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FMRP Links Optimal Codons to Mrna Stability in Neurons

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FMRP Links Optimal Codons to Mrna Stability in Neurons FMRP links optimal codons to mRNA stability in neurons Huan Shua,1, Elisa Donnardb, Botao Liua, Suna Junga, Ruijia Wanga, and Joel D. Richtera aProgram in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605; and bBioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA 01605 Edited by Lynne E. Maquat, University of Rochester School of Medicine and Dentistry, Rochester, NY, and approved October 12, 2020 (received for review May 8, 2020) Fragile X syndrome (FXS) is caused by inactivation of the FMR1 Here, we have used ribosome profiling and RNA sequencing gene and loss of encoded FMRP, an RNA binding protein that re- (RNA-seq) to investigate translational dysregulation in the presses translation of some of its target transcripts. Here we use FMRP KO cortex and found that FMRP coordinates the link ribosome profiling and RNA sequencing to investigate the dysre- between RNA destruction and codon usage bias (codon opti- gulation of translation in the mouse brain cortex. We find that mality). We find that the apparent dysregulation of translational most changes in ribosome occupancy on hundreds of mRNAs are activity (i.e., ribosome occupancy) in FMRP KO cortex can be largely driven by dysregulation in transcript abundance. Many accounted for by commensurate changes in steady-state RNA down-regulated mRNAs, which are mostly responsible for neuro- levels. Down-regulated mRNAs in FMRP KO cortex are nal and synaptic functions, are highly enriched for FMRP binding enriched for those that encode factors involved in neuronal and targets. RNA metabolic labeling demonstrates that, in FMRP- synaptic functions and are highly enriched for FMRP binding deficient cortical neurons, mRNA down-regulation is caused by targets. These observations suggest that, in the cortex, FMRP elevated degradation and is correlated with codon optimality. directly or indirectly regulates RNA stability. Indeed, RNA Moreover, FMRP preferentially binds mRNAs with optimal codons, metabolic profiling by 5-ethynyl uridine (5-EU) incorporation suggesting that it stabilizes such transcripts through direct inter- and whole-transcriptome sequencing reveals widespread RNA actions via the translational machinery. Finally, we show that the degradation in FMRP KO cortical neurons, while synthesis and paradigm of genetic rescue of FXS-like phenotypes in FMRP- processing rates remained substantially unchanged. Of the ∼700 Cpeb1 BIOCHEMISTRY deficient mice by deletion of the gene is mediated by res- mRNAs that degraded significantly faster in FMRP KO cortex toration of steady-state RNA levels and consequent rebalancing of compared to WT, those enriched for optimal codons were par- translational homeostasis. Our data establish an essential role of ticularly affected. This widespread codon-dependent RNA de- FMRP in codon optimality-dependent mRNA stability as an impor- struction in FMRP-deficient neurons involves a massive tant factor in FXS. reshuffling of the identities of stabilizing or destabilizing codons. FMRP | codon optimality | RNA decay | ribosome profiling | CPEB1 Moreover, FMRP can distinguish between optimal and nonop- timal codon-containing mRNAs, probably through the associ- ated translational machinery. Finally, we demonstrate that, in a ragile X syndrome (FXS) is the most common form of genetic rescue paradigm of FXS where a double deficiency of Finherited intellectual disability caused by a single gene mu- FMRP and CPEB1 mitigates the disorder in mice, restoration of tation (1). FXS is caused by a trinucleotide repeat expansion in RNA levels drives the recovery of ribosome occupancy. These the FMR1 locus, which results in transcriptional silencing and loss of its protein product, FMRP (2). In FMRP-deficient mice, Significance protein synthesis in the brain is elevated by ∼15 to 20% (3, 4), indicating that FMRP represses translation. In the mouse brain, FMRP binds mostly to coding regions of ∼850 to 1,000 mRNAs Fragile X syndrome (FXS), the most prevalent monogenic form (5–9) and cosediments with polyribosomes (5). FMRP has been of autism, is caused by the loss of FMRP, an RNA binding pro- proposed to repress translation by impeding ribosome translo- tein. In the absence of FMRP, translation is dysregulated, but cation (5, 10–13). This hypothesis is based on the evidence that restoration of translational homeostasis rescues the syndrome ribosomes associated with many of these mRNAs are resistant to and thus could suggest new treatments for the disorder. Using puromycin treatment in vitro, which causes premature polypep- ribosome profiling and RNA metabolic profiling, we show that, in the FMRP-deficient mouse brain, there are few specific tide release and thus is an indirect measure of ribosome trans- translational disturbances. Instead, there is widespread imbal- location, and that