Destabilization Pathways Research Articles

Widespread destabilization of C. elegans microRNAs by the E3 ubiquitin ligase EBAX-1.

MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to form complexes that direct mRNA repression. miRNAs are also the subject of regulation. For example, some miRNAs are destabilized through a pathway in which pairing to specialized transcripts recruits the ZSWIM8 E3 ubiquitin ligase, which polyubiquitinates AGO, leading to its degradation and exposure of the miRNA to cellular nucleases. Here, we found that 22 miRNAs in C. elegans are sensitive to loss of EBAX-1, the ZSWIM8 ortholog in nematodes, implying that these 22 miRNAs might be subject to this pathway of target-directed miRNA degradation (TDMD). The impact of EBAX-1 depended on the developmental stage, with the greatest effect on the miRNA pool (14.5%) observed in L1 larvae and the greatest number of different miRNAs affected (17) observed in germline-depleted adults. The affected miRNAs included the miR-35-42 family, as well as other miRNAs among the least stable in the worm, suggesting that TDMD is a major miRNA destabilization pathway in the worm. The excess miR-35-42 molecules that accumulated in ebax-1 mutants caused increased repression of their predicted target mRNAs and underwent 3' trimming over time. In general, however, miRNAs sensitive to EBAX-1 loss had no consistent pattern of either trimming or tailing. Replacement of the 3' region of miR-43 substantially reduced EBAX-1 sensitivity, a result that differed from that observed previously for miR-35. Together, these findings broaden the implied biological scope of TDMD-like regulation of miRNA stability in animals, and indicate that a role for miRNA 3' sequences is variable in the worm.

Study in the stabilization of proteins encapsulated in PLGA delivery system: Effects of additives on protein encapsulation, release, and stability

Therapeutic proteins/peptides face severe stability impairment in biotechnology drug delivery systems prepared by the W/O/W solvent evaporation method. Screening of protein stabilizers for poly (D, l-lactide-co-glycolide) (PLGA) nanoparticles can improve the low encapsulation efficiency and undesirable release behavior of proteins caused by PLGA carriers, which is a crucial step to achieve a high proportion of continuous release of encapsulated protein in the target time and give full play to the therapeutic effect. Therefore, model proteins were selected for stability testing to simulate the specific destabilization pathways. Through the comprehensive study of protein conformation, recovery rate, encapsulation rate, and drug release behavior, the effects of additives on the stability of protein were synthetically characterized, and their action links and mechanisms were also further studied. The results showed that different denaturation environments were intertwined during the preparation and release of peptide-loaded NPs, jointly inducing protein denaturation and incomplete release behavior. After optimization with stabilizers, urea and lactose increased the encapsulation efficiency of proteins to more than 95% (P < 0.001). Except for the P188, all other stabilizers showed great potential for optimizing the release and stability of the encapsulated protein as a whole, increasing the release proportion of protein from 44% to 60 %-75% (P < 0.0001). The improvement of the drug release curve demonstrated the long-term stabilizing effect of the above-mentioned co-solubilizing additives on the therapeutic protein.

Antibody-mediated blockade of the IL23 receptor destabilizes intratumoral regulatory T cells and enhances immunotherapy

Regulatory T cells (Treg) can impede antitumor immunity and currently represent a major obstacle to effective cancer immunotherapy. Targeting tumor-infiltrating regulatory Treg while sparing systemic Treg represents an optimal approach to this problem. Here, we provide evidence that the interleukin 23 receptor (IL23R) expressed by tumor-infiltrating Treg promotes suppressive activity. Disruption of the IL23R results in increased responsiveness of destabilized Treg to the IL12 cytokine, the production of γ-interferon, and the recruitment of CD8 T cells that inhibit tumor growth. Since the Treg destabilization pathway that is initiated by IL23R blockade is distinct and independent from the destabilization pathway coupled to glucocorticoid-induced TNFR-related protein (GITR) activation, we examined the impact of the coordinate induction of the two destabilization pathways on antitumor immune responses. Combined GITR and IL23R antibody treatment of mice inoculated with MC38 tumors resulted in robust and synergistic antitumor responses. These findings indicate that the delineation of independent Treg destabilization pathways may allow improved approaches to the development of combination immunotherapy for cancers.

A Stilbenoid Isorhapontigenin as a Potential Anti-Cancer Agent against Breast Cancer through Inhibiting Sphingosine Kinases/Tubulin Stabilization.

