Quantitative Nucleotide Level Analysis of Regulation of Translation in Response to Depolarization of Cultured Neural Cells

Abstract

Studies on regulation of gene expression have contributed substantially to understanding mechanisms for the long-term activity-dependent alterations in neural connectivity that are thought to mediate learning and memory. Most of these studies, however, have focused on the regulation of mRNA transcription. Here, we utilized high-throughput sequencing coupled with ribosome footprinting to globally characterize the regulation of translation in primary mixed neuronal-glial cultures in response to sustained depolarization. We identified substantial and complex regulation of translation, with many transcripts demonstrating changes in ribosomal occupancy independent of transcriptional changes. We also examined sequence-based mechanisms that might regulate changes in translation in response to depolarization. We found that these are partially mediated by features in the mRNA sequence—notably upstream open reading frames and secondary structure in the 5′ untranslated region—both of which predict downregulation in response to depolarization. Translationally regulated transcripts are also more likely to be targets of FMRP and include genes implicated in autism in humans. Our findings support the idea that control of mRNA translation plays an important role in response to neural activity across the genome.

Widespread Alterations in Translation Elongation in the Brain of Juvenile Fmr1 Knockout Mice

“…A critical aspect is that neuronal activity may be tightly coupled to translational regulation. Several recent studies found translational repression of neuronal mRNAs following fear conditioning in vivo (Cho et al, 2015) and of FMRP binding partners following KCl depolarization in vitro (Dalal et al, 2017). Given extensive evidence of cortical hyperexcitability (Gibson et al, 2008; Hays et al, 2011) and dys-regulation of GABAergic neurotransmission in Fmr1 -KO mice (Paluszkiewicz et al, 2011), it is possible that the downregulation of RFApm we observed in FMRP binding partners (Figure 2C) is linked to increased cortical activity.…”

Cell type specific profiling of alternative translation identifies novel protein isoforms in the mouse brain

“…We found that many alternative TISs show differential ribosomal occupancy in response to KCl treatment, which is known to stimulate neurons by phosphorylating cyclic adenosine 3',5'-monophosphate response element binding protein (CREB) via an influx of Ca2+ through L-type Ca+2 channels 72 . Previously, we have demonstrated that KCl stimulation of neural cultures modulates the ribosomal occupancy of specific CDSs 39 . Others have shown that KCl-stimulation of neuronal cultures enhances translation initiation by promoting the expression and activity of eukaryotic initiation factor 4E and enhance initiation 73 .…”

“…Neuronal activity induces de novo protein synthesis-both from existing mRNAs and via induction of a specific transcriptional program [37][38][39] . However, it is unclear if it can also alter TIS usage on a given transcript.…”

“…Neuron/glia mix cultures from the cortices of P0 FVB mouse pups were generated as described 39 . After 7 days in vitro, cells were treated with 2 µg/ml HHT ((LKT lab) and compared to parallel cultures treated with DMSO 39 (Sigma) for 2 m, and then with 100ug/ml CHX (Sigma) for 7 m at 37 0 C before being lysed for RF 12 .…”

“…RF RF was performed as described 12,39 . Briefly, the cell lysates were treated with RNAseI and subjected to sucrose-cushion ultracentrifugation for pelleting monosomes.…”