Control Of Protein Stability Research Articles

The scaffolding function of LSD1 controls DNA methylation in mouse ESCs

Lysine-specific histone demethylase 1 (LSD1), which demethylates mono- or di- methylated histone H3 on lysine 4 (H3K4me1/2), is essential for early embryogenesis and development. Here we show that LSD1 is dispensable for mouse embryonic stem cell (ESC) self-renewal but is required for mouse ESC growth and differentiation. Reintroduction of a catalytically-impaired LSD1 (LSD1MUT) recovers the proliferation capability of mouse ESCs, yet the enzymatic activity of LSD1 is essential to ensure proper differentiation. Indeed, increased H3K4me1 in Lsd1 knockout (KO) mouse ESCs does not lead to major changes in global gene expression programs related to stemness. However, ablation of LSD1 but not LSD1MUT results in decreased DNMT1 and UHRF1 proteins coupled to global hypomethylation. We show that both LSD1 and LSD1MUT control protein stability of UHRF1 and DNMT1 through interaction with HDAC1 and the ubiquitin-specific peptidase 7 (USP7), consequently, facilitating the deacetylation and deubiquitination of DNMT1 and UHRF1. Our studies elucidate a mechanism by which LSD1 controls DNA methylation in mouse ESCs, independently of its lysine demethylase activity.

ASB3 expression aggravates inflammatory bowel disease by targeting TRAF6 protein stability and affecting the intestinal microbiota.

Ubiquitination is a key process that controls protein stability. We determined the ubiquitination of TRAF6 by ASB3 in intestinal epithelial cells during colonic inflammation. Inflammatory bowel disease patients exhibit upregulated ASB3 expression at focal sites, supporting the involvement of degradation of TRAF6, which promotes TLR-Myd88/TRIF-independent NF-κB aberrant activation and intestinal microbiota imbalance. Sustained inflammatory signaling in intestinal epithelial cells and dysregulated protective probiotic immune responses mediated by ASB3 collectively contribute to the exacerbation of inflammatory bowel disease. These findings provide insights into the pathogenesis of inflammatory bowel disease and suggest a novel mechanism by which ASB3 increases the risk of colitis. Our results suggest that future inhibition of ASB3 in intestinal epithelial cells may be a novel clinical strategy.

The biophysics of water in cell biology: perspectives on a keystone for both marine sciences and cancer research.

The biophysics of water, has been debated over more than a century. Although its importance is still underestimated, significant breakthroughs occurred in recent years. The influence of protein condensation on water availability control was documented, new findings on water-transport proteins emerged, and the way water molecules rearrange to minimize free energy at interfaces was deciphered, influencing membrane thermodynamics. The state of knowledge continued to progress in the field of deep-sea marine biology, highlighting unknown effects of high hydrostatic pressure and/or temperature on interactions between proteins and ligands in extreme environments, and membrane structure adaptations. The role of osmolytes in protein stability control under stress is also discussed here in relation to fish egg hydration/buoyancy. The complexity of water movements within the cell is updated, all these findings leading to a better view of their impact on many cellular processes. The way water flow and osmotic gradients generated by ion transport work together to produce the driving force behind cell migration is also relevant to both marine biology and cancer research. Additional common points concern water dynamic changes during the neoplastic transformation of cells and tissues, or embryo development. This could improve imaging techniques, early cancer diagnosis, and understanding of the molecular and physiological basis of buoyancy for many marine species.

Abstract 4508: Defining PIM kinase protein regulation and PROTAC for pre-clinical testing

