Baker, K. E. | bioRxiv Science

bioRxiv2022

Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response

Endoplasmic Reticulum (ER) stress, caused by the accumulation of misfolded proteins in the ER, elicits a homeostatic mechanism known as the Unfolded Protein Response (UPR). The UPR reprograms gene expression to promote adaptation to chronic ER stress. The UPR comprises an acute phase involving inhibition of bulk protein synthesis and a chronic phase of transcriptional induction coupled with the partial recovery of protein synthesis. However, the role of transcriptional regulation in the acute phase of the UPR is not well understood. Here we analyzed the fate of newly synthesized mRNA encoding the protective and homeostatic transcription factor X-box binding protein 1 (XBP1) during this acute phase. We have previously shown that global translational repression induced by the acute UPR was characterized by decreased translation and increased stability of XBP1 mRNA. We demonstrate here that this stabilization is independent of new transcription. In contrast, we show XBP1 mRNA newly synthesized during the acute phase accumulates with long poly(A) tails and escapes translational repression. Inhibition of nascent RNA polyadenylation during the acute phase decreased cell survival with no effect in unstressed cells. Furthermore, during the chronic phase of the UPR, levels of XBP1 mRNA with long poly(A) tails decreased in a manner consistent with co-translational deadenylation. Finally, additional pro-survival, transcriptionally-induced mRNAs show similar regulation, supporting the broad significance of the pre-steady state UPR in translational control during ER stress. We conclude that the biphasic regulation of poly(A) tail length during the UPR represents a previously unrecognized pro-survival mechanism of mammalian gene regulation.

molecular biology↗

bioRxiv2017

Translational Profiling Of Microglia Reveals Artifacts Of Cell Sorting

Microglia are the resident innate immune population of the central nervous system that constantly survey and influence their local environment. Transcriptomic profiling has led to significant advances in our understanding of microglia in several disease states, but tissue dissociation and purification of microglia is known to lead to cellular activation. Here we use RiboTag translational RNAseq profiling to demonstrate that commonly used cell sorting methods lead to a fundamental alteration of the microglial transcriptome, with several transcripts that can be used to mark artifacts of isolation. Microglial RiboTag RNAseq profiling after peripheral immune challenge with lipopolysaccharide demonstrates unique transcriptional targets that are not evident using cell sorting methodology. Finally, we applied our technique to reveal novel shared and distinct pathways when comparing microglial transcriptomes after peripheral challenge with bacterial or viral mimetics. This study has broad implications for approaches that examine microglial transcriptomes in normal and pathological states.\n\nSummaryKang et al. demonstrate artifactual induction of microglial transcripts associated with cell sorting. Using RiboTag translational profiling methodology, several markers of cell sorting artifact were revealed. Furthermore, RiboTag isolation unveiled changes in microglial transcriptomes following systemic inflammation that would otherwise have been masked by artifacts of cell sorting.

neuroscience↗