examine the patterns within the known RNA repertoire, we utilize the suggested encoder to establish scatter plots of RNA sequences. Dimension reduction using PHATE21 is then employed to map the embeddings onto a two-dimensional plane. We evaluate the impact of the learning process by considering both pretrained and randomly initialized RNAErnie embeddings, as well as 3mer statistical embeddings22 for visualization.

Figure 3a shows the results, where the pretrained RNAErnie embedding space effectively organizes RNA types into distinct clusters based on their structural and functional properties. We also use a random model for comparing encoding effects, establishing a baseline for comparison with other encoding methods. This comparison allows us to evaluate the effectiveness of each method in enhancing the encoding process. The random model exhibits a less-defined clustering structure, and the 3mer embeddings lack distinguishable features. This indicates that RNAErnie captures structural and functional information beyond the primary structure of RNA, enabling grouping based on similar properties. To investigate the diversity of non-coding RNAs (ncRNAs), we categorize them using sequence ontology at various levels. Figure 3b illustrates selected classes of ncRNA, such as ribosomal RNA (rRNA), long ncRNA (lncRNA) and small ncRNA (sncRNA). Figure 3c shows the high-level ontology relationships between ncRNA, transcript, messenger RNA (mRNA) and intron RNA. Figure 3d represents the low-level ontology of small regulatory ncRNA. RNAErnie effectively discriminates between classes at different ontology levels, while the 3mer statistical embeddings struggle to separate them. This suggests that RNAErnie captures structural or functional similarities rather than relying solely on the length of ncRNAs. Note that the random approach seems to outperform RNAErnie in differentiating between classes across various ontology levels. This finding suggests that RNAErnie might be less effective in capturing the ontology patterns of low-level, small regulatory ncRNA classes. We believe that this limitation in identifying low-level ontology patterns may stem from several factors, including the complexity and heterogeneity of classes at this level or potential biases in our training dataset. Further research and detailed analysis are needed to identify the specific causes behind RNAErnie’s reduced efficacy in discerning patterns in low-level ontology.

Fig. 3: RNAErnie captures multilevel ontology patterns.
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Left, RNAErnie embeddings. Middle, randomly initialized embeddings. Right, 3mer statistical embeddings. a, Scatter plot showcasing all ncRNA types within a subset of RNAcentral, utilizing different embedding methods. b, Embedding projections of selected ncRNA classes, including rRNA, lncRNA, RNase P RNA, ribozyme, sncRNA, signal recognition particle RNA (SRP RNA), antisense RNA and Y RNA. c, Distribution of embeddings based on high-level sequence ontology, encompassing ncRNA, transcript, mRNA and intron RNA. d, Detailed distribution of embeddings for low-level small regulatory ncRNA classes, such as Piwi-interacting RNA (piRNA), miRNA, transfer-messenger RNA (tmRNA) and small interfering RNA (siRNA).

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In total, these findings demonstrate that RNAErnie constructs scatter plots by capturing the structural and functional characteristics of ncRNAs, going beyond nucleic acid statistics alone.

Supervised domain adaptation on downstream tasks

In this section, we demonstrate the effectiveness of RNAErnie in three essential supervised learning tasks: RNA sequence classification, RNA–RNA interaction and RNA secondary structure prediction.

To reveal the effectiveness of the designs in RNAErnie, we conducted a series of ablation studies using variant models derived from RNAErnie. These models vary in complexity, beginning with Ernie-base, which lacks RNA-specific pretraining and includes standard fine-tuning. RNAErnie−− employs base-level masking during pretraining, and RNAErnie− adds subsequence-level masking to the mix. The complete RNAErnie model further integrates motif-level masking and is fine-tuned using either TBTH or FBTH architectures. Extending this, RNAErnie+ represents the apogee of complexity within this family, including all three levels of masking and a STACK architecture for pretraining. Lastly, the RNAErnie without chunk model is tailored for long RNA sequences by truncating and discarding segments to contend with computational constraints, aimed at the efficient classification of long non-coding and protein-encoding transcripts.

In addition, we also bring pretrained models from existing literature, including RNABERT23, RNA-MSM24 and RNA-FM14 for comparison.

RNA sequence classification

We evaluate the performance of our proposed sequence-classification models on the benchmark nRC25. This dataset consists of ncRNA sequences selected from the Rfam database release 12 (ref. 26). nRC is composed of a balanced collection of sequences, with 20% non-redundant samples for each of the 13 classes. It has 6,320 training sequences and 2,600 testing sequences labelled with 13 classes.