Isorhapontigenin (ISO), a tetrahydroxylated stilbenoid, is an analog of resveratrol (Rsv). The various biological activities of Rsv and its derivatives have been previously reported in the context of both cancer and inflammation. However, the anti-cancer effect of ISO against breast cancer has not been well established, despite being an orally bioavailable dietary polyphenol. In this study, we determine the anti-cancer effects of ISO against breast cancer using MCF7, T47D, and MDA-MB-231 cell lines. We observed that ISO induces breast cancer cell death, cell cycle arrest, oxidative stress, and the inhibition of cell proliferation. Additionally, sphingosine kinase inhibition by ISO controlled tubulin polymerization and cancer cell growth by regulating MAPK/PI3K-mediated cell cycle arrest in MCF7 cells. Interestingly, SPHK1/2 gene silencing increased oxidative stress, cell death, and tubulin destabilization in MCF7 cells. This suggests that the anti-cancer effect of ISO can be regulated by SPHK/tubulin destabilization pathways. Overall, ISO successfully induced breast cancer cell death and cell growth arrest, suggesting this phytochemical is a better alternative for breast cancer treatment. Further studies in animal models could confirm the potency and usability of ISO over Rsv for targeting breast cancer, potentially posing an alternative candidate for improved therapy in the near future.

Freezing of Biologicals Revisited: Scale, Stability, Excipients, and Degradation Stresses.

Although many biotech products are successfully stored in the frozen state, there are cases of degradation of biologicals during freeze storage. These examples are discussed in the Perspective to emphasize the fact that stability of frozen biologicals should not be taken for granted. Frozen-state degradation (predominantly, aggregation) has been linked to crystallization of a cryoprotector in many cases. Other factors, for example, protein unfolding (either due to cold denaturation or interaction of protein molecules with ice crystals), could also contribute to the instability. As a hypothesis, additional freezing-related destabilization pathways are introduced in the paper, that is, air bubbles formed on the ice crystallization front, and local pressure and mechanical stresses due to volume expansion during water-to-ice transformation. Furthermore, stability of frozen biologicals can depend on the sample size, via its impact on the freezing kinetics (i.e., cooling rates and freezing time) and cryoconcentration effects, as well as on the mechanical stresses associated with freezing. We conclude that, although fundamentals of freezing processes are fairly well described in the current literature, there are important gaps to be addressed in both scientific foundations of the freezing-related manufacturing processes and implementation of the available knowledge in practice.

The stabilization of biopharmaceuticals: current understanding and future perspectives

Proteins have many therapeutic advantages over small synthetic drugs in terms of their high specificity and activity. Accordingly, the global biopharmaceutical market grows rapidly and has driven the continuous increase of R&D investment in protein-based drug products, thus the number of approved protein drugs also continuously increases. However, during their manufacture, transport, and storage, proteins are often exposed to various detrimental environments that can cause protein destabilization, resulting in undesirable drug properties such as partial or complete loss of biological activity, altered solubility, and immunogenicity. Therefore, the thorough evaluation/monitoring and optimization of process variables and product formulations are critical to ensure product quality during its appropriate shelf-life. However, because of (i) the complexity of protein structures, (ii) multiple degradation pathways, and (iii) various intrinsic and extrinsic factors that can affect the stability, protein stabilization is not straightforward and remains a big challenge in the clinical development and commercialization of protein-based drug products, although significant progress has been made. Considering that a better understanding of the various instability mechanisms and factors is important for the quality control of protein drug products and their successful clinical usage, this review briefly overviews protein destabilization pathways and deals with general issues such as controlling the factors of protein stabilization, especially during manufacturing, shipping and storage.

Exaptive origins of regulated mRNA decay in eukaryotes.

Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense‐mediated decay (NMD) and motif‐specific transcript destabilization by CCCH‐type zinc finger RNA‐binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of “professional” innate and adaptive immunity systems allowed NMD and the motif‐triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post‐transcriptional gene regulation in this lineage might have been derived through a similar exaptation route.

