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

Abstract

Translation canonically begins at a single AUG and terminates at the stop codon, generating one protein species per transcript. However, some transcripts may use alternative initiation sites or sustain translation past their stop codon, generating multiple protein isoforms. Through other mechanisms such as alternative splicing, both neurons and glia exhibit remarkable transcriptional diversity, and these other forms of post-transcriptional regulation are impacted by neural activity and disease. Here, using ribosome footprinting, we demonstrate that alternative translation is likewise abundant in the central nervous system and modulated by stimulation and disease. First, in neuron/glia mixed cultures we identify hundreds of transcripts with alternative initiation sites and confirm the protein isoforms corresponding to a subset of these sites by mass spectrometry. Many of them modulate their alternative initiation in response to KCl stimulation, indicating activity-dependent regulation of this phenomenon. Next, we detect several transcripts undergoing stop codon readthrough thus generating novel C-terminally-extended protein isoforms in vitro. Further, by coupling Translating Ribosome Affinity Purification to ribosome footprinting to enable cell-type specific analysis in vivo, we find that several of both neuronal and astrocytic transcripts undergo readthrough in the mouse brain. Functional analyses of one of these transcripts, Aqp4, reveals readthrough confers perivascular localization, indicating readthrough can be a conserved mechanism to modulate protein function. Finally, we show that AQP4 readthrough is disrupted in multiple gliotic disease models. Our study demonstrates the extensive and regulated use of alternative translational events in the brain and indicates that some of these events alter key protein properties. SignificanceHere we examine the extent and function of unusual protein variants resulting from alternative modes of mRNA translation in the mouse brain. We unexpectedly find that hundreds of neural transcripts use non-canonical translation initiation sites generating N-terminal protein variants, and dozens undergo stop codon readthrough generating C-terminally-extended protein variants.Further, we find that many transcripts modulate their use of non-canonical initiation in response to neuronal stimulation and at least one transcript modules its readthrough in response to diseases or conditions involving gliosis. Thus, our results demonstrate a novel use of alternative translation in homeostatic functions as well as in disease pathogenesis in the brain.However, an 80S complex may assemble at an upstream translation initiation site (uTIS) if the 5'UTR has an optimal AUG (or near-cognate NUG) or at a downstream translation initiation site (dTIS) if the aTIS is suboptimal 9 . Similarly, in conventional termination, the 80S complex concludes translation at the first in-frame stop codon. For some transcripts, however, it may read past that stop codon at some frequency and conclude translation at a se...

Stem Cell Quiescence: Dynamism, Restraint, and Cellular Idling

“…Though TRAP can provide a proxy for protein levels, protein abundance also depends on the rate of translation and protein degradation. Recently, TRAP was combined with ribosome footprinting, which can define the exact location of a ribosome on the transcript and aides in addressing translation rates and provide an even better proxy for protein levels (Sapkota et al, 2018).…”

Identification of compounds enhancingAqp4stop codon readthrough

“…We began by evaluating a dual luciferase assay that we have previously used to quantify readthrough (Sapkota et al, 2019) for its suitability as a screening approach. This assay utilizes a dual luciferase vector with an Aqp4 test cassette cloned in between Renilla and Firefly luciferases in such a way that the former is expressed constitutively, but the latter only if the Aqp4 stop codon is read past by ribosomes.…”

“…Viruses use stop codon readthrough to increase their protein repertoire from a limited amount of genetic material (Firth and Brierley, 2012). Higher-order organisms, from yeasts to humans, also use a programmed readthrough in a subset of transcripts (Dunn et al, 2013;Loughran et al, 2014;Sapkota et al, 2019). Studies on a few of these transcripts have shown that the C-terminally extended protein variants arising from readthrough can gain new properties (Loughran et al, 2018;Schueren et al, 2014).…”

“…In the case of Aqp4, which encodes an astrocyte-specific water channel protein Aquaporin 4 (AQP4), we and others have recently shown that the readthrough-extended AQP4 is exclusively perivascular in the brain whereas the normal-length AQP4 is restricted away from blood vessels (De Bellis et al, 2017;Sapkota et al, 2019). Importantly, AQP4, specifically its perivascular pool, is a key mediator of the glymphatic system, which clears amyloid beta and other waste products from the brain (Iliff et al, 2012;Mestre et al, 2018).…”

“…We have previously utilized a dual-luciferase assay to quantify stop codon readthrough of fragments from several brain transcripts (Sapkota et al, 2019). Here, we adapt the same assay to screen compounds for their ability to modulate Aqp4 stop codon readthrough.…”