ribosomes transit at faster rates in FMRP- ance of RNA levels. This imbalance is caused by destabilization knockout (KO) brain extracts compared to WT (5, 10). FMRP of the FMRP targets and other mRNAs based on codon opti- also regulates translation directly or indirectly at the level of mality. Rebalancing the transcriptome may therefore be a key initiation (14, 15), RNA splicing (13), editing (16, 17), nuclear 6 – to correcting syndrome-related pathophysiologies. This study export (18, 19), and m A modifications (18 20). However, the establishes a role for FMRP in linking codon optimality to RNA relationship of these molecular impairments to the etiology of stability. FXS, if any, is unknown. Translation is also controlled by the supply and demand of Author contributions: H.S., E.D., and J.D.R. designed research; H.S., B.L., and S.J. per- available tRNAs (21). When particular codons represented in formed research; E.D. contributed new reagents/analytic tools; H.S., E.D., and R.W. ana- mRNA are not met by a sufficient supply of charged tRNA, ri- lyzed data; and H.S., E.D., and J.D.R. wrote the paper. bosome transit slows or stalls, which in turn causes RNA de- The authors declare no competing interest. struction (22–27). Control of specific translation and RNA This article is a PNAS Direct Submission. destruction by codon bias varies with tissue (24), time of develop- Published under the PNAS license. ment (23), and cell stress (28). Additionally, in yeast, transacting 1To whom correspondence may be addressed. Email: [email protected]. factors can influence codon bias-mediated RNA destruction (22), This article contains supporting information online at https://www.pnas.org/lookup/suppl/ indicating a more complex regulation than simple codon–tRNA doi:10.1073/pnas.2009161117/-/DCSupplemental. balance. www.pnas.org/cgi/doi/10.1073/pnas.2009161117 PNAS Latest Articles | 1of12 Downloaded by guest on September 30, 2021 results indicate that a primary consequence of FMRP depletion moderately (10 to 15%) higher rate of protein synthesis in from the brain is the uncoupling of codon bias from the RNA FMRP-deficient brain (3, 4, 30). Using Xtail (31), an algorithm destruction machinery. This uncoupling may be a general that tests for differential ROs (DROs) between samples, we mechanism that underlies FXS, and restoration of the RNA identified 431 mRNAs with DROs between FK and WT (false stability landscape could be a key to ameliorating the disorder. discovery rate [FDR] < 0.05; Fig. 1B, SI Appendix, Fig. S1A, and Dataset S1). Consistent with FMRP acting as a translation re- Results pressor, 80% of these mRNAs (345 of 431) have increased RO. Steady-State RNA Level Changes Drive Translational Buffering in To determine the underlying cause of DRO, we analyzed our Fragile X Brain Cortex. To identify mRNAs that are translation- RPF and RNA-seq data separately. DRO can result from ally dysregulated in FMRP-deficient mouse cortex, we per- translational dysregulation driven by differential RPF but with formed ribosome profiling (29) and RNA-seq from WT and little change in RNA levels; conversely, translational buffering FMRP KO (FK) animals (Fig. 1A). Ribosome occupancy (RO), occurs when RPFs are unchanged but the DRO is driven by defined as ribosome protected fragments (RPFs) normalized to dysregulated RNA levels (32). The RPF changes in the DRO mRNA levels, is a measure of translational activity (29) and has mRNAs are subtle; however, the changes in RNA levels strongly served as a proxy for protein synthesis. Accumulating evidence oppose the changes in RO (Fig. 1B). When taken as a group, suggests that one mechanism whereby FMRP inhibits translation these mRNAs with altered ROs have stronger changes at RNA is by stalling ribosome transit (5, 10, 12, 13), and indeed there is a levels than at RPF levels (SI Appendix, Fig. S1A). A B all DRO mRNAs WT FK log2FC 2 ribosome profiling RNA-seqNA-se 1 AAAA AAAA 0 AA AA −1 RNase treatment RPF RO RNA −2 random fragmentation AAAA RPF extraction AA C n = 5, P-value = 3.19 x 10-11 RO down (86) RNA up (12) library generation deep sequencing RNA down (93) RO up (345) RPF RNA n = 75 mRNA P-value = 5.89 x 10-110 stop codon start codon D downregulated RNAs fold enrichment 0 5 10 15 20 25 30 35 plasma membrane bounded cell projection morphogenesis chemical synaptic transmission brain development regulation of AMPA receptor activity transcription from RNA polymerase II promoter histone H3−K4 methylation positive regulation of calcium ion−dependent exocytosis 0246810 -log10(P) E n = 58 F P-value = 1.01 x 10-104 RPF RO RNA 1.00 RNA up (12) action 0.75 FMRP targets e fr 0.50 no RNA down (93) yes ulativ 0.25 cum 0.00 FMRP targets −0.4 0.0 0.4 −0.4 0.0 0.4 −0.4 0.0 0.4 (842) log2FC, FK vs WT Fig. 1. Steady-state RNA level changes drive translational buffering in Fragile X brain cortex. (A) Illustration of the experimental pipeline of ribosome profiling and RNA-seq for WT and FK mouse brain cortices.
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