Abstract ED05-03: Rexinoid nuclear receptors and cancer prevention

Abstract This session will present on the use of retinoid X receptor (RXR) agonists (rexinoids) for cancer prevention. Several areas will be highlighted. Using lung cancer as a model, both preclinical and clinical evidence for use of RXR-selective ligands in cancer prevention will be discussed. Emphasis will be placed on the pharmacology of newer rexinoids. Lung cancer is the most common cause of cancer death for men and women. Innovative ways to combat lung cancer are needed. A previously unrecognized antineoplastic pathway that triggers proteasomal degradation of different cyclins was found. Both retinoic acid receptor (RAR) and RXR agonists (among other agents) cause degradation of cyclins. This confers check point arrest and repair of carcinogenic damage of DNA in bronchial epithelial cells. Mechanisms responsible for this induced proteasomal degradation will be presented. The critical receptor that confers cyclin destabilization is RARβ. Yet, silencing of RARβ and of the novel isoform RARβ1' likely accounts for clinical resistance to classical retinoids in lung carcinogenesis. It was explored whether RXR/RAR heterodimer complex activation with a rexinoid triggers G1 cyclin destabilization. This was found and this result implied that the same pathway would be engaged in vivo. To establish if this occurred in the lung, transgenic mice were independently engineered with human surfactant C-driven wild-type cyclin E or a proteasome-degradation resistant cyclin E species. This was done because human pre-malignant and malignant lung lesions deregulate cyclin expression. Intriguingly, these mice recapitulated frequent features of lung carcinogenesis found in patients. These changes were enhanced by the expression of degradation-resistant cyclin E species. This finding was built upon by showing that a rexinoid can chemoprevent lung cancers in vinyl carbamate-induced lung tumors within strain A/J mice. The epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) erlotinib also repressed cyclin D1 expression, but through different mechanisms. The rexinoid bexarotene was combined with erlotinib. Effects on transgenic lung cancer cell growth and cyclin D1 expression were studied when KRAS or KRAS/p53 mutations were present or not. Over the past decade, this work was confirmed by extending analyses into the cancer clinic through a series of five clinical trials that moved this work through successive phase 0, phase I, and phase II trials. In window of opportunity trials, pharmacodynamic responses (cyclin repression and induced necrosis and inflammatory responses) were seen when intratumoral drug levels were comparable to those detected in vitro. Cyclin D1 levels were measured in pre-treatment versus post-treatment tumor biopsies. A phase II trial in heavily pre-treated stage IV non-small cell lung cancer (NSCLC) cases was performed. EGFR expression and KRAS mutations were examined in these cases. Findings revealed substantial cyclin repression and reduction of lung cancer growth by combined treatment with a rexinoid and EGFR-TKI. Cyclins were repressed while necrosis and inflammatory responses were induced in the window of opportunity trial independent of KRAS or activating EGFR mutations. This refractory NSCLC trial had 3 major clinical responses (2 had KRAS or EGFR mutations) with prolonged survival, as will be discussed. Median survival was 22 weeks (16 weeks for controls). Hypertriglyceridemia or rash significantly increased median overall survival to 24 weeks. This regimen revealed anti-tumor activity independent of the presence of EGFR or KRAS mutations in NSCLCs. Notably, these findings were independently replicated in the lung cancer BATTLE trial. An analogous approach was used to uncover a distinct rexinoid-induced protein destabilization pathway that involves the deubiquitinase USP18/UBP43, as will be discussed. Taken together, these findings reveal that rexinoid-induced cyclin destabilization is a previously unrecognized antineoplastic pathway. Thus, additional rexinoid trials for cancer therapy and prevention are warranted. Citation Format: Ethan Dmitrovsky. Rexinoid nuclear receptors and cancer prevention. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr ED05-03.

CARHSP1 Is Required for Effective Tumor Necrosis Factor Alpha mRNA Stabilization and Localizes to Processing Bodies and Exosomes

