Engineered tRNAs suppress nonsense mutations in cells and in vivo.

Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases1. Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation2-7. However, tRNA-based gene therapy has not yielded an optimal combination of clinical efficacy and safety and there is presently no treatment for individuals with nonsense mutations. Here we introduce a strategy based on altering native tRNAs into  efficient suppressor tRNAs (sup-tRNAs) by individually fine-tuning their sequence to the physico-chemical properties of the amino acid that they carry. Intravenous and intratracheal lipid nanoparticle (LNP) administration of sup-tRNA in mice restored the production of functional proteins with nonsense mutations. LNP-sup-tRNA formulations caused no discernible readthrough at endogenous native stop codons, as determined by ribosome profiling. At clinically important PTCs in the cystic fibrosis transmembrane conductance regulator gene (CFTR), the sup-tRNAs re-established expression and function in cell systems and patient-derived nasal epithelia and restored airway volume homeostasis. These results provide a framework for the development of tRNA-based therapies with a high molecular safety profile and high efficacy in targeted PTC suppression.

Posterior cerebral artery stenosis related to implanted intracranial electrodes for temporal lobe epilepsy: A case report.

INTRODUCTION: Intracranial electroencephalography is a crucial diagnostic technique for epilepsy surgery, though it is associated with a range of complications, including infection, intracranial hemorrhage, increased intracranial pressure, and cerebral infarction. This case study presents an uncommon occurrence of stenosis of the left posterior cerebral artery (PCA) following intracranial electrode implantation. CASE PRESENTATION: A woman in her thirties with drug-resistant focal impaired awareness seizures underwent implantation of subdural and depth electrodes on the bilateral temporal lobes to lateralize seizure onset. A left anterior-temporal lobectomy was performed based on the evaluation results. Following the resection of the hippocampus, stenosis of the left PCA, with a pinched appearance, was observed. Postoperatively, extensive cerebral edema in the bilateral temporal lobes and a defect in the left PCA were detected on magnetic resonance (MR) imaging. MR imaging performed the day after surgery showed cerebral infarction in the left medial temporal lobe and left lateral thalamus. A video review indicated that surgical manipulation was not the cause of vascular stenosis. MR angiography one week later confirmed the recanalization of the PCA. DISCUSSION: We surmised that the subdural electrodes inserted along the middle skull base might have induced the PCA stenosis or spasms. The patient did not experience any significant sequelae, with no episodes of seizures for more than five years after surgery. CONCLUSION: It is essential to note that subdural grid electrodes placed in the medial temporal lobe can cause vascular stenosis, albeit with an extremely rare occurrence.

Continuous Repetition Motor Imagery Training and Physical Practice Training Exert the Growth of Fatigue and Its Effect on Performance.

Continuous repetition of motor imagery leads to mental fatigue. This study aimed to examine whether fatigue caused by motor imagery training affects improvement in performance and the change in corticospinal excitability. The participants were divided into "physical practice training" and "motor imagery training" groups, and a visuomotor task (set at 50% of maximal voluntary contraction in participants) was performed to assess the training effect on fatigue. The measurements were recorded before and after training. Corticospinal excitability at rest was measured by transcranial magnetic stimulation according to the Neurophysiological Index. Subjective mental fatigue and muscle fatigue were assessed by using the visual analog scale and by measuring the pinch force, respectively. Additionally, the error area was evaluated and calculated at pre-, mid-, and post-terms after training, using a visuomotor task. After training, muscle fatigue, subjective mental fatigue, and decreased corticospinal excitability were noted in both of the groups. Moreover, the visuomotor task decreased the error area by training; however, there was no difference in the error area between the mid- and post-terms. In conclusion, motor imagery training resulted in central fatigue by continuous repetition, which influenced the improvement in performance in the same manner as physical practice training.

Identification and validation of a gray matter volume network in Alzheimer's disease.

