Contextual fear conditioning induces differential alternative splicing

Abstract

The process of memory consolidation requires transcription and translation to form long-term memories. Significant effort has been dedicated to understanding changes in hippocampal gene expression after contextual fear conditioning. However, alternative splicing by differential transcript regulation during this time period has received less attention. Here, we use RNA-seq to determine exon-level changes in expression after contextual fear conditioning and retrieval. Our work reveals that a short variant of Homer1, Ania-3, is regulated by contextual fear conditioning. The ribosome biogenesis regulator Las1l, small nucleolar RNA Snord14e, and the RNA-binding protein Rbm3 also change specific transcript usage after fear conditioning. The changes in Ania-3 and Las1l are specific to either the new context or the context-shock association, while the changes in Rbm3 occur after context or shock only. Our analysis revealed novel transcript regulation of previously undetected changes after learning, revealing the importance of high throughput sequencing approaches in the study of gene expression changes after learning.

Keywords: Alternative splicing; Fear conditioning; Homer1; RNA-seq; Transcription.

Fig. 1

Bin-specific regulation of Homer1 (Ania-3), Las1l, and Rbm3. (A) (left) diffSplice result showing the predicted significant bin changes of the Homer1 gene in red on a log₂ scale. Bins 16–18 indicate the Ania-3 isoform. (right) qPCR validation of the change in Bin18 in an independent cohort of mice. Bin 21 expression was compared as a control. (B) (left) diffSplice result showing the predicted significant bin changes of the Las1l gene in red on a log₂ scale. (right) qPCR validation of the change in Bin 15 in an independent cohort of mice. Expression of Bin 17 was used as a control. (C) (left) diffSplice result showing the predicted significant bin changes of the Rbm3 gene in red on a log₂ scale. (right) qPCR validation of the changes in Bin 2 and Bin 22 an independent cohort of mice. Expression of Bin 7 was used as a control. HC = homecage, FC = fear conditioned. * denotes a p-value of <0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Alternative splicing changes variably in response to either component of fear conditioning alone. The same primers used in Fig. 1 were used to test gene expression after context only (n = 8) or immediate shock controls (n = 8) or homecage animals (n = 7) and analyzed by ANOVA. Ania-3 and Las1l changes in response to the context only control and Rbm3 changes with all manipulations. This may suggest Ania-3 and Las1l are markers of contextual novelty while Rbm3 responds to many stimuli. No control bins change with either context-only or immediate shock controls. HC = homecage, CTX = context only, SH = shock only. * denotes significance below an alpha of 0.05.