Tumor necrosis factor alpha (TNF-α) is a critical mediator of inflammation, and its production is tightly regulated, with control points operating at nearly every step of its biosynthesis. We sought to identify uncharacterized TNF-α 3' untranslated region (3'UTR)-interacting proteins utilizing a novel screen, termed the RNA capture assay. We identified CARHSP1, a cold-shock domain-containing protein. Knockdown of CARHSP1 inhibits TNF-α protein production in lipopolysaccharide (LPS)-stimulated cells and reduces the level of TNF-α mRNA in both resting and LPS-stimulated cells. mRNA stability assays demonstrate that CARHSP1 knockdown decreases TNF-α mRNA stability from a half-life (t(1/2)) of 49 min to a t(1/2) of 22 min in LPS-stimulated cells and from a t(1/2) of 29 min to a t(1/2) of 24 min in resting cells. Transfecting CARHSP1 into RAW264.7 cells results in an increase in TNF-α 3'UTR luciferase expression in resting cells and CARHSP1 knockdown LPS-stimulated cells. We examined the functional effect of inhibiting Akt, calcineurin, and protein phosphatase 2A and established that inhibition of Akt or calcineurin but not PP2A inhibits CARHSP1 function. Subcellular analysis establishes CARHSP1 as a cytoplasmic protein localizing to processing bodies and exosomes but not on translating mRNAs. We conclude CARHSP1 is a TNF-α mRNA stability enhancer required for effective TNF-α production, demonstrating the importance of both stabilization and destabilization pathways in regulating the TNF-α mRNA half-life.

Tumor necrosis factor superfamily molecules in acute coronary syndromes

Accumulating evidence suggests that inflammatory pathways play an essential role in all stages of atherogenesis. Inflammatory processes are not only involved in plaque progression, but seem also to play a critical role in plaque rupture. Members of the tumor necrosis factor (TNF) superfamiliy are potent regulators of inflammation and cell survival and consist of 20 ligands that signal through 29 different receptors. Several lines of evidence suggest that TNF-related molecules are involved in the development of acute coronary syndromes (ACS). Most, convincing evidence exists for CD40 ligand-CD40 interaction, but several other members of the TNF superfamily seem also to be involved in this immune-mediated promotion of plaque instability, including LIGHT, receptor activator of nuclear factor κB ligand, and TNF-α. These plaque destabilization pathways involve the bidirectional interaction between platelets and endothelial cells/monocytes, activation of vascular smooth muscle cells, and co-stimulatory effects on T cells, promoting inflammation, thrombus formation, matrix degradation, and apoptosis. TNF-related pathways could contribute to the non-resolving inflammation that characterizes atherosclerosis, representing pathogenic loops that are operating during plaque rupture and the development of ACS. These TNF-related molecules could also represent attractive new targets for therapy in this disorder.

Phospholipase Cγ Activation Drives Increased Production of Autotaxin in Endothelial Cells and Lysophosphatidic Acid-Dependent Regression

We previously reported that vascular endothelial growth factor (VEGF)-dependent activation of phospholipase Cgamma1 (PLCgamma) regulated tube stability by competing with phosphoinositide 3-kinase (PI3K) for their common substrate. Here we describe an additional mechanism by which PLCgamma promoted regression of tubes and blood vessels. Namely, it increased the level of autotaxin (ATX), which is a secreted form of lysophospholipase D that produces lysophosphatidic acid (LPA). LPA promoted motility of endothelial cells, leading to disorganization/regression of tubes in vitro. Furthermore, mice that under- or overexpressed members of this intrinsic destabilization pathway showed either delayed or accelerated, respectively, regression of blood vessels. We conclude that endothelial cells can be instructed to engage a PLCgamma-dependent intrinsic destabilization pathway that results in the production of soluble regression factors such as ATX and LPA. These findings are likely to potentiate ongoing efforts to prevent, manage, and eradicate numerous angiogenesis-based diseases such as proliferative diabetic retinopathy and solid tumors.

Macrocyclic Conjugation Pathways in Porphyrins

Macrocyclic aromaticity is the most important concept in porphyrinoid chemistry. Bond resonance energy (BRE) for any pi-bond linking adjacent pyrrolic or other rings represents the stabilization energy due to macrocyclic aromaticity. We found that a main conjugation pathway associated with macrocyclic aromaticity can be traced by choosing a pi-bond with a larger BRE at every bifurcation of the pi-network. All pi-bonds located along the main conjugation pathway are intensified with large positive BREs compared with those located along the bypasses. On the other hand, a main destabilization pathway associated with macrocyclic antiaromaticity can be traced by choosing a pi-bond with a smaller BRE at every bifurcation of the pi-network. Macrocyclic conjugation pathways thus determined are fully consistent with the chemical shifts of protons attached to the macrocycle.