Abstract The PIM kinase family are serine/threonine kinases known to be overexpressed in castration-resistant prostate cancer (CRPC). PIM1, an isoform of PIM, is known to regulate cell survival, migration, and proliferation. Uniquely, PIM kinases are constitutively active due to the lack of a regulatory domain. Therefore, PIM protein levels are reflected in PIM activity and are regulated by protein degradation and synthesis. PIM inhibitors have been developed, targeting the PIM kinase activity and inhibiting phosphorylation of downstream targets, but have had limited efficacy. Our lab and others demonstrate that PIM inhibitor treatment increases total PIM protein levels, suggesting that PIM kinases control protein stability via autophosphorylation. To elucidate the mechanism of autophosphorylation dependent regulation of PIM1, we used phospho-proteomics to identify potential autophosphorylation sites on PIM1. We have identified overexpression of deubiquitinase USP28 inhibiting degradation of PIM1, leading to the investigation of the role of F-box protein Fbw7 in regulating PIM1. Dominant-negative expression of Fbw7 results in increased PIM1 total protein. Additionally, coimmunoprecipitation of Fbw7 knockdown cells resulted in decreased PM1 ubiquitination compared to control. Therefore, we have identified Fbw7 as a putative E3 ligase responsible for PIM1 degradation. To address the limited efficacy of PIM inhibitors, we have developed the first PIM-PROTAC (PIMTAC) with our collaborators. Proteolysis targeting chimera (PROTAC) is a promising approach to eliminating the pro-tumorigenic effects of PIM. The PIMTAC selectively targets PIM kinases for degradation and we have demonstrated in vitro efficacy. PIMTAC successfully maintains degradation of all isoforms of PIM for over 72 hours. In vitro assessment of anti-tumor efficacy demonstrated increased apoptotic death with PIMTAC alone compared to PIM inhibitor or docetaxel. Furthermore, dual treatment of PIMTAC and docetaxel had more apoptotic death compared to PIM inhibitor with docetaxel. We aim to develop the PIMTAC for pre-clinical testing. Citation Format: Hope Liou, John Brognard, Rolf Swenson, Pedro Torres-Ayuso, Noel Warfel. Defining PIM kinase protein regulation and PROTAC for pre-clinical testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4508.

Insights into a functional model of key deubiquitinases UBP12/13 in plants.

Understanding the complexities of protein ubiquitination is crucial, as it plays a multifaceted role in controlling protein stability, activity, subcellular localization, and interaction, which are central to diverse biological processes. Deubiquitinases (DUBs) serve to reverse ubiquitination, but research progress in plant DUBs is noticeably limited. Among existing studies, UBIQUITIN-SPECIFIC PROTEASE 12 (UBP12) and UBP13 have garnered attention for their extensive role in diverse biological processes in plants. This review systematically summarizes the recent advancements in UBP12/13 studies, emphasizing their function, and their substrate specificity, their relationship with E3 ubiquitin ligases, and the similarities and differences with their mammalian orthologue, USP7. By unraveling the molecular mechanisms of UBP12/13, this review offers in-depth insights into the ubiquitin-proteasome system (UPS) in plants and aims to catalyze further explorations and comprehensive understanding in this field.

Ubiquitin ligase RNF20 coordinates sequential adipose thermogenesis with brown and beige fat-specific substrates

In mammals, brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) execute sequential thermogenesis to maintain body temperature during cold stimuli. BAT rapidly generates heat through brown adipocyte activation, and further iWAT gradually stimulates beige fat cell differentiation upon prolonged cold challenges. However, fat depot-specific regulatory mechanisms for thermogenic activation of two fat depots are poorly understood. Here, we demonstrate that E3 ubiquitin ligase RNF20 orchestrates adipose thermogenesis with BAT- and iWAT-specific substrates. Upon cold stimuli, BAT RNF20 is rapidly downregulated, resulting in GABPα protein elevation by controlling protein stability, which stimulates thermogenic gene expression. Accordingly, BAT-specific Rnf20 suppression potentiates BAT thermogenic activity via GABPα upregulation. Moreover, upon prolonged cold stimuli, iWAT RNF20 is gradually upregulated to promote de novo beige adipogenesis. Mechanistically, iWAT RNF20 mediates NCoR1 protein degradation, rather than GABPα, to activate PPARγ. Together, current findings propose fat depot-specific regulatory mechanisms for temporal activation of adipose thermogenesis.

Targeted Protein Degradation Systems: Controlling Protein Stability Using E3 Ubiquitin Ligases in Eukaryotic Species.

This review explores various methods for modulating protein stability to achieve target protein degradation, which is a crucial aspect in the study of biological processes and drug design. Thirty years have passed since the introduction of heat-inducible degron cells utilizing the N-end rule, and methods for controlling protein stability using the ubiquitin-proteasome system have moved from academia to industry. This review covers protein stability control methods, from the early days to recent advancements, and discusses the evolution of techniques in this field. This review also addresses the challenges and future directions of protein stability control techniques by tracing their development from the inception of protein stability control methods to the present day.

A pepper RING-finger E3 ligase, CaFIRF1, negatively regulates the high-salt stress response by modulating the stability of CaFAF1.

