The Role of Upstream Open Reading Frames in Regulating Neuronal Protein Synthesis

[摘要] Spatial and temporal control of protein synthesis in response to activity is required for neuronal function and plasticity. mRNA structure and sequence provide a powerful platform for such regulation, but how such information is utilized in neurons is incompletely understood. In my thesis, I explore how functional elements within 5’leaders (traditionally termed 5’UTR or untranslated region) of mRNAs act as cis-regulatory elements to influence basal and activity-dependent translation in neurons.First, I identified a specific role for upstream open reading frames (uORFs) in regulating mRNA translation during neuronal differentiation. uORFs are regions within the 5’ leader that undergo translation. Using ribosome profiling (RP), an emerging next-generation sequencing technique which utilizes a modified RNA-sequencing library preparation to detect regions of mRNA occupied by actively translating ribosomes, I identified thousands of uORFs in human neuroblastoma cells. A portion of these uORFs demonstrated clear usage shifts with differentiation. Highly conserved uORFs exhibited increased GC content and were associated with cumulatively repressed CDSs. Importantly, changes in the translational efficiency of these conserved uORFs across differentiation were inversely correlated with CDS translation on these same transcripts. These data demonstrate uORF usage is common in neuroblastoma cells and that specific uORFs act as regulators of cell state-specific translation in neuronal differentiation. Next, I investigated the function of CGG repeats in the 5’ leader of FMR1. All humans have a conserved CGG-trinucleotide repeat (typically 20-45 repeats) in FMR1 that can become unstable and expand intergenerationally.Large expansions (>200 CGG repeats) cause Fragile X Syndrome, a common cause of intellectual disability, by silencing FMR1, leading to loss of the fragile X protein, FMRP. Intermediate (55-200 CGGs) expansions, in contrast, are transcribed and cause an age-related neurodegenerative condition known as Fragile-X Associated Tremor/Ataxia Syndrome (FXTAS). Our lab discovered that this repeat facilitates Repeat Associated Non-AUG translation (RANT), whereby ribosomes initiate at non-AUG codons upstream of the repeat to produce toxic homopolymeric proteins that drive pathogenesis in FXTAS. FMR1 avidly supports RANT at normal repeat sizes, suggesting that it might serve as a regulatory uORF to control FMRP synthesis. To address this, I expressed nanoluciferase reporters in rat hippocampal neurons. Using this strategy, I found that RANT exhibits a strong negative effect on FMRP synthesis at both normal and expanded repeats. FMRP is a key synaptic protein that is rapidly synthesized in response to mGluR activity. Importantly, preventing RANT or removing the repeat itself blocked this mGluR-induced response. This suggests that FMR1 relies on these two elements to appropriately scale synaptic FMRP synthesis. Using non-cleaving antisense oligonucleotides (ASOs) that target the RANT initiation sites, I found that blocking RANT could decrease toxic protein production and prevent neuronal death. In a line of iPSC-derived neurons from a patient with a large CGG repeat (>200) that still generates FMR1 mRNA but has deficits in FMRP, treatment with the ASO increased endogenous FMRP expression by 50%. These findings define a native function for RANT and CGG repeats in regulating FMRP synthesis, and delineate RANT as a therapeutic target in Fragile X-associated disorders.

[发布机构] University of Michigan

[效力级别] upstream open reading frame

[关键词] mRNA translation;upstream open reading frame;RAN translation;ribosome profiling;antisense oligonucleotide;activity-dependent translation;Molecular;Cellular and Developmental Biology;Science;Neuroscience