Chimeric RNA isoforms generated by diverse mechanisms from two C-type lectins modulate innate immunity in arthropods

Abstract

Chimeric RNA formation represents a critical mechanism for expanding protein functional diversity, yet its role in invertebrate immune adaptation remains poorly characterized. Here, we report that two C-type lectin genes (MnLec2 and MnLec3) from distinct genomic loci in the oriental river prawn Macrobrachium nipponense undergo positionally flexible chimeric RNA formation via alternative trans-splicing and transcriptional slippage, generating 11 structurally diverse chimeric isoforms (MnLec1, MnLec4–13) with bidirectional exon joining. Crucially, pathogen challenges reprogram chimeric RNA frequencies to shift immune equilibrium, universally suppressing detrimental MnLec9 while promoting protective MnLec7 formation. Functional dissection confirms dual-action pathogen suppression, recombinant MnLec7 (rMnLec7) suppresses white spot syndrome virus replication by upregulating antimicrobial peptides and RNAi effectors, while accelerating Vibrio parahaemolyticus clearance and improving survival. Conversely, suppression of MnLec9 removes its immunosuppressive activity, synergistically enhancing host defense. This coordinated isoform rebalancing enables effective pathogen clearance. Thus, positional flexibility in chimeric RNA formation generates antagonistic isoforms that maintain immune homeostasis and deploy targeted defense upon infection, revealing an adaptive transcriptional strategy in arthropods.

Chimeric RNA formation, driven by chromosomal rearrangements (genomic events) or aberrant RNA processing (e.g., trans-splicing), generates novel transcripts by merging sequences from distinct parental genes into chimeric constructs (1, 2). This process facilitates the emergence of proteins with neofunctionalized domains pivotal to evolutionary innovation, particularly in immune adaptation (3). In arthropods, lectins function as key pattern recognition receptors that orchestrate innate immunity against pathogens (4). However, evidence for lectin diversification through chimeric RNA events remains scarce, obscuring their functional evolution in immune pathways. Here, we report that the oriental river prawn, Macrobrachium nipponense, exhibits positional flexibility in generating chimeric C-type lectin RNAs. MnLec2 and MnLec3 from distinct genomic loci produce diverse chimeric transcripts through transcriptional and posttranscriptional mechanisms, yielding up to 11 chimeric lectin isoforms. These chimeric RNAs demonstrate divergent expression levels following pathogen challenge, implicating important roles in immune regulation.

Results

Diverse Chimeric RNAs Arise from Positional Flexibility Between MnLec2 and MnLec3.

Transcriptomic analysis of M. nipponense, combined with sequencing of cDNA clones derived from mRNA, revealed that two C-type lectin genes, MnLec2 and MnLec3, underwent extensive chimeric RNA formation, generating a diverse repertoire of 11 chimeric lectin isoforms (MnLec1, MnLec4–13). Sequencing of multiple cDNA clones demonstrated remarkable positional heterogeneity in these chimeric transcripts. While canonical cis-splicing produced mature MnLec2 and MnLec3 mRNAs (Fig. 1A), two principal chimeric RNA generation mechanisms were identified. Intact exon (IE)-type chimeras arose from trans-splicing of intact exons at the posttranscriptional level, joining full-length exons from parental genes. MnLec4 combined MnLec2 exons 1–3 with MnLec3 exons 2–4, MnLec9 contained MnLec2 exons 1–3 with MnLec3 exon 4, and MnLec12 joined MnLec3 exon 1 with MnLec2 exons 2–4 (Fig. 1 B and C). Broken exon (BE)-type chimeras formed via transcriptional slippage or noncanonical trans-splicing. Transcriptional slippage initially created chimeric preprocessed RNA with hybrid exons that matured after intron removal. All BE-type isoforms (except MnLec10) contained short homologous sequences (SHS) shared by MnLec2 and MnLec3. Transcriptional slippage occurred at SHS sites during transcription, producing diverse chimeric preprocessed RNAs. MnLec7 comprised MnLec2 exons 1–2, a broken exon containing SHS, and MnLec3 exon 4. MnLec1, MnLec6, and MnLec8 shared MnLec2 exons 1–3 but possessed distinct broken exons with different SHSs. MnLec5 consisted of MnLec3 exons 1–2, a broken exon, and MnLec2 exon 4. MnLec11 and MnLec13 contained MnLec3 exons 1–3 plus a broken exon (Fig. 1 D and E). Notably, MnLec10 represented a mechanistic outlier, forming without SHS via noncanonical trans-splicing at AG/AG sites within exons (combining MnLec2 exons 1–2, a broken exon and MnLec3 exon 4; Fig. 1 F and G). Critically, chimeric RNAs formed bidirectionally (MnLec2→MnLec3: e.g., MnLec4/7/9; MnLec3→MnLec2: e.g., MnLec5/12). High sequence similarity between MnLec2 and MnLec3 facilitated SHS dependent transcriptional slippage (Fig. 1H). SHS mediated broken-exon formation in most BE-type isoforms (MnLec10 excluded). Genomic DNA sequencing confirmed MnLec2 and MnLec3 resided at distinct loci, establishing that chimerism arose from RNA-level mechanisms, not genomic fusion.