Controlling protein stability or degradation via the ubiquitin-26S proteasome system is a crucial mechanism in plant cellular responses to stress conditions. Previous studies have revealed that the pepper FANTASTIC FOUR-like gene, CaFAF1, plays a positive role in salt tolerance and that, in this process, CaFAF1 protein degradation is delayed. Here, we sought to isolate the E3 ligases potentially responsible for modulating CaFAF1 protein stability in response to salt stress. The pepper RING-type E3 ligase CaFIRF1 (Capsicum annuum FAF1 Interacting RING Finger protein 1) was found to interact with and ubiquitinate CaFAF1, leading to the degradation of CaFAF1 proteins. In response to high-salt treatments, CaFIRF1-silenced pepper plants exhibited tolerant phenotypes. In contrast, co-silencing of CaFAF1 and CaFIRF1 led to increased sensitivity to high-salt treatments, revealing that CaFIRF1 functions upstream of CaFAF1. A cell-free degradation analysis showed that high-salt treatment suppressed CaFAF1 protein degradation via the 26S proteasome pathway, in which CaFIRF1 is functionally involved. In addition, an in vivo ubiquitination assay revealed that CaFIRF1-mediated ubiquitination of CaFAF1 proteins was reduced by high-salt treatment. Taken together, these findings suggest that the degradation of CaFAF1 mediated by CaFIRF1 has a critical role in pepper plant responses to high salinity.

Contribution of a surface salt bridge to the protein stability of deep-sea Shewanella benthica cytochrome c'

Two homologous cytochromes c', SBCP and SVCP, from deep-sea Shewanella benthica and Shewanella violacea respectively exhibit only nine surface amino acid substitutions, along with one at the N-terminus. Despite the small sequence difference, SBCP is thermally more stable than SVCP. Here, we examined the thermal stability of SBCP variants, each containing one of the nine substituted residues in SVCP, and found that the SBCP K87V variant was the most destabilized. We then determined the X-ray crystal structure of the SBCP K87V variant at a resolution of 2.1 Å. The variant retains a four-helix bundle structure similar to the wild-type, but notable differences are observed in the hydration structure around the mutation site. Instead of forming of the intrahelical salt bridge between Lys-87 and Asp-91 in the wild-type, a clathrate-like hydration around Val-87 through a hydrogen bond network with the nearby amino acid residues is observed. This network potentially enhances the ordering of surrounding water molecules, leading to an entropic destabilization of the protein. These results suggest that the unfavorable hydrophobic hydration environment around Val-87 and the inability to form the Asp-91-mediated salt bridge contribute to the observed difference in stability between SBCP and SVCP. These findings will be useful in future protein engineering for controlling protein stability through the manipulation of surface intrahelical salt bridges.

Transcriptional and post-translational regulation of plant autophagy.

In response to changing environmental conditions, plants activate cellular responses to enable them to adapt. One such response is autophagy, in which cellular components, for example proteins and organelles, are delivered to the vacuole for degradation. Autophagy is activated by a wide range of conditions, and the regulatory pathways controlling this activation are now being elucidated. However, key aspects of how these factors may function together to properly modulate autophagy in response to specific internal or external signals are yet to be discovered. In this review we discuss mechanisms for regulation of autophagy in response to environmental stress and disruptions in cell homeostasis. These pathways include post-translational modification of proteins required for autophagy activation and progression, control of protein stability of the autophagy machinery, and transcriptional regulation, resulting in changes in transcription of genes involved in autophagy. In particular, we highlight potential connections between the roles of key regulators and explore gaps in research, the filling of which can further our understanding of the autophagy regulatory network in plants.

Controlling protein stability with SULI, a highly sensitive tag for stabilization upon light induction

Optogenetics tools for precise temporal and spatial control of protein abundance are valuable in studying diverse complex biological processes. In the present study, we engineer a monomeric tag of stabilization upon light induction (SULI) for yeast and zebrafish based on a single light-oxygen-voltage domain from Neurospora crassa. Proteins of interest fused with SULI are stable upon light illumination but are readily degraded after transfer to dark conditions. SULI shows a high dynamic range and a high tolerance to fusion at different positions of the target protein. Further studies reveal that SULI-mediated degradation occurs through a lysine ubiquitination-independent proteasome pathway. We demonstrate the usefulness of SULI in controlling the cell cycle in yeast and regulating protein stability in zebrafish, respectively. Overall, our data indicate that SULI is a simple and robust tool to quantitatively and spatiotemporally modulate protein levels for biotechnological or biomedical applications.

