Long-read RNA sequencing reveals sex-associated isoform distribution in soma and neuropil of the rat hippocampal CA1

Abstract

Sex-associated differences in hippocampal function are well-recognized, yet the molecular mechanisms underlying these variations remain incompletely understood. In the CA1 region, the spatial organization of the transcriptome between the soma and neuropil supports local mRNA translation, a mechanism essential for synaptic plasticity. Using long-read direct RNA sequencing, we conducted a targeted comparison of the CA1 soma and neuropil transcriptomes between male and estrus-phase female rats. We investigated differences in isoform usage and transcript composition, including UTR extensions and poly(A) tail lengths, that characterize the CA1 molecular landscape under basal conditions. Long-read direct RNA sequencing (Oxford Nanopore) was performed on soma and neuropil-enriched fractions microdissected from the dorsal CA1 region. This approach enabled full-length transcript identification, isoform-level resolution, and detection of previously unannotated RNA species. Overall gene and isoform numbers were comparable between males and estrus-phase females. Approximately 80% of detected transcripts were classified as protein-coding, and ~ 8% corresponded to unannotated species. Females and male-associated differences were more pronounced in the somatic compartment, whereas transcript profiles in the neuropil showed limited divergence. Differential transcript expression identified 892 neuropil-enriched and 467 somatic-enriched transcripts in estrus-phase females, compared with 973 neuropil-enriched and 541 somatic-enriched transcripts in males. Regardless of sex, both somatic and neuropil-enriched transcripts exhibited longer 5′ UTRs. Furthermore, somatic transcripts exhibited longer 3′ UTRs, while shorter poly(A) tails were a hallmark of the neuropil fraction. A distinct sex-specific feature is the significantly shorter CDS observed in female neuropil-enriched transcripts. Functional enrichment revealed lipid metabolism and translation-related processes among female-enriched neuropil transcripts. Conversely, male-enriched somatic transcripts were associated with memory, regulation of cellular activity, and neuronal projection development. Isoform-level analyses further revealed sex and stratum-specific enrichment of RNAs encoding receptors, scaffolding proteins, ribosomal components, and transcriptional regulators. Under basal conditions, the CA1 region of males and estrus-phase females is associated with distinct patterns of isoform usage and compartment-specific transcript distribution. These findings provide new molecular context for localized transcript regulation in the hippocampus and underscore the value of long-read sequencing for resolving isoform diversity within specific physiological states.