Abstract
Long-lasting forms of synaptic plasticity that underlie learning and memory require new transcription and translation for their persistence. The remarkable polarity and compartmentalization of neurons raises questions about the spatial and temporal regulation of gene expression within neurons. Alternative cleavage and polyadenylation (APA) generates mRNA isoforms with different 3′ untranslated regions (3′UTRs) and/or coding sequences. Changes in the 3′UTR composition of mRNAs can alter gene expression by regulating transcript localization, stability and/or translation, while changes in the coding sequences lead to mRNAs encoding distinct proteins. Using specialized 3′ end deep sequencing methods, we undertook a comprehensive analysis of APA following induction of long-term potentiation (LTP) of mouse hippocampal CA3-CA1 synapses. We identified extensive LTP-induced APA changes, including a general trend of 3′UTR shortening and activation of intronic APA isoforms. Comparison with transcriptome profiling indicated that most APA regulatory events were uncoupled from changes in transcript abundance. We further show that specific APA regulatory events can impact expression of two molecules with known functions during LTP, including 3′UTR APA of Notch1 and intronic APA of Creb1. Together, our results reveal that activity-dependent APA provides an important layer of gene regulation during learning and memory.
Highlights
Pre-mRNA cleavage and polyadenylation (C/P) is a nearly universal 3′ end processing mechanism for protein-coding genes in eukaryotes, and is coupled to transcription termination[7]
We note that a higher percentage of polyA site (PAS) were associated with the AAUAAA hexamer in our RNA samples than the 42% previously reported in the mouse genome[12], presumably because APA transcripts in brain preferentially use distal PASs4,22,24–26, which are more frequently associated with AAUAAA than proximal PASs13
By dividing intronic PAS isoforms into 5 groups based on the intron locations of their PASs, i.e., first intron (+1), second (+2), last (−1), second to last (−2), and middle, we found that PASs located in the 5′-most intron (+1) had the highest increase in usage as compared to those in the 3′-most intron 3 hr post longterm potentiation (LTP) (P = 0.008, Wilcoxon rank sum test)
Summary
Pre-mRNA cleavage and polyadenylation (C/P) is a nearly universal 3′ end processing mechanism for protein-coding genes in eukaryotes, and is coupled to transcription termination[7]. In keeping with the preferential expression of distal PAS isoforms in neurons, 3′UTRs play a important role in compartmentalized gene expression by directing the localization and regulated translation of mRNAs within dendrites and axons and at synapses[30,31,32,33]. Using deep sequencing of 3′ ends of transcripts, we systematically characterized APA regulation following LTP induction in acute mouse hippocampal slices. We examined both 3′UTR APA and intronic APA events at different time points post LTP, and analyzed the interplay between APA and gene expression regulation. APA events are largely uncoupled from gene expression changes, and constitute a distinct layer of gene regulation during LTP
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