Associated Water Dynamics Might Be a Key Factor Affecting Protein Stability in the Crowded Milieu.

Over the past 20 years, the most studied and debated aspect of macromolecular crowding is how it affects protein stability. Traditionally, it is explained by a delicate balance between the stabilizing entropic effect and the stabilizing or destabilizing enthalpic effect. However, this traditional crowding theory cannot explain experimental observations like (i) negative entropic effect and (ii) entropy-enthalpy compensation. Herein, we provide experimental evidence that associated water dynamics plays a crucial role in controlling protein stability in the crowded milieu for the first time. We have correlated the modulation of associated water dynamics with the overall stability and its individual components. We showed that rigid associated water would stabilize the protein through entropy but destabilize it through enthalpy. In contrast, flexible associated water destabilizes the protein through entropy but stabilizes through enthalpy. Consideration of entropic and enthalpic modulation through crowder-induced distortion of associated water successfully explains the negative entropic part and entropy-enthalpy compensation. Furthermore, we argued that the relationship between the associated water structure and protein stability should be better understood by individual entropic and enthalpic components instead of the overall stability. Although a huge effort is necessary to generalize the mechanism, this report provides a unique way of understanding the relationship between protein stability and associated water dynamics, which might be a generic phenomenon and should trigger much research in this area.

TMAO: Protecting proteins from feeling the heat

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolyte trimethylamine N-oxide (TMAO) is used by marine animals to counteract the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and urea denaturation has been extensively studied, but unlike the case of other osmolytes, the ability of TMAO to protect proteins from high temperature has not been quantified. To reveal the effect of TMAO on folded and unfolded protein ensembles and the hydration shell at different temperatures, we study a mutant of the well-characterized, fast-folding model protein B (PRB). We carried out, in total, >190 μs all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations show increased thermal stability of PRB in the presence of TMAO. Partly structured, compact ensembles are favored over the unfolded state. TMAO forms two shells near the protein: an outer shell away from the protein surface has altered H-bond lifetimes of water molecules and increases hydration of the protein to help stabilize it; a less-populated inner shell with an opposite TMAO orientation closer to the protein surface binds exclusively to basic side chains. The cooperative cosolute effect of the inner and outer shell TMAO has a small number of TMAO molecules “herding” water molecules into two hydration shells at or near the protein surface. The stabilizing effect of TMAO on our protein saturates at 1 M despite higher TMAO solubility, so there may be little evolutionary pressure for extremophiles to produce higher intracellular TMAO concentrations, if true in general.

Osmolyte induced protein stabilization: modulation of associated water dynamics might be a key factor.

The mechanism of protein stabilization by osmolytes remains one of the most important and long-standing puzzles. The traditional explanation of osmolyte-induced stability through the preferential exclusion of osmolytes from the protein surface has been seriously challenged by the observations like the concentration-dependent reversal of osmolyte-induced stabilization/destabilization. The more modern explanation of protein stabilization/destabilization by osmolytes considers an indirect effect due to osmolyte-induced distortion of the water structure. It provides a general mechanism, but there are numerous examples of protein-specific effects, i.e., a particular osmolyte might stabilize one protein, but destabilize the other, that could not be rationalized through such an explanation. Herein, we hypothesized that osmolyte-induced modulation of associated water might be a critical factor in controlling protein stability in such a medium. Taking different osmolytes and papain as a protein, we proved that our proposal could explain protein stability in osmolyte media. Stabilizing osmolytes rigidify associated water structures around the protein, whereas destabilizing osmolytes make them flexible. The strong correlation between the stability and the associated water dynamics, and the fact that such dynamics are very much protein specific, established the importance of considering the modulation of associated water structures in explaining the osmolyte-induced stabilization/destabilization of proteins. More interestingly, we took another protein, bromelain, for which a traditionally stabilizing osmolyte, sucrose, acts as a stabilizer at higher concentrations but as a destabilizer at lower concentrations. Our proposal successfully explains such observations, which is probably impossible by any known mechanisms. We believe this report will trigger much research in this area.

F-box receptor mediated control of substrate stability and subcellular location organizes cellular development of Aspergillus nidulans.

