Activity-Dependent Regulation of Alternative Cleavage and Polyadenylation During Hippocampal Long-Term Potentiation

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.

Alternative 3′ UTRs Modify the Localization, Regulatory Potential, Stability, and Plasticity of mRNAs in Neuronal Compartments

“…We found that following an epoch of enhanced neuronal activity, the 3 0 UTR isoforms in both compartments, the somata and the neuropil, were altered, with an overall shortening of 3 0 UTRs observed. A shortening of 3 0 UTRs was recently reported in hippocampal slices following a chemical-LTP protocol (Fontes et al, 2017). Most of the activity-dependent changes in 3 0 UTR isoforms were transcription dependent, consistent with alternative polyadenylation identified as an important mechanism for diversifying 3 0 UTRs in both dendrites (Fontes et al, 2017) and developing axons (Shigeoka et al, 2016;Zhang et al, 2016).…”

“…A shortening of 3 0 UTRs was recently reported in hippocampal slices following a chemical-LTP protocol (Fontes et al, 2017). Most of the activity-dependent changes in 3 0 UTR isoforms were transcription dependent, consistent with alternative polyadenylation identified as an important mechanism for diversifying 3 0 UTRs in both dendrites (Fontes et al, 2017) and developing axons (Shigeoka et al, 2016;Zhang et al, 2016). Surprisingly, though, a substantial fraction ($25%) of activity-induced changes in 3 0 UTR isoforms were independent of transcription.…”

“…When analyzed at the population level, we found that elevated activity resulted in a global and significant shortening of the 3 0 UTR isoforms in both compartments, especially pronounced for the neuropil-localized isoforms ( Figure 8C). Approximately 14% of the 3 0 UTRs that were regulated and 18% of the 3 0 UTRs that were shortened by bicuculline were also regulated by a chemical long-term potentiation (LTP) protocol in a recent study (Fontes et al, 2017); this degree of overlap is not significantly higher than one would predict by chance ( Figure S8E). We validated the activity-dependent changes in 3 0 UTRs for several transcripts in each compartment using qRT-PCR ( Figure S8F).…”

Selective roles of vertebrate PCF11 in premature and full-length transcript termination

“…To quantify APA, DEXseq (https://doi.org/doi:10.18129/B9.bioc.DEXSeq) was employed (following previously published workflows for APA analysis (Rot et al, 2017;Fontes et al, 2017). Genes from the analysis set which had at least two alternative active PAS (7718 genes) were subject to differential PAS usage quantification with DEXSeq.…”

Alternative polyadenylation analysis in animals and plants: newly developed strategies for profiling, processing and validation

“…The bioinformatics pipelines for APA processing described above have been well tested by the scientific community 24 , 32 - 35 . Figure 2 demonstrates the bioinformatics pipelines we have developed to analyze our WTTS-seq datasets 1 , 14 , 36 - 37 .…”