Identification of functional, endogenous programmed −1 ribosomal frameshift signals in the genome of Saccharomyces cerevisiae

In viruses, programmed −1 ribosomal frameshifting (−1 PRF) signals direct the translation of alternative proteins from a single mRNA. Given that many basic regulatory mechanisms were first discovered in viral systems, the current study endeavored to: (i) identify −1 PRF signals in genomic databases, (ii) apply the protocol to the yeast genome and (iii) test selected candidates at the bench. Computational analyses revealed the presence of 10 340 consensus −1 PRF signals in the yeast genome. Of the 6353 yeast ORFs, 1275 contain at least one strong and statistically significant −1 PRF signal. Eight out of nine selected sequences promoted efficient levels of PRF in vivo. These findings provide a robust platform for high throughput computational and laboratory studies and demonstrate that functional −1 PRF signals are widespread in the genome of Saccharomyces cerevisiae. The data generated by this study have been deposited into a publicly available database called the PRFdb. The presence of stable mRNA pseudoknot structures in these −1 PRF signals, and the observation that the predicted outcomes of nearly all of these genomic frameshift signals would direct ribosomes to premature termination codons, suggest two possible mRNA destabilization pathways through which −1 PRF signals could post-transcriptionally regulate mRNA abundance.

Recent trends in stabilizing protein structure upon encapsulation and release from bioerodible polymers.

Sustained release of pharmaceutical proteins from biocompatible polymers offers new opportunities in the treatment and prevention of disease. The manufacturing of such sustained-release dosage forms, and also the release from them, can impose substantial stresses on the chemical integrity and native, three-dimensional structure of proteins. Recently, novel strategies have been developed towards elucidation and amelioration of these stresses. Non-invasive technologies have been implemented to investigate the complex destabilization pathways that can occur. Such insights allow for rational approaches to protect proteins upon encapsulation and release from bioerodible systems. Stabilization of proteins when utilizing the most commonly employed procedure, the water-in-oil-in-water (w/o/w) double emulsion technique, requires approaches that are based mainly on either increasing the thermodynamic stability of the protein or preventing contact of the protein with the destabilizing agent (e.g. the water/oil interface) by use of various additives. However, protein stability is still often problematic when using the w/o/w technique, and thus alternative methods have become increasingly popular. These methods, such as the solid-in-oil-in-oil (s/o/o) and solid-in-oil-in-water (s/o/w) techniques, are based on the suspension of dry protein powders in an anhydrous organic solvent. It has become apparent that protein structure in the organic phase is stabilized because the protein is "rigidified" and therefore unfolding and large protein structural perturbations are kinetically prohibited. This review focuses on strategies leading to the stabilization of protein structure when employing these different encapsulation procedures.

The vascular smooth muscle type I angiotensin II receptor mRNA is destabilized by cyclic AMP-elevating agents.

Although processes involved in mRNA degradation play a significant role in dictating steady state mRNA levels, the influence of cell surface signaling on mRNA stability control is understood incompletely. In this study, the effects of cAMP-elevating agents on type I angiotensin II receptor (AT1-R) mRNA levels were assessed in cultured rat aortic vascular smooth muscle cells (VSMCs). AT1-R mRNA levels are rapidly reduced by forskolin treatment, in which the maximal effect yields an 80% reduction in AT1-R mRNA levels after 6 hr of treatment. The rate of AT1-R mRNA decay in response to forskolin is greater than its apparent intrinsic decay, as assessed in the presence of the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, suggesting forskolin treatment destabilizes the AT1-R mRNA. Nuclear run-on analysis indicates forskolin treatment does not affect transcription of the AT1-R gene in VSMCs, implying induced AT1-R mRNA destabilization accounts for the entire effect of forskolin in decreasing AT1-R mRNA levels. Dose-effect studies that assessed AT1-R mRNA levels and cAMP production were conducted using forskolin and the beta-adrenergic receptor agonist isoproterenol as agonists. Isoproterenol is almost 3 orders of magnitude more potent at eliciting the reduction in AT1-receptor mRNA levels than it is at stimulating cAMP production. Similarly, forskolin elicits reductions in AT1-R mRNA, which occur at concentrations that fail to elicit a detectable production of cAMP. However, protein kinase A activity is stimulated maximally by isoproterenol and forskolin concentrations that do not stimulate detectable cAMP production. These data provide evidence that the mechanism for down-regulation of AT1-R mRNA levels by cAMP-elevating agents in VSMCs occurs via a PKA-regulated mRNA destabilization pathway.

Capacitation mechanisms, and the role of capacitation as seen in eutherian mammals.