Table 1 presents the sequence-classification results for RNAErnie on the nRC dataset. The table includes several baseline methods as well as different variants of the RNAErnie models. The baseline values are all taken from cited literature except the pretrained models: RNABERT, RNA-MSM and RNA-FM. Analysing the performance of the models, we observe that the baseline methods achieve varying levels of accuracy. Notably, ncRDense demonstrates decent performance, achieving high accuracy, recall, precision, F1 score and Matthews correlation coefficient (MCC) values. Turning our attention to the RNAErnie variants, we can see that they consistently outperform most of the baseline models across all evaluation metrics. Although ncRDense can beat the first two (that is, Ernie-base and RNAErnie−−), RNAErnie−, RNAErnie and RNAErnie+ show better performance in all five dimensions.

Table 1 Performance of RNAErnie on sequence classification for the nRC dataset
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In the hierarchy of the RNAErnie model family, performance metrics improve incrementally with complexity of design. The foundational model, Ernie-base, establishes a baseline that is modestly surpassed by RNAErnie−− through the introduction of base-level masking in pretraining. Furthermore, RNAErnie− incorporates subsequence-level masking and delivers notably enhanced accuracy, recall, precision, F1 score and MCC values, endorsing the value of a more comprehensive masking strategy. The full RNAErnie model integrates base, subsequence and motif-level masking, achieving superior performance over its predecessors across all metrics and illustrating the cumulative benefits of multilevel masking. The apex model, RNAErnie+, which employs an exhaustive masking regimen in conjunction with a two-stage fine-tuning architecture, outperforms all variants in our experiments.

RNA–RNA interaction

We evaluate the performance of our model on one of the most representative benchmark datasets, DeepMirTar27,28, which is used for predicting the interaction between microRNAs (miRNAs) and mRNAs. This dataset consists of 13,860 positive pairs and 13,860 negative pairs. The miRNA sequences in DeepMirTar are all shorter than 26 nts, and the mRNA sequences are shorter than 53 nts. Because most of the target sites are believed to be located at the 3′ untranslated region, DeepMirTar only considers them. Furthermore, two seeds were taken into consideration: the non-canonical seed, which pairs at position 2-7 or 3-8, permitting G-U couplings and up to one bulged or mismatched nt; and the canonical seed, which is the precise W-C pairing of 2-7 or 3-8 nts of the miRNA. Given that RNA types (miRNA, mRNA) are fixed here, we do not test RNAErnie+ version which uses a two-stage pipeline here.

Table 2 presents the performance comparison between the proposed RNAErnie models and baseline methods from existing literature, such as Miranda29, RNAhybrid30, PITA31, TargetScan v.7.0 (ref. 32), TarPmiR33 and DeepMirTar27. The baseline values are all taken from cited literature except the pretrained models: RNABERT, RNA-MSM and RNA-FM. These are evaluated on the RNA–RNA interaction prediction task using the DeepMirTar dataset. DeepMirTar emerges as a strong baseline, exhibiting high scores across all metrics. The Ernie-base model and the RNAErnie variations are then assessed, with the RNAErnie model demonstrating superior performance and particularly excelling in accuracy, precision, F1 score and area under the curve (AUC). This variation achieves an impressive accuracy score of 0.9872, a competitive precision score of 0.9901, an F1 score of 0.9873 and the highest AUC score of 0.9976, indicating excellent overall performance and discriminative power.

Table 2 Performance of RNAErnie on RNA–RNA interaction prediction task using the DeepMirTar dataset
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Overall, the results suggest that the RNAErnie model, particularly the RNAErnie variation, outperforms the existing methods and the Ernie-base model in the RNA–RNA interaction prediction task. These findings highlight the potential of the RNAErnie model in accurately predicting RNA–RNA interactions.

RNA secondary structure prediction

This section presents a comprehensive comparison between our pretrained RNAErnie model and several baseline models, including the state-of-the-art UFold model34, in the context of RNA secondary structure prediction tasks. The experiments are conducted using commonly used benchmarks employed in state-of-the-art models. These benchmarks include:

  • RNAStralign35: This dataset comprises 37,149 RNA structures from eight RNA families, with lengths ranging from approximately 100 to 3,000 base pairs (bp).

  • ArchiveII36: This dataset consists of 3,975 RNA structures from ten RNA families, with lengths ranging from approximately 100 to 2,000 bp.

