Abstract
Sleep deprivation is a global health problem adversely affecting health as well as causing decrements in learning and performance. Sleep deprivation induces significant changes in gene transcription in many brain regions, with the hippocampus particularly susceptible to acute sleep deprivation. However, less is known about the impacts of sleep deprivation on post-transcriptional gene regulation. To identify the effects of sleep deprivation on the translatome, we took advantage of the RiboTag mouse line to express HA-labeled Rpl22 in CaMKIIα neurons to selectively isolate and sequence mRNA transcripts associated with ribosomes in excitatory neurons. We found 198 differentially expressed genes in the ribosome-associated mRNA subset after sleep deprivation. In comparison with previously published data on gene expression in the hippocampus after sleep deprivation, we found that the subset of genes affected by sleep deprivation was considerably different in the translatome compared with the transcriptome, with only 49 genes regulated similarly. Interestingly, we found 478 genes differentially regulated by sleep deprivation in the transcriptome that were not significantly regulated in the translatome of excitatory neurons. Conversely, there were 149 genes differentially regulated by sleep deprivation in the translatome but not in the whole transcriptome. Pathway analysis revealed differences in the biological functions of genes exclusively regulated in the transcriptome or translatome, with protein deacetylase activity and small GTPase binding regulated in the transcriptome and unfolded protein binding, kinase inhibitor activity, neurotransmitter receptors and circadian rhythms regulated in the translatome. These results indicate that sleep deprivation induces significant changes affecting the pool of actively translated mRNAs.
Highlights
Sleep deprivation is a widespread problem affecting more than one-third of U.S adults and 70% of teenagers (Center for Disease Control and Prevention, 2017) leading to significant impairments in memory and performance
It has been difficult to identify the subset of genes most affected at the protein level as most studies measuring protein abundance after sleep deprivation in a specific brain region have focused on individual proteins [25, 26], Translating ribosome affinity purification combined with unbiased RNA sequencing (TRAP-Seq) has become a powerful technique for identification of the translatome, the set of mRNA transcripts bound to ribosomes, in a cell-type specific manner [27,28,29,30,31]
Isolation of ribosome‐associated RNA transcripts from hippocampal CaMKIIα expressing neurons To isolate ribosome-associated mRNA transcripts associated in excitatory neurons, we used the RiboTag mouse with ribosomal protein Rpl22 labeled with a hemagglutinin epitope ([28]; Jackson Laboratory Stock 011029) crossed with CaMKIIα mice (Jackson Laboratory Stock 005359) to generate mice with expression of the HA-tagged Rpl22 in neurons expressing CaMKIIα (Fig. 1a), a technique previously used to identify translating mRNAs during hippocampal synaptic plasticity [32]
Summary
Sleep deprivation is a widespread problem affecting more than one-third of U.S adults and 70% of teenagers (Center for Disease Control and Prevention, 2017) leading to significant impairments in memory and performance. It has been difficult to identify the subset of genes most affected at the protein level as most studies measuring protein abundance after sleep deprivation in a specific brain region have focused on individual proteins [25, 26], Translating ribosome affinity purification combined with unbiased RNA sequencing (TRAP-Seq) has become a powerful technique for identification of the translatome, the set of mRNA transcripts bound to ribosomes, in a cell-type specific manner [27,28,29,30,31]. The CaMKIIα-Cre transgene has been used to express the HA-tagged ribosomal protein Rpl at endogenous levels in excitatory neurons [32] Taking advantage of this technique, we investigated the effects of sleep deprivation downstream of transcription at the level of the ribosome
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