OBJECTIVE: This study aims to identify and validate a gray matter volume network in patients with Alzheimer's disease (AD). METHODS: To identify a disease-related network, a principal component analysis-based algorithm, Scaled Subprofile Model, was applied to gray matter volume data derived from structural T1-weighted magnetic resonance imaging of the training sample that consisted of nine patients with AD (women, four; dementia, seven; mild cognitive impairment, two; age, 66.7 ± 8.8 [mean ± SD] years) with positive 18F-flutemetamol amyloid positron emission tomography and eight age-matched healthy controls obtained on-site. The network expression scores were calculated by topographic profile rating in the validation sample obtained via the Open Access Series of Imaging Studies and comprised 12 patients with AD dementia (women, four; age, 70.0 ± 3.7 years) and 12 age-matched healthy controls. RESULTS: A significant network from the training sample, for which subject expression differed between the groups (permutation test, P = 0.006; sensitivity and specificity, 100%; area under the curve, 1), was identified. This network was represented by the principal components 1, 2, and 3 and showed a relative decrease in the inferior parietal lobule including angular gyrus, inferior temporal gyrus, premotor cortex, amygdala, hippocampus, and precuneus. It significantly differed between the groups with a sensitivity, specificity, and area under the curve of 83%, 91%, and 0.85, respectively, in the validation sample (P = 0.003). CONCLUSIONS: An AD-related gray matter volume network that captured relevant regions was identified in amyloid positron emission tomography-positive patients and validated in an independent sample.

Efficacy of combined hand exercise intervention in patients with chemotherapy-induced peripheral neuropathy: a pilot randomized controlled trial.

PURPOSE: Previous evidence regarding the impact of exercise interventions on chemotherapy-induced peripheral neuropathy often focuses on lower-extremity functions, such as muscle strength and balance ability, while their effects on upper extremities remain unknown. We aimed to evaluate the efficacy of combined hand exercise intervention on upper-extremity function, symptoms, and quality-of-life in patients with chemotherapy-induced peripheral neuropathy (CIPN). METHODS: After screening 341 patients, 42 were randomly assigned to either the intervention (n = 21) or control (n = 21) group. Participants were evaluated at baseline (T0) and after one (T1) and two (T2) chemotherapy cycles. The primary outcome was upper-extremity function measured using the Michigan Hand Outcomes Questionnaire (MHQ) at T2. The intention-to-treat and as-treated populations were compared using a mixed-effect model. RESULTS: In the intention-to-treat analysis, the decline in activities of daily living of MHQ was significantly suppressed in the intervention group compared with that in the control group at T2 (difference: 7.23; 95% confidence interval: 0.35-14.10). Similarly, in the as-treated analysis, the decline in activities of daily living of MHQ was significantly suppressed in the intervention group compared with that in the control group at T2 (difference: 13.09; 95% confidence interval: 5.68-20.49). Pain also significantly improved in the intervention group compared with that in the control group at T2 (difference: 13.21; 95% confidence interval: - 22.91 to - 3.51). CONCLUSION: The combined hand exercise intervention may improve upper-extremity function, such as by suppressing decline in ADL, and reduce pain in patients with CIPN.

Ultrasonic Heating Detects Lipiodol Deposition within Liver Tumors after Transarterial Embolization: An In Vivo Approach.

Computed tomography (CT) is the standard method to evaluate Lipiodol deposition after transarterial embolization (TAE) for a long period. However, iodine but not Lipiodol can be observed on CT. A minimally invasive other method to detect Lipiodol has been needed to evaluate accurate evaluation after procedure. The purpose of this study was to evaluate the efficacy of using the rate of change in sound velocity caused by ultrasonic heating to reflect Lipiodol accumulation after TAE in a rat liver tumor model. We analyzed the association of this developed technique with CT images and histological findings. Eight rats bearing N1S1 cells were prepared. After confirmation of tumor development in a rat liver, Lipiodol was injected via the hepatic artery. Seven days after TAE, CT scan and sound velocity changes caused by ultrasonic heating were measured, and then the rats were sacrificed. An ultrasonic pulse-echo method was used to measure the sound velocity. The temperature coefficient of the sound velocity in each treated tumor was evaluated and compared with the mean CT value and the histological Lipiodol accumulation ratio. Pearson's correlation coefficients were calculated to assess the correlation between the measured values. The correlation coefficient (r) of the mean CT value and histological Lipiodol accumulation ratio was 0.835 (p = 0.010), which was considered statistically significant. Also, those of the temperature coefficient of the sound velocity and the histological Lipiodol accumulation ratio were statistically significant (r = 0.804; p = 0.016). To our knowledge, this is the first study that reported the efficacy of ultrasonic heating to detect Lipiodol accumulation in rat liver tumors after TAE. Our results suggest that the rate of change in sound velocity caused by ultrasonic heating can be used to evaluate Lipiodol accumulation in liver tumors after TAE, and thus could represent an alternative to CT in this application. This new innovative technique is easy to treat and less invasive in terms of avoiding radiation compared with CT.

A single dose of self-transcribing and replicating RNA-based SARS-CoV-2 vaccine produces protective adaptive immunity in mice.

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4+ T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 µg and 10 µg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.

A Study on the Effect of Mental Practice Using Motor Evoked Potential-Based Neurofeedback.

This study aimed to investigate whether the effect of mental practice (motor imagery training) can be enhanced by providing neurofeedback based on transcranial magnetic stimulation (TMS)-induced motor evoked potentials (MEP). Twenty-four healthy, right-handed subjects were enrolled in this study. The subjects were randomly allocated into two groups: a group that was given correct TMS feedback (Real-FB group) and a group that was given randomized false TMS feedback (Sham-FB group). The subjects imagined pushing the switch with just timing, when the target circle overlapped a cross at the center of the computer monitor. In the Real-FB group, feedback was provided to the subjects based on the MEP amplitude measured in the trial immediately preceding motor imagery. In contrast, the subjects of the Sham-FB group were provided with a feedback value that was independent of the MEP amplitude. TMS was applied when the target, moving from right to left, overlapped the cross at the center of the screen, and the MEP amplitude was measured. The MEP was recorded in the right first dorsal interosseous muscle. We evaluated the pre-mental practice and post-mental practice motor performance in both groups. As a result, a significant difference was observed in the percentage change of error values between the Real-FB group and the Sham-FB group. Furthermore, the MEP was significantly different between the groups in the 4th and 5th sets. Therefore, it was suggested that TMS-induced MEP-based neurofeedback might enhance the effect of mental practice.

Comparison of cerebral activation between motor execution and motor imagery of self-feeding activity.

Motor imagery is defined as an act wherein an individual contemplates a mental action of motor execution without apparent action. Mental practice executed by repetitive motor imagery can improve motor performance without simultaneous sensory input or overt output. We aimed to investigate cerebral hemodynamics during motor imagery and motor execution of a self-feeding activity using chopsticks. This study included 21 healthy right-handed volunteers. The self-feeding activity task comprised either motor imagery or motor execution of eating sliced cucumber pickles with chopsticks to examine eight regions of interest: pre-supplementary motor area, supplementary motor area, bilateral prefrontal cortex, premotor area, and sensorimotor cortex. The mean oxyhemoglobin levels were detected using near-infrared spectroscopy to reflect cerebral activation. The mean oxyhemoglobin levels during motor execution were significantly higher in the left sensorimotor cortex than in the supplementary motor area and the left premotor area. Moreover, significantly higher oxyhemoglobin levels were detected in the supplementary motor area and the left premotor area during motor imagery, compared to motor execution. Supplementary motor area and premotor area had important roles in the motor imagery of self-feeding activity. Moreover, the activation levels of the supplementary motor area and the premotor area during motor execution and motor imagery are likely affected by intentional cognitive processes. Levels of cerebral activation differed in some areas during motor execution and motor imagery of a self-feeding activity. This study was approved by the Ethical Review Committee of Nagasaki University (approval No. 18110801) on December 10, 2018.

Corticospinal excitability during motor imagery is diminished by continuous repetition-induced fatigue.

Application of continuous repetition of motor imagery can improve the performance of exercise tasks. However, there is a lack of more detailed neurophysiological evidence to support the formulation of clear standards for interventions using motor imagery. Moreover, identification of motor imagery intervention time is necessary because it exhibits possible central fatigue. Therefore, the purpose of this study was to elucidate the development of fatigue during continuous repetition of motor imagery through objective and subjective evaluation. The study involved two experiments. In experiment 1, 14 healthy young volunteers were required to imagine grasping and lifting a 1.5-L plastic bottle using the whole hand. Each participant performed the motor imagery task 100 times under each condition with 48 hours interval between two conditions: 500 mL or 1500 mL of water in the bottle during the demonstration phase. Mental fatigue and a decrease in pinch power appeared under the 1500-mL condition. There were changes in concentration ability or corticospinal excitability, as assessed by motor evoked potentials, between each set with continuous repetition of motor imagery also under the 1500-mL condition. Therefore, in experiment 2, 12 healthy volunteers were required to perform the motor imagery task 200 times under the 1500-mL condition. Both concentration ability and corticospinal excitability decreased. This is the first study to show that continuous repetition of motor imagery can decrease corticospinal excitability in addition to producing mental fatigue. This study was approved by the Institutional Ethics Committee at the Nagasaki University Graduate School of Biomedical and Health Sciences (approval No. 18121302) on January 30, 2019.

Cerebral haemodynamics during motor imagery of self-feeding with chopsticks: differences between dominant and non-dominant hand.

Purpose: Motor imagery is defined as a dynamic state during which a subject mentally simulates a given action without overt movements. Our aim was to use near-infrared spectroscopy to investigate differences in cerebral haemodynamics during motor imagery of self-feeding with chopsticks using the dominant or non-dominant hand.Materials and methods: Twenty healthy right-handed people participated in this study. The motor imagery task involved eating sliced cucumber pickles using chopsticks with the dominant (right) or non-dominant (left) hand. Activation of regions of interest (pre-supplementary motor area, supplementary motor area, pre-motor area, pre-frontal cortex, and sensorimotor cortex was assessed.Results: Motor imagery vividness of the dominant hand tended to be significantly higher than that of the non-dominant hand. The time of peak oxygenated haemoglobin was significantly earlier in the right pre-frontal cortex than in the supplementary motor area and left pre-motor area. Haemodynamic correlations were detected in more regions of interest during dominant-hand motor imagery than during non-dominant-hand motor imagery.Conclusions: Haemodynamics might be affected by differences in motor imagery vividness caused by variations in motor manipulation.

Changing Artificial Playback Speed and Real Movement Velocity Do Not Differentially Influence the Excitability of Primary Motor Cortex during Observation of a Repetitive Finger Movement.

Action observation studies have investigated whether changing the speed of the observed movement affects the action observation network. There are two types of speed-changing conditions; one involves "changes in actual movement velocity," and the other is "manipulation of video speed." Previous studies have investigated the effects of these conditions separately, but to date, no study has directly investigated the differences between the effects of these conditions. In the "movement velocity condition," increased velocity is associated with increased muscle activity; however, this change of muscle activities is not shown in the "video speed condition." Therefore, a difference in the results obtained under these conditions could be considered to reflect a difference in muscle activity of actor in the video. The aim of the present study was to investigate the effects of different speed-changing conditions and spontaneous movement tempo (SMT) on the excitability of primary motor cortex (M1) during action observation, as assessed by motor-evoked potentials (MEPs) amplitudes induced by transcranial magnetic stimulation (TMS). A total of 29 healthy subjects observed a video clip of a repetitive index or little finger abduction movement under seven different speed conditions. The video clip in the movement velocity condition showed repetitive finger abduction movements made in time with an auditory metronome, at frequencies of 0.5, 1, 2, and 3 Hz. In the video speed condition, playback of the 1-Hz movement velocity condition video clip was modified to show movement frequencies of 0.5, 2, or 3 Hz (Hz-Fake). TMS was applied at the time of maximal abduction and MEPs were recorded from two right-hand muscles. There were no differences in M1 excitability between the movement velocity and video speed conditions. Moreover, M1 excitability did not vary across the speed conditions for either presentation condition. Our findings suggest that changing playback speed and actual differences in movement velocity do not differentially influence M1 excitability during observation of a simple action task, such as repetitive finger movement, and that it is not affected by SMT. In simple and meaningless observational task, people might not be able to recognize the difference in muscle activity of actor in the video.

Primary Motor Cortex Activation during Action Observation of Tasks at Different Video Speeds Is Dependent on Movement Task and Muscle Properties.

The aim of the present study was to investigate how the video speed of observed action affects the excitability of the primary motor cortex (M1), as assessed by the size of motor-evoked potentials (MEPs) induced by transcranial magnetic stimulation (TMS). Twelve healthy subjects observed a video clip of a person catching a ball (Experiment 1: rapid movement) and another 12 healthy subjects observed a video clip of a person reaching to lift a ball (Experiment 2: slow movement task). We played each video at three different speeds (slow, normal and fast). The stimulus was given at two points of timing in each experiment. These stimulus points were locked to specific frames of the video rather than occurring at specific absolute times, for ease of comparison across different speeds. We recorded MEPs from the first dorsal interosseous muscle (FDI) and abductor digiti minimi muscle (ADM) of the right hand. MEPs were significantly different for different video speeds only in the rapid movement task. MEPs for the rapid movement task were higher when subjects observed an action played at slow speed than normal or fast speed condition. There was no significant change for the slow movement task. Video speed was effective only in the ADM. Moreover, MEPs in the ADM were significantly higher than in the FDI in a rapid movement task under the slow speed condition. Our findings suggest that the M1 becomes more excitable when subjects observe the video clip at the slow speed in a rapid movement, because they could recognize the elements of movement in others. Our results suggest the effects of manipulating the speed of the viewed task on the excitability of the M1 during passive observation differ depending on the type of movement task observed. It is likely that rehabilitation in the clinical setting will be more efficient if the video speed is changed to match the task's characteristics.

Translation regulation by ribosomes: Increased complexity and expanded scope.

The primary function of ribosomes is to decode mRNAs into polypeptide chains; however, this description is overly simplistic. Accumulating evidence shows that ribosomes themselves can affect the relative efficiency with which various mRNAs are translated and indicates that these effects can be modulated by ribosome heterogeneity. The notion that ribosomes have regulatory capabilities was elaborated more than a decade ago in the ribosome filter hypothesis. Various lines of evidence support this idea and have shown that the translation of some mRNAs is affected by discrete binding interactions with rRNA or ribosomal proteins. Recent work from our laboratory has demonstrated that base-pairing of the Hepatitis C Virus (HCV) internal ribosome entry site (IRES) to 18S rRNA is required for IRES function, but only in the context of more complex ribosomal interactions. The HCV IRES provides an example of the ribosome filter that involves multiple binding interactions between mRNAs and ribosomal subunits.

Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo.

Degeneracy in eukaryotic translation initiation is evident in the initiation strategies of various viruses. Hepatitis C virus (HCV) provides an exceptional example--translation of the HCV RNA is facilitated by an internal ribosome entry site (IRES) that can autonomously bind a 40S ribosomal subunit and accurately position it at the initiation codon. This binding involves both ribosomal protein and 18S ribosomal RNA (rRNA) interactions. In this study, we evaluate the functional significance of the rRNA interaction and show that HCV IRES activity requires a 3-nt Watson-Crick base-pairing interaction between the apical loop of subdomain IIId in the IRES and helix 26 in 18S rRNA. Mutations of these nucleotides in either RNA dramatically disrupted IRES activity. The activities of the mutated HCV IRESs could be restored by compensatory mutations in the 18S rRNA. The effects of the 18S rRNA mutations appeared to be specific inasmuch as ribosomes containing these mutations did not support translation mediated by the wild-type HCV IRES, but did not block translation mediated by the cap structure or other viral IRESs. The present study provides, to our knowledge, the first functional demonstration of mRNA-rRNA base pairing in mammalian cells. By contrast with other rRNA-binding sites in mRNAs that can enhance translation as independent elements, e.g., the Shine-Dalgarno sequence in prokaryotes, the rRNA-binding site in the HCV IRES functions as an essential component of a more complex interaction.

Determinants of initiation codon selection during translation in mammalian cells.

Factors affecting translation of mRNA contribute to the complexity of eukaryotic proteomes. In some cases, translation of a particular mRNA can generate multiple proteins. However, the factors that determine whether ribosomes initiate translation from the first AUG codon in the transcript, from a downstream codon, or from multiple sites are not completely understood. Various mRNA properties, including AUG codon-accessibility and 5' leader length have been proposed as potential determinants that affect where ribosomes initiate translation. To explore this issue, we performed studies using synthetic mRNAs with two in-frame AUG codons-both in excellent context. Open reading frames initiating at AUG1 and AUG2 encode large and small isoforms of a reporter protein, respectively. Translation of such an mRNA in COS-7 cells was shown to be 5' cap-dependent and to occur efficiently from both AUG codons. AUG codon-accessibility was modified by using two different elements: an antisense locked nucleic acid oligonucleotide and an exon-junction complex. When either element was used to mask AUG1, the ratio of the proteins synthesized changed, favoring the smaller (AUG2-initiated) protein. In addition, we observed that increased leader length by itself changed the ratio of the proteins and favored initiation at AUG1. These observations demonstrate that initiation codon selection is affected by various factors, including AUG codon-accessibility and 5' leader length, and is not necessarily determined by the order of AUG codons (5'→3'). The modulation of AUG codon accessibility may provide a powerful means of translation regulation in eukaryotic cells.

UPF1 association with the cap-binding protein, CBP80, promotes nonsense-mediated mRNA decay at two distinct steps.

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that in mammals generally occurs upon recognition of a premature termination codon (PTC) during a pioneer round of translation. This round involves newly synthesized mRNA that is bound at its 5' end by the cap-binding protein (CBP) heterodimer CBP80-CBP20. Here we show that precluding the binding of the NMD factor UPF1 to CBP80 inhibits NMD at two steps: the association of SMG1 and UPF1 with the two eukaryotic release factors (eRFs) during SURF complex formation at a PTC, and the subsequent association of SMG1 and UPF1 with an exon-junction complex. We also demonstrate that UPF1 binds PTC-containing mRNA more efficiently than the corresponding PTC-free mRNA in a way that is promoted by the UPF1-CBP80 interaction. A unifying model proposes a choreographed series of protein-protein interactions occurring on an NMD target.

The 5' leader of the mRNA encoding the marek's disease virus serotype 1 pp14 protein contains an intronic internal ribosome entry site with allosteric properties.

We demonstrate the presence of a functional internal ribosome entry site (IRES) within the 5' leader (designated 5L) from a variant of bicistronic mRNAs that encode the pp14 and RLORF9 proteins from Marek's disease virus (MDV) serotype 1. Transcribed as a 1.8-kb family of immediate-early genes, the mature bicistronic mRNAs have variable 5' leader sequences due to alternative splicing or promoter usage. Consequently, the presence or absence of the 5L IRES in the mRNA dictates the mode of pp14 translation and leads to the production of two pp14 isoforms that differ in their N-terminal sequences. Real-time reverse transcription-quantitative PCR indicates that the mRNA variants with the 5L IRES is two to three times more abundant in MDV-infected and transformed cells than the mRNA variants lacking the 5L IRES. A common feature to all members of the 1.8-kb family of transcripts is the presence of an intercistronic IRES that we have previously shown to control the translation of the second open reading frame (i.e., RLORF9). Investigation of the two IRESs residing in the same bicistronic reporter mRNA revealed functional synergism for translation efficiency. In analogy with allosteric models in proteins, we propose IRES allostery to describe such a novel phenomenon. The functional implications of our findings are discussed in relation to host-virus interactions and translational control.

Chapter 9. Studying nonsense-mediated mRNA decay in mammalian cells.

Nonsense-mediated decay (NMD) in eukaryotic cells largely functions as a quality control mechanism by degrading faulty mRNAs that terminate translation prematurely. In recent years it has become evident that NMD also eliminates a subset of naturally occurring mRNA during proper gene expression. The mechanism of NMD in mammalian cells can be distinguished from the mechanism in, for example, Saccharomyces cerevisiae or Caenorhabditis elegans, by its apparent restriction to newly synthesized mRNA during a pioneer round of translation. This dependence can be explained by the need for at least one exon-exon junction complex (EJC) that is deposited on newly synthesized mRNA during the process of pre-mRNA splicing. Additionally, mammalian-cell NMD is promoted by the cap-binding protein heterodimer CBP80/20 that also typifies newly synthesized mRNA. When translation terminates sufficiently upstream of an EJC, the NMD factor Up-frameshift (Upf)1 is thought to join the stable EJC constituent NMD factors Upf2 and Upf3 or Upf3X (also called Upf3a or Upf3b, respectively), and undergo phosphorylation. Phosphorylation appears to trigger translational repression and mRNA decay. Although there are established rules for what generally defines an NMD target in mammalian cells, as with any rule there are exceptions and, thus, the need to experimentally verify individual mRNAs as bona fide targets of NMD. This chapter provides guidelines and protocols for how to define NMD targets using cultured mammalian cells.

Failsafe nonsense-mediated mRNA decay does not detectably target eIF4E-bound mRNA.

Nonsense-mediated mRNA decay (NMD) generally eliminates messenger RNAs that prematurely terminate translation and occurs in all eukaryotes that have been studied, although with mechanistic variations. In mammals, NMD seems to be restricted to newly synthesized mRNA that is bound by the cap-binding heterodimer CBP80-CBP20 (CBP80/20) and typically has at least one exon junction complex (EJC) situated downstream of the nonsense codon and added post-splicing. However, mammalian NMD can also target spliced mRNA lacking an EJC downstream of the nonsense codon. Here we provide evidence that this additional pathway, known as failsafe NMD, likewise seems to be restricted to CBP80/20-bound mRNA and does not detectably target its subsequently remodeled product, eIF4E-bound mRNA. Our studies, including analyses of factor dependence, reveal important shared features of the two mammalian-cell NMD pathways as well as fundamental differences between NMD in mammals and Saccharomyces cerevisiae.