  • bpRNA-1m37: This dataset contains 13,419 RNA structures from 2,588 RNA families, with sequence similarity removed using an 80% sequence-identity cut-off. The lengths of the sequences range from approximately 100 to 500 bp. The dataset is randomly split into three subsets: TR0 (10,814 structures) for training, TV0 (1,300 structures) for validation and TS0 (1,305 structures) for testing.

We train our model on the entire RNAStralign dataset, as well as the TR0 subset and other augmented mutated datasets, following the approach used in UFold. Subsequently, we evaluate performance on the ArchiveII600 dataset, which is a subset of ArchiveII with lengths less than 600 bp, and the TS0 dataset.

Table 3 presents a comparative analysis of the performance of various methods on the RNA secondary structure prediction task using the ArchiveII and TS0 datasets. The table presents the results of several baseline methods, including RNAstructure, RNAsoft, RNAfold, MXfold2, Mfold, LinearFold, Eternafold, E2Efold, Contrafold and Contextfold. Each method is assessed based on its precision, recall and F1 score for both the ArchiveII600 and TS0 datasets. The baseline values are all taken from cited literature except the pretrained models: RNABERT, RNA-MSM and RNA-FM. Among the RNAErnie variations, RNAErnie+ achieves the highest scores in precision, recall and F1 score, indicating its superior performance in RNA secondary structure prediction. Notably, RNAErnie+ achieves a remarkable precision score of 0.886, a high recall score of 0.870 and an impressive F1 score of 0.875 on the ArchiveII600 dataset. These results highlight the effectiveness of RNAErnie+ in accurately predicting RNA secondary structures.

Table 3 Performance of RNAErnie on RNA secondary structure prediction task using the ArchiveII600 and TS0 datasets
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Discussion

Our method, RNAErnie, outperforms existing advanced techniques across seven RNA sequence datasets encompassing over 17,000 major RNA motifs, 20 RNA classes/types and 50,000 RNA sequences. Evaluation using 30 mainstream RNA sequence technologies confirms the generalization and robustness of RNAErnie. We employed accuracy, precision, recall, F1 score, MCC and AUC as evaluation metrics to ensure a fair comparison of RNA sequence-analysis methods. Currently, little research exists on applying transformer architectures with enhanced external knowledge to RNA sequence data analysis. Our from-scratch RNAErnie framework integrates RNA sequence embedding and a self-supervised learning strategy, resulting in superior performance, interpretability and generalization potential for downstream RNA tasks. Additionally, RNAErnie is adaptable to other tasks through modification of the output and supervision signals. RNAErnie is publicly available and serves as an effective tool for understanding type-guided RNA analysis and advanced applications.

The RNAErnie model, despite its innovations in RNA sequence analysis, confronts several challenges. First, the model is constrained by the size of the RNA sequences it can analyse, as sequences longer than 512 nts are dropped, potentially omitting vital structural and functional information. The chunking method developed to handle longer sequences might result in the further loss of information about long-range interactions. Second, the focus of this study is narrow, centred only on the RNA domain and not extending to tasks like RNA-protein prediction or binding-site identification. Additionally, the model encounters difficulties in considering three-dimensional structural motifs of RNAs, such as loops and junctions, which are essential for understanding RNA functions.

More importantly, the existing post hoc architectural design has potential limitations, including heightened inference overhead. An alternative approach involves designing a specialized loss function that incorporates RNA type information and pretraining the model in an end-to-end fashion. We have experimented with this concept and engaged in preliminary pretraining. Our findings indicate that although this method proves beneficial for discriminative tasks such as sequence classification, it unfortunately leads to suboptimal token representations with performance degradation in reconstruction of structures. Detailed information is provided Supplementary Information Section C.6. Our future work will go deeper into this issue and explore solutions.

Methods

This section provides a comprehensive overview of the design features associated with each component of RNAErnie. We will explore the specific characteristics of each element and discuss their collaborative functionality in enabling the accomplishment of diverse downstream tasks.

Overall design

In this work, we present RNAErnie, an approach for large-scale pretraining of RNA sequences based on the ERNIE framework38, which incorporates multilayer and multihead transformer blocks39.