Fungal growth and development are coordinated with specific secondary metabolism. This coordination requires 8 of 74 F-box proteins of the filamentous fungus Aspergillus nidulans. F-box proteins recognize primed substrates for ubiquitination by Skp1-Cul1-Fbx (SCF) E3 ubiquitin RING ligases and degradation by the 26S proteasome. 24 F-box proteins are found in the nuclear fraction as part of SCFs during vegetative growth. 43 F-box proteins interact with SCF proteins during growth, development or stress. 45 F-box proteins are associated with more than 700 proteins that have mainly regulatory roles. This corroborates that accurate surveillance of protein stability is prerequisite for organizing multicellular fungal development. Fbx23 combines subcellular location and protein stability control, illustrating the complexity of F-box mediated regulation during fungal development. Fbx23 interacts with epigenetic methyltransferase VipC which interacts with fungal NF-κB-like velvet domain regulator VeA that coordinates fungal development with secondary metabolism. Fbx23 prevents nuclear accumulation of methyltransferase VipC during early development. These results suggest that in addition to their role in protein degradation, F-box proteins also control subcellular accumulations of key regulatory proteins for fungal development.

A Class of Disulfide Compounds Suppresses Ferroptosis by Stabilizing GPX4.

Ferroptosis is a nonapoptotic form of cell death characterized by iron-dependent lipid peroxidation and has been implicated in multiple pathological conditions. Glutathione peroxidase 4 (GPX4) plays an essential role in inhibiting ferroptosis by eliminating lipid peroxide using glutathione (GSH) as a reductant. In this study, we found Ellman's reagent DTNB and a series of disulfide compounds, including disulfiram (DSF), an FDA-approved drug, which protect cells from erastin-induced ferroptosis. Mechanistically, DTNB or DSF is conjugated to multiple cysteine residues in GPX4 and disrupts GPX4 interaction with HSC70, an adaptor protein for chaperone mediated autophagy, thus preventing GPX4 degradation induced by erastin. In addition, DSF ameliorates concanavalin A induced acute liver injury by suppressing ferroptosis in a mouse model. Our work reveals a novel regulatory mechanism for GPX4 protein stability control. We also discover disulfide compounds as a new class of ferroptosis inhibitors and suggest therapeutic repurposing of DSF in treating ferroptosis-related diseases.

Regulation of CLB6 expression by the cytoplasmic deadenylase Ccr4 through its coding and 3’ UTR regions

RNA stability control contributes to the proper expression of gene products. Messenger RNAs (mRNAs) in eukaryotic cells possess a 5’ cap structure and the 3’ poly(A) tail which are important for mRNA stability and efficient translation. The Ccr4-Not complex is a major cytoplasmic deadenylase and functions in mRNA degradation. The CLB1-6 genes in Saccharomyces cerevisiae encode B-type cyclins which are involved in the cell cycle progression together with the cyclin-dependent kinase Cdc28. The CLB genes consist of CLB1/2, CLB3/4, and CLB5/6 whose gene products accumulate at the G2-M, S-G2, and late G1 phase, respectively. These Clb protein levels are thought to be mainly regulated by the transcriptional control and the protein stability control. Here we investigated regulation of CLB1-6 expression by Ccr4. Our results show that all CLB1-6 mRNA levels were significantly increased in the ccr4Δ mutant compared to those in wild-type cells. Clb1, Clb4, and Clb6 protein levels were slightly increased in the ccr4Δ mutant, but the Clb2, Clb3, and Clb5 protein levels were similar to those in wild-type cells. Since both CLB6 mRNA and Clb6 protein levels were most significantly increased in the ccr4Δ mutant, we further analyzed the cis-elements for the Ccr4-mediated regulation within CLB6 mRNA. We found that there were destabilizing sequences in both coding sequence and 3’ untranslated region (3’ UTR). The destabilizing sequences in the coding region were found to be both within and outside the sequences corresponding the cyclin domain. The CLB6 3’ UTR was sufficient for mRNA destabilization and decrease of the reporter GFP gene and this destabilization involved Ccr4. Our results suggest that CLB6 expression is regulated by Ccr4 through the coding sequence and 3’ UTR of CLB6 mRNA.