Fig. 1.

Mechanisms and structural diversity of chimeric RNAs derived from MnLec2 and MnLec3. (A) Canonical cis-splicing generates mature MnLec2 and MnLec3 mRNA transcripts. (B) Alternative trans-splicing yields IE-type chimeric RNAs (MnLec4, MnLec9, MnLec12). (C) Posttranscriptional trans-splicing model is illustrated using MnLec9 as representative. (D) Transcriptional slippage is dependent on SHS generating BE-type chimeric RNAs (MnLec7, MnLec1, MnLec6, MnLec8, MnLec5, MnLec11, MnLec13). (E) Transcriptional slippage mechanism is exemplified by MnLec7 formation. (F) Noncanonical trans-splicing produces MnLec10. (G) Proposed model shows MnLec10 formation via AG/AG splicing sites within exons. Structural annotations: exons (colored boxes; lengths in nucleotides), introns (lines), SHS (green domains), unresolved regions (dashed lines). (H) Sequence alignment of MnLec2 and MnLec3. Identical residues (black), conserved substitutions (blue), nonconserved residues (red).

Pathogen Induction Patterns of MnLec2-MnLec3 Chimeric Lectins Reveal Functional Diversity.

Pathogen challenge universally modulated chimeric RNA frequencies in M. nipponense. White spot syndrome virus (WSSV), Decapod iridescent virus 1 (DIV1), Vibrio parahaemolyticus, and Staphylococcus aureus consistently altered chimeric RNA frequencies, suppressing MnLec1/MnLec9 and elevating MnLec6/MnLec7, with all pathogens increasing MnLec7 and decreasing MnLec9 (Fig. 2 A–C). Functional characterization showed recombinant MnLec7 (rMnLec7) suppressed WSSV infection by reducing VP28 transcription (Fig. 2D) and protein levels (Fig. 2E), and via upregulation of antimicrobial peptides (AMPs, anti-lipopolysaccharide factor 1–4 (ALF1–4), Crustin 2 (Crus2), Crus4–6, and Crus9; Fig. 2F) and RNAi effectors (Argonaute1–2, Dicer1–2; Fig. 2G), which lowered viral copies (Fig. 2H) and enhanced survival rates (Fig. 2I). Conversely, rMnLec9 exacerbated WSSV pathogenesis, elevating VP28 (Fig. 2 D and E), suppressing immune effectors (Fig. 2 F and G), increasing viral load (Fig. 2H), and mortality (Fig. 2I). Against V. parahaemolyticus, rMnLec7 induced AMPs (Fig. 2J), accelerated bacterial clearance (Fig. 2K), and improved survival rates (Fig. 2L), whereas rMnLec9 suppressed AMPs (Fig. 2J), impaired clearance (Fig. 2K), and increased mortality (Fig. 2L). Thus, chimeric RNA positional flexibility generated antagonistic isoforms. rMnLec7 enhanced immunity, while rMnLec9 suppressed it.

Fig. 2.

Functional divergence of MnLec2-MnLec3 chimeric lectins in antiviral and antibacterial immunity. (A–C) Differential induction of chimeric lectins (MnLec1, MnLec6–9) in M. nipponense following challenge with WSSV, DIV1, V. parahaemolyticus, or S. aureus. Relative frequencies are determined by PCR cloning/sequencing and normalized to healthy controls (dashed line = 1). The color legend shown in panel 2B applies to 2C. (D and E) Expression levels of VP28 gene (RT–qPCR) and protein (Western blot) in hemocytes at 48 h post-injection (hpi). Prawns receive WSSV preincubated with rMnLec7 or rMnLec9. GAPDH serves as a loading control. (F and G) Transcript levels of AMP genes (ALF1–4, Crus2, Crus4–6, and Crus9) and RNAi effectors (Argo1–2, Dicer1–2) in WSSV-challenged prawns. (H) WSSV genomic copies in hemocytes at 48 hpi. (I) Survival rates of WSSV-infected prawns are monitored over 6 d (log-rank test). (J) Expression of AMPs in prawns injected with V. parahaemolyticus plus rMnLec7 or rMnLec9 at 48 hpi. (K) In vivo bacterial clearance assay. Hemolymph bacterial loads are quantified at 2, 15, and 30 min postinjection. (L) Survival rates of V. parahaemolyticus–infected prawns (6 d). Asterisks denote statistical significance versus control (*P < 0.05, **P < 0.01, ***P < 0.001). Data represent mean ± SD (n = 3 biological replicates for D–L); β-actin and GAPDH serve as internal controls.