RNAErnie transformer

The basic block of the RNAErnie transformer shares the same architectural configuration as ERNIE38, employing a 12-layer transformer and a hidden state dimension of Dh = 768. Consider an input RNA sequence denoted as x = (x1, x2, ⋯ , xL), where each element xi ∈ {‘A’, ‘U’, ‘C’, ‘G’} and L represents the length of the sequence. An RNAErnie block first tokenizes RNA bases in the sequence and subsequently feeds them into the transformer. This process enables us to extract token embeddings \({{{\bf{h}}}}=({h}_{1},{h}_{2},\cdots \,,{h}_{L})\in {{\mathbb{R}}}^{L\times {D}_{{\mathrm{h}}}}\), where Dh represents the dimension of the hidden representations for the tokens. Given the embeddings for every token in the RNA sequence, the RNAErnie basic block transforms the series of token embeddings into a lower-dimensional vector (that is, 768 dimensions) using trainable parameters38 and then outputs the embedding of the RNA sequence. The total number of trainable parameters in RNAErnie is approximately 105 million.

Pretraining datasets

Basically, like many other pretraining based approaches, the RNAErnie approach is structured into two main phases: pretraining and fine-tuning. In the pretraining phase, which is agnostic to any specific task, RNAErnie is meticulously trained on a vast corpus of 23 million ncRNA sequences obtained from the RNAcentral database16. This self-supervised autoregressive training phase allows RNAErnie to capture sequential distributions and patterns within the RNA sequences, thereby acquiring a comprehensive understanding of their structural and functional information. In the subsequent task-specific fine-tuning phase, the pretrained RNAErnie model is either fine-tuned with downstream modules or used to generate sequence embeddings (features) that complement a lightweight prediction layer. Regarding the tokenization of RNA bases, the sequences are tokenized to represent ‘A’, ‘T/U’, ‘C’ and ‘G’, with the initial token of each sequence reserved for the special classification embedding ([CLS]). Additionally, an indication embedding ([IND]) is appended to each RNA sequence, followed by indication classes (for example, ‘miRNA’, ‘mRNA’, ‘lnRNA’) derived from the RNAcentral database, as depicted in Extended Data Fig. 1. The inclusion of the indication embedding encourages the model to cluster similar RNA sequences in a latent space, facilitating retrieval-based learning40.

Motif-aware pretraining strategies

To integrate both subsequence and motif-level knowledge into the representation of RNA sequences, we introduce a motif-ware multilevel masking strategy to pretrain the RNAErnie basic block, as opposed to directly incorporating motif embedding. In addition, the RNAErnie approach follows the standard routine of pretraining with all three levels of masking tasks, learning to predict the masked tokens and also capture contextualized representations of the input RNA sequence. Specifically, the procedure of RNAErnie pretraining with motif-aware multilevel masking strategies is as follows.

Base-level masking

In the initial stage of the learning process, we employ base-level masking as a crucial component. Specifically, we randomly mask 15% of the nucleobases within an RNA sequence. Among the masked positions, 10% are preserved without any alterations, and the remaining 10% are replaced with other nucleobases. The model takes the remaining nucleobases as input and is tasked with predicting the masked positions. This stage primarily focuses on acquiring fundamental token representations; capturing intricate higher-level biological insights proves to be a challenging endeavour.

Subsequence-level masking

Next, we incorporate the masking of random subsequences, which are short and contiguous segments of nucleobases within an RNA sequence. Previous studies, such as refs. 41 and 42, have demonstrated the efficacy of contiguous token masking in enhancing pretrained models for span-selection tasks. Additionally, it is important to consider that the functionality of nucleobases often manifests within the context of sequential arrangements. By predicting these subsequences as a whole, we encourage the model to capture a deeper understanding of the biological information inherent in the relationships between consecutive nucleobases. In our research, we specifically mask subsequences with lengths ranging from 4 to 8 bp.

Motif-level masking

In the final stage of pretraining, we employ motif-level masking as part of our approach. RNA motifs, characterized as recurrent structural elements with a high concentration of information, have been extensively observed in atomic-resolution RNA structures17. These motifs are widely recognized for their crucial involvement in various biological activities, such as the formation of RNA tertiary structures19, interaction with dsRNA-binding proteins (RBPs) and participation in complex formation with proteins18. To incorporate these motifs into our model as so-called biological priors, we gather them from multiple sources:

  • ATtRACT43: This resource provides comprehensive information on 370 RBPs and 1,583 RBP consensus binding motifs. The data is extracted and carefully curated from experimentally validated sources such as CISBP-RNA, SpliceAid-F and RBPDB databases.

  • SpliceAid44: We gather information from SpliceAid, which encompasses 2,220 target sites associated with 62 human splicing proteins. Additionally, it includes expression data from 320 tissues per cell.

  • We also extract the most frequently occurring contiguous nucleobase sequences, ranging from 4 to 8 bp, by scanning the entirety of the RNAcentral database.

By incorporating motifs from these diverse sources, we aim to capture a comprehensive representation of RNA structural elements for our analysis.

Type-guided fine-tuning strategy

Given the RNAErnie basic block pretrained with motif-aware multilevel masking strategies, we need to combine the basic blocks of the RNAErnie transformer with task-specific heads—for example, a fully connected layer for RNA classification—into a neural network for the downstream task and further train the neural network subject to labelled datasets for the downstream application in a supervised learning manner. Here, we introduce our proposed type-guided fine-tuning strategy in two parts: neural architectures for tasks and domain-adaptation strategies.

Neural architectures for fine-tuning

To adapt various downstream tasks, the RNAErnie approach follows the surgical fine-tuning strategies20 and offers three sets of neural network architectures as follows.

FBTH

In the FBTH architecture, given RNA sequences and their labels for a downstream task, the RNAErnie approach simply extracts embeddings of RNA sequences from a pretrained RNAErnie basic block and then leverages the embeddings as inputs to train a separate task-specific head subject to the downstream tasks. In this way, the parameters in the RNAErnie backbone are frozen, while the head is trainable. According to ref. 20, this architecture would work well when the downstream tasks are out-of-distribution of pretraining datasets.

TBTH

In the TBTH architecture, the RNAErnie approach directly combines the RNAErnie basic block and the task-specific head to construct an end-to-end neural network for downstream tasks and then trains the neural network using the labelled datasets in a supervised learning manner. In this way, the parameters in both the RNAErnie backbone and the head are trainable. According to ref. 20, this architecture would work well when the downstream tasks and pretraining datasets are in the same distribution.

STACK

In the STACK architecture, the RNAErnie approach first leverages an RNAErnie basic block to predict the top-K most possible coarse-grained RNA types (that is, the K coarse-grained RNA types with the highest probabilities) using the input RNA sequence. Then it stacks an additional layer of K downstream modules with shared parameters for fine-tuning, where every downstream module refers to a TBTH/FBTH network and is fed with the RNA sequence and a predicted RNA type for the downstream task. The K downstream modules output K prediction results, and the RNAErnie approach outputs the ensemble of K results as the final outcome.

More specifically, in the STACK architecture, the RNAErnie basic block first predicts the indication of an RNA sequence following the [IND] marker by estimating the probability of the masked indication token, denoted as p(xIND∣x; θ). From these predictions, the RNAErnie approach selects the top-K indications, denoted as \({I}_{k}\in {{{\mathcal{I}}}}\) for k = 1, ⋯ , K, along with their corresponding probabilities σ1, …, σK. Each selected indication is then appended to the end of the RNA sequence, resulting in K parallel inputs to the downstream module. Then the downstream module takes the K parallel inputs simultaneously, enabling ensemble learning through soft majority voting. Specifically, the RNAErnie approach calculates the weighted sum for soft majority voting as follows:

$$\bar{q}=\mathop{\sum }\limits_{k=1}^{K}{\sigma }_{k}{q}_{k},$$
(1)

where qk could be either scalar, vector or matrix outputs from the downstream module for various downstream tasks (for example, logit vectors for classification tasks or pair-wise feature maps for structural analysis), while \(\bar{q}\) refers to the weight sum.

Note that although we consider the stacking architecture part of our key contributions, FBTH and TBTH sometimes deliver better performance.

Domain adaptation to downstream tasks

Upon completion of the pretraining phase, the RNAErnie basic block is prepared for type-guided fine-tuning, enabling its application to various downstream tasks. It is important to emphasize that RNAErnie has the potential to accommodate a diverse array of tasks, extending beyond the examples provided below, through appropriate FBTH, TBTH and STACK architectures.

RNA sequence classification

RNA sequence classification is a pivotal task that assigns RNA sequences to specific categories. In other words, it maps an RNA sequence x of length L to scalar labels, which refer to different categories. RNA sequence classification is crucial for understanding their functions and their roles in various biological processes. Accurate classification of RNA sequences enables researchers to identify ontology and predict functions, which facilitates the development of new therapies and treatments for RNA-related diseases.

Our work leverages STACK with TBTH to classify RNA sequences. It stacks K classification modules: the RNAErnie basic block combined with a trainable MLP as a prediction head. However, the computational complexity of transformers, which exhibit a quadratic time complexity of \({{{\mathcal{O}}}}({n}^{2}d)\), where n denotes the sequence length, posed challenges when processing excessively long RNA sequences. To discern lncRNA amidst protein-coding transcripts, we employed a chunk strategy. This strategy entails the division of lengthy RNA sequences into more manageable segments, which are independently fed into the RNAErnie approach. Subsequently, we aggregate the segment-level logits to obtain the sequence-level logit and employ an MLP for classification purposes.

RNA–RNA interaction prediction

RNA–RNA interaction prediction refers to the estimation of interactions between two RNA sequences, such as miRNA and mRNA, circular RNA and lncRNA. This task maps two RNA sequences, xa of length L1 and xb of length L2, to binary labels 0/1, where 0 indicates no interaction between the two RNA sequences and 1 indicates interaction. Accurate prediction of RNA–RNA interactions can provide valuable insights into RNA-mediated regulatory mechanisms and enhance our understanding of biological processes, including gene expression, splicing and translation45.

Our work employs a TBTH architecture, which combines the RNAErnie basic block with a hybrid neural network inspired by ref. 46. This hybrid neural network acts as the interaction prediction head, sequentially incorporating several components: a convolutional neural network, a bidirectional long short-term memory network and a MLP. Because the types of interacting RNA are fixed, it is unnecessary to employ the STACK architecture for the purpose of RNA–RNA interaction analysis.

RNA secondary structure prediction

RNA secondary structure prediction determines the probable arrangement of bp within an RNA sequence, which can fold back onto itself and form specific pairings. It maps an RNA sequence x of length L to a 0/1 matrix with shape L × L, where element i, j means whether nt i forms bp with nt j. The secondary structure of RNA plays a critical role in understanding its interactions with other molecules and its functional importance. This prediction technique is a valuable tool in molecular biology, aiding in the identification of potential targets for drug design and enhancing our understanding of gene expression and regulation mechanisms.

Our work utilizes the STACK architecture with FBTH to fold RNA sequences. We combined the RNAErnie basic block with a folding neural network inspired by the methodology described in ref. 47. It computes four distinct folding scores—helix stacking, unpaired region, helix opening and helix closing—for each pair of nt bases. Subsequently, we utilize a Zuker-style dynamic programming approach48 to predict the most favourable secondary structure. This is achieved by maximizing the cumulative scores of adjacent loops, following a systematic and rigorous computational procedure. To facilitate the training of our deep neural network, we adopt the max-margin framework. Within this framework, the network minimizes the structured hinge loss function while incorporating thermodynamic regularization.

Hyperparameters and configurations

During the pretraining phase, our model underwent approximately 2,580,000 steps of training, with a batch size set to 50 and a maximum sequence length for ERNIE limited to 512. We utilized the AdamW optimizer, which was regulated by a learning-rate schedule involving anneal warm-up and decay. The initial learning rate was set at 1 × 10−4, with a minimum learning rate of 5 × 10−5. The learning-rate scheduler was designed to warm up during the first 5% of the steps and then decay in the final 5% of the steps. In terms of masking strategies, we maintained a proportion of 1:1:1 across the three different masking levels, with the training algorithm randomly selecting one strategy for each training session. The pretraining was conducted on four Nvidia Tesla V100 32 GB graphics processing units, taking around 250 hours to reach convergence.

Here, in additional to the hyperparameters for pretraining, we introduce the configurations of variant pretrained models derived from RNAErnie and used in experiments:

  • Ernie-base: this model represents the vanilla ERNIE architecture without any pretraining on RNA sequence datasets. It underwent standard fine-tuning.

  • RNAErnie−−: in this model, only base-level masking was employed during the pretraining phase of the RNAErnie family. It was then fine-tuned using the standard approach.

  • RNAErnie−: the RNAErnie family model with both base and subsequence-level masking during pretraining, followed by standard fine-tuning.

  • RNAErnie: this model encompasses the complete set of masking strategies, including base, subsequence and motif-level masking during pretraining. It was fine-tuned using the TBTH or FBTH architecture.

  • RNAErnie+: the most comprehensive model in the RNAErnie family, incorporating all three levels of masking during pretraining and the STACK architecture.

  • RNAErnie without chunk: this model truncates RNA sequences and discards any remaining segments when classifying long RNA sequences, specifically lncRNA (for example, in lnRC_H and lnRC_M datasets) alongside protein-encoding transcripts.