G protein‐coupled receptor regulation of the alpha‐arrestin ARRDC3

Adaptor proteins are an important class of proteins that help to maintain proper spatiotemporal control of cell signaling. One of the best‐studied families of adaptor proteins are the ubiquitously expressed beta‐arrestins, which are known to be key regulators of G protein‐coupled receptor (GPCR) function. Recently another family of arrestins, termed the alpha‐arrestins, were discovered and shown to be expressed in mammalian cells. While alpha‐ and beta‐arrestins share minimal sequence homology, they are predicted to be structurally similar, and therefore may be subject to common regulatory mechanisms. However, while GPCR regulation of beta‐arrestin function has been well characterized, it remains entirely unknown how GPCRs might regulate alpha‐arrestins, such as ARRDC3. We recently showed that ARRDC3 is a multifunctional adaptor that distinctly regulates protease‐activated receptor‐1 (PAR1) signaling and trafficking, but it remains unknown how these distinct ARRDC3 functions may be regulated. PAR1 is a GPCR for the protease thrombin and is known to regulate a diverse array of physiological functions in health and disease, thus, it is important to better understand the molecular mechanisms that control PAR1 function. The objective of this study is to understand the mechanisms that control ARRDC3 multifaceted functions. We hypothesize that GPCRs regulate ARRDC3 function via distinct processes that enable diverse functions. Using biochemical and microscopy‐based techniques, we show that PAR1 may regulate ARRDC3 through modulation of post‐translational modifications, including ubiquitination. We also show that ubiquitin is critical for regulating distinct ARRDC3 functions, including regulation of GPCR signaling and trafficking, through various mechanisms that may include control of protein stability, subcellular localization, and protein‐protein interactions. Additionally, ARRDC3 has previously been shown to act as a tumor suppressor in highly invasive triple‐negative breast cancers. Thus, in future studies we will assess the role of ubiquitination on ARRDC3 tumor suppressor function in vitro in vivo. The results of these studies will advance our understanding of how alpha‐arrestins may contribute to GPCR regulation and may lead to the identification of novel targets for therapeutic development to treat metastatic breast cancer.

A dual conditional CRISPR-Cas9 system to activate gene editing and reduce off-target effects in human stem cells

The CRISPR-Cas9 system has emerged as a powerful and efficient tool for genome editing. An important drawback of the CRISPR-Cas9 system is the constitutive endonuclease activity when Cas9 endonuclease and its sgRNA are co-expressed. This constitutive activity results in undesirable off-target effects that hinder studies using the system, such as probing gene functions or its therapeutic use in humans. Here, we describe a convenient method that allows temporal and tight control of CRISPR-Cas9 activity by combining transcriptional regulation of Cas9 expression and protein stability control of Cas9 in human stem cells. To achieve this dual control, we combined the doxycycline-inducible system for transcriptional regulation and FKBP12-derived destabilizing domain fused to Cas9 for protein stability regulation. We showed that approximately 5%–10% of Cas9 expression was observed when only one of the two controls was applied. By combining two systems, we markedly lowered the baseline Cas9 expression and limited the exposure time of Cas9 endonuclease in the cell, resulting in little or no undesirable on- or off-target effects. We anticipate that this dual conditional CRISPR-Cas9 system can serve as a valuable tool for systematic characterization and identification of genes for various pathological processes.

Multilayered regulations of alternative splicing, NMD, and protein stability control temporal induction and tissue-specific expression of TRIM46 during axon formation

The gene regulation underlying axon formation and its exclusiveness to neurons remains elusive. TRIM46 is postulated to determine axonal fate. We show Trim46 mRNA is expressed before axonogenesis, but TRIM46 protein level is inhibited by alternative splicing of two cassette exons coupled separately to stability controls of Trim46 mRNA and proteins, effectively inducing functional knockout of TRIM46 proteins. Exon 8 inclusion causes nonsense-mediated mRNA decay of Trim46 transcripts. PTBP2-mediated exon 10 skipping produces transcripts encoding unstable TRIM46 proteins. During axonogenesis, transcriptional activation, decreased exon 8 inclusion, and enhanced exon 10 inclusion converge to increase TRIM46 proteins, leading to its neural-specific expression. Genetic deletion of these exons alters TRIM46 protein levels and shows TRIM46 is instructive though not always required for AnkG localization nor a determinant of AnkG density. Therefore, two concurrently but independently regulated alternative exons orchestrate the temporal induction and tissue-specific expression of TRIM46 proteins to mediate axon formation.