Capacitation, the process whereby spermatozoa are rendered capable of interacting with and fertilizing the egg, was discovered more than 40 years ago. However, our understanding of it is still far from satisfactory. Several factors conspire to obfuscate studies of capacitation mechanisms: the inherent functional heterogeneity of sperm populations, the range of functions used as parameters of capacitation (whence the endpoint of the process has become conceptually uncertain), and the several profound differences between model in vitro fertilization (IVF) systems and the situation in vivo in the female reproductive tract. Recent investigations in the author's laboratory have shown that bicarbonate/CO2, an essential component for successful IVF, causes rapid changes in lipid architecture of the sperm plasma membrane and slower changes in surface coating. These changes are accompanied by membrane destabilization and cell death. Evidence suggests that bicarbonate's actions are mediated through cyclic nucleotide signalling. Of particular note is the heterogeneity in rate of response to bicarbonate shown by individual cells in the sperm populations. Taken together with other observations, the findings suggest that capacitation is a series of positive destabilizing events that eventually lead to cell death. The 'capacitated' state would then be a window of destabilization within which spermatozoa can undergo a zona-induced acrosome reaction and display hyperactivated motility. Further along the destabilization pathway, spontaneous acrosome reactions would occur before total membrane degeneration. In vivo, capacitation would be a conflict between destabilization and sperm survival. Concentrations of bicarbonate are maintained low in the cauda epididymidis, where sperm survive for long periods, and one may speculate that hormonal control of local bicarbonate/CO2 in oviducal 'storage' sites in the female tract could allow 'safe' sequestering of live spermatozoa until around the time of ovulation; the environment may then change to produce a 'capacitating' effect, whence, due to the inherent functional heterogeneity of the sequestered population, small numbers of capacitated spermatozoa are released sequentially. In this way, a succession of spermatozoa in the correct physiological state may be provided for the freshly ovulated egg.

An amino-terminal tetrapeptide specifies cotranslational degradation of beta-tubulin but not alpha-tubulin mRNAs.

The steady-state level of alpha- and beta-tubulin synthesis is autoregulated by a posttranscriptional mechanism that selectively alters alpha- and beta-tubulin mRNA levels in response to changes in the unassembled tubulin subunit concentration. For beta-tubulin mRNAs, previous efforts have shown that this is the result of a selective mRNA degradation mechanism which involves cotranslational recognition of the nascent amino-terminal beta-tubulin tetrapeptide as it emerges from the ribosome. Site-directed mutagenesis is now used to determine that the minimal sequence requirement for conferring the full range of beta-tubulin autoregulation is the amino-terminal tetrapeptide MR(E/D)I. Although tubulin-dependent changes in alpha-tubulin mRNA levels are shown to result from changes in cytoplasmic mRNA stability, transfection of wild-type and mutated alpha-tubulin genes reveals that alpha- and beta-tubulin mRNA degradation is not mediated through a common pathway. Not only does the amino-terminal alpha-tubulin tetrapeptide MREC fail to confer regulated mRNA degradation, neither wild-type alpha-tubulin transgenes nor an alpha-tubulin gene mutated to encode an amino-terminal MREI yields mRNAs that are autoregulated. Further, although slowing ribosome transit accelerates the autoregulated degradation of endogenous alpha- and beta-tubulin mRNAs, degradation of alpha-tubulin transgene mRNAs is not enhanced, and in one case, the mRNA is actually stabilized. We conclude that, despite similarities, alpha- and beta-tubulin mRNA destabilization pathways utilize divergent determinants to link RNA instability to tubulin subunit concentrations.

Autoregulatory control of beta-tubulin mRNA stability is linked to translation elongation.

Tubulin synthesis in animal cells is controlled in part by an autoregulatory mechanism that modulates the stability of ribosome-bound tubulin mRNAs. For beta tubulin, the initial recognition event for this selective RNA instability has previously been shown to be a cotranslational binding (presumably by tubulin itself) to the nascent amino-terminal beta-tubulin tetrapeptide just after it emerges from the ribosome. Although this "autoregulation" of tubulin expression is thus obligatorily linked to the translation process, the mechanism of how a cotranslational protein-protein binding event ultimately triggers RNA degradation is unknown. Using protein synthesis inhibitors to slow and ultimately to block translation elongation, we now show that the mRNA destabilization pathway requires ongoing ribosome translocation.