Discussion

Chimeric RNA formation can generate chimeric RNAs at the genomic or posttranscriptional level through trans-splicing. However, our study reveals that positional flexibility in chimeric RNA formation between MnLec2 and MnLec3 generates up to 11 chimeric lectins via at least two distinct mechanisms. IE-type chimeric RNAs (e.g., MnLec4, MnLec9) arise from canonical trans-splicing of intact exons at the posttranscriptional level, producing mature chimeric RNA. In contrast, BE-type chimeric RNAs (e.g., MnLec1, MnLec7) depend on SHS from two homologous genes and first produce chimeric preprocessed RNA at the transcriptional level. Previous research observed “trans-splicing via microhomology” in Drosophila chimeric RNAs (5), while transcriptional slippage generates immune receptor diversity in mollusks (6). SHS acts as a molecular guide for exon joining, akin to microhomology-mediated end joining (7). Here, BE-type chimeric RNAs form through transcriptional slippage (evidenced by chimeric preprocessed RNA detection) followed by splicing. Notably, MnLec10 represents a mechanistic outlier, its broken exon 3 lacks SHS and likely forms via noncanonical trans-splicing at AG/AG sites within exons, indicating SHS-independent plasticity. While SHS are the primary drivers of BE-type chimeric RNA formation, the SHS-independent generation of MnLec10 (containing BE) via noncanonical trans-splicing reveals the existence of auxiliary mechanisms. Such events are likely stochastic and less efficient due to rare alignment of splice-compatible sites, analogous to Alu-element-driven exon shuffling in vertebrates (8). Thus, although SHS dominates chimeric RNA production, trans-splicing provides secondary routes for structural innovation. The positional independence of MnLec2 and MnLec3 enables combinatorial flexibility through transcriptional/posttranscriptional mechanisms, expanding immune receptor diversification beyond gene duplications. Although current events occur between homologous genes, the mechanistic basis—trans-splicing for IE-types and transcriptional slippage for BE-types—could theoretically enable chimeric RNA formation between nonhomologous genes if they share microhomology or splice-compatible sites. Nevertheless, the requirement for SHS or splice-site compatibility inherently biases diversification toward homologous families, balancing evolutionary innovation with functional feasibility.

The universal reprogramming of chimeric isoforms by diverse pathogens exemplifies the “focused efficacy” paradigm of invertebrate innate immunity. Unlike antigen-specific adaptive responses in vertebrates, this reflects pattern-specific recalibration. MnLec7 recognizes conserved pathogen-associated molecular patterns to broadly enhance defense, while suppressing MnLec9 removes immunosuppressive vulnerability. Despite the limited repertoire of germline-encoded receptors in innate immunity, the host can generate multiple chimeric RNA transcripts to combat foreign pathogens (9). Functional analyses confirm synergistic pathogen suppression, rMnLec7 enhances immunity by suppressing WSSV replication and accelerating bacterial clearance through upregulation of AMPs/RNAi effectors. Concurrently, pathogen-induced MnLec9 downregulation ablates its proinfection activity, synergistically augmenting host defense. Thus, positional flexibility in chimeric RNA formation establishes functional antagonism, MnLec9 acts as a “homeostatic brake” under steady-state conditions, akin to Drosophila PGRP-LB (10), while infection triggers coordinated rebalancing (upregulation of MnLec7 and downregulation of MnLec9) for targeted defense. This consolidates bidirectional immune regulation through chimeric RNA formation involving two homologous genes from distinct loci. Our study establishes chimeric RNA formation as a transcriptional strategy for expanding invertebrate immune capacity, bridging structural diversity to functional antagonism. Future studies should explore whether pathogens actively manipulate chimeric RNA frequencies to evade defense, as seen in viral hijacking of mammalian RNA splicing (11).

Materials and Methods

Detailed experimental procedures are provided in SI Appendix.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix.