Real-time single-molecule imaging of transcriptional regulatory networks in living cells.

Gene regulatory networks drive the specific transcriptional programmes responsible for the diversification of cell types during the development of multicellular organisms. Although our knowledge of the genes involved in these dynamic networks has expanded rapidly, our understanding of how transcription is spatiotemporally regulated at the molecular level over a wide range of timescales in the small volume of the nucleus remains limited. Over the past few decades, advances in the field of single-molecule fluorescence imaging have enabled real-time behaviours of individual transcriptional components to be measured in living cells and organisms. These efforts are now shedding light on the dynamic mechanisms of transcription, revealing not only the temporal rules but also the spatial coordination of underlying molecular interactions during various biological events.

Influence of the Pringle maneuver during partial hepatectomy on the neuromuscular block induced by intermittent and continuous dosing of rocuronium.

PURPOSE: The Pringle maneuver (PM) is a common procedure in hepatectomy that is known to interrupt drug elimination. The purpose of this study was to examine the influence of PM on the duration of action of rocuronium administered by intermittent bolus dosing, the continuous rocuronium infusion dose required for maintenance of a moderate neuromuscular block, and changes in plasma concentrations of rocuronium. METHODS: Twenty-seven adult patients undergoing partial hepatectomy with PM were enrolled in this study. The duration of action of 0.2 mg/kg rocuronium boluses (DUR), and the continuous rocuronium infusion dose required for maintenance of the height of the first twitch of the train-of-four (T1) at 10-20% of the control value (%T1), respectively, were electromyographically monitored on the adductor digiti minimi muscle. The effects of PM on DUR, %T1, and the plasma concentration of rocuronium were measured. RESULTS: The DUR was significantly prolonged during PM [mean: 42.2 (SD: 8.0) min, P < 0.001] compared to baseline [29.7 (6.3) min]. It was prolonged even after completion of the PM [46.2 (10.5) min, P < 0.001]. The plasma concentration of rocuronium measured at every reappearance of T1 was comparable between before and during PM. %T1 [15.5 (5.6)%] was significantly depressed after the start of PM [6.5 (3.9)%, P < 0.001], with persistence of the depression even after completion of PM. However, there were no significant changes in the plasma concentration of rocuronium. CONCLUSIONS: Rocuronium-induced neuromuscular block is significantly augmented during PM. However, the augmentation is not associated with an increase in plasma rocuronium concentration.

Preanesthetic ultrasonography assessment of inferior vena cava diameter in the supine position, left lateral tilt position, and with the left uterine displacement maneuver in full-term pregnant women: A randomized cross-over design study.

Preanesthetic ultrasonography assessment of inferior vena cava diameter (IVCD) in the supine position (SP), left lateral tilt position (LLT), and with the left uterine displacement maneuver (LUD) in full-term pregnant women: a randomized cross-over design study. AIM: We pre-anesthetically measured IVCD using ultrasonography in the SP, LLT, and the LUD in full-term pregnant women, using a cross-over design, to evaluate the effectiveness of LLT and LUD on increasing IVCD, and the presence of inter-individual differences among patients in the effect of posture on IVCD. METHODS: Twenty-two parturients scheduled for elective cesarean section under spinal anesthesia were recruited. All patients were sequentially placed in the SP, LLT, and with LUD before spinal anesthesia induction. Indices of IVCD, measured by subxiphoid ultrasonography, including maximum IVCD (IVCDmax), minimum IVCD (IVCDmin), and collapsibility index (CI) were recorded in each of the postures. Mean or median values of all measurements were compared among the postures. RESULTS: The mean values of IVCDmax observed with both LLT and LUD were significantly larger than those in the SP, respectively (SP vs. LLT: p < 0.05, SP vs. LUD: p < 0.01), although there were no significant differences between IVCDmax with LLT and LUD. There were no significant differences in IVCDmin and CI between any of the postures. IVCDmax was highest with LUD in 11 patients (55%), in the LLT in seven patients (35%) and in the SP in two patients (10%). CONCLUSIONS: LLT and LUD might be equally effective in enlarging the narrowed IVCD as compared to SP. However, both LLT and LUD might not necessarily be appropriate treatments to relieve IVC compression in some cases.

An improved imaging system that corrects MS2-induced RNA destabilization.

The MS2 and MS2-coat protein (MS2-MCP) imaging system is widely used to study messenger RNA (mRNA) spatial distribution in living cells. Here, we report that the MS2-MCP system destabilizes some tagged mRNAs by activating the nonsense-mediated mRNA decay pathway. We introduce an improved version, which counteracts this effect by increasing the efficiency of translation termination of the tagged mRNAs. Improved versions were developed for both yeast and mammalian systems.

Comparison of two electromyography-based neuromuscular monitors, AF-201P and TetraGraph, in rocuronium-induced neuromuscular block: A prospective comparative study.

BACKGROUND: The study aimed to compare the responses obtained simultaneously from the newly developed electromyography (EMG)-based neuromuscular monitors, AF-201P and TetraGraph™, during rocuronium-induced neuromuscular block. METHODS: Twenty patients were enrolled in this study. During total intravenous general anesthesia, train-of-four (TOF) responses following 0.9-mg/kg-rocuronium administration were monitored at the abductor digiti minimi muscle with AF-201P and TetraGraph on the contralateral arms. Sugammadex 2 mg/kg was administered when both devices showed TOF counts (TOFC) = 2. The primary outcome was time from rocuronium administration to the first appearance of the post-tetanic count (PTC) response (first PTC). The secondary outcomes were supramaximal current, baseline compound muscle action potential, onset time, time to TOFC = 1, time to TOFC = 2, and time from sugammadex administration to TOF ratio ≥ 0.9. We used the paired t-test and Wilcoxon signed-rank test to analyze parametric and non-parametric data, respectively. P < 0.05 defined statistical significance. RESULTS: A total of 19 patients were analyzed. The supramaximal current was significantly lower with AF-201P than TetraGraph (31.7 ± 13.2 vs. 43.2 ± 8.2, p = .002). The time to first PTC (24.9 ± 9.4 vs. 27.3 ± 8.9 min, p = .026), time to TOFC = 1 (42.3 ± 9.0 vs. 45.1 ± 10.4 min, p = .03), and time to TOFC = 2 (52.0 ± 10.5 vs. 54.6 ± 11.7 min, p = .014) were significantly faster with AF-201P than with TetraGraph. There were no significant differences in the other outcomes between the devices. CONCLUSIONS: AF-201P showed faster recovery of rocuronium-induced neuromuscular block compared with TetraGraph.

A comparison between the adductor pollicis muscle and the abductor digiti minimi muscle using electromyography AF-201P in rocuronium-induced neuromuscular block: a prospective comparative study.

BACKGROUND: The AF-201P, a new electromyography (EMG)-based neuromuscular monitor has been developed recently. The aim of this clinical study was to compare two ulnar nerve innervated muscles: the adductor pollicis (AP) muscle and the abductor digiti minimi (ADM) muscle during the recovery from rocuronium-induced neuromuscular block by using EMG AF-201P. METHODS: Twenty patients undergoing surgery with general anesthesia were enrolled in the study. During total intravenous general anesthesia, train-of-four (TOF) and post-tetanic counts (PTC) responses following 0.9 mg/kg rocuronium administration were concurrently monitored at the AP and the ADM muscles with EMG AF-201P on the opposite arms. At the end of the surgery, sugammadex 2 mg/kg was administered when TOF counts of 2 (TOFC2) was observed at both muscles. The primary outcome of the study was time from administration of rocuronium to first appearance of PTC response (first PTC). The secondary outcomes of the study were time from administration of rocuronium to TOF count of 1 (TOFC1), time from first PTC to TOFC1 (PTC-TOF time), time to TOFC2, and time from administration of sugammadex to TOF ratio ≥ 0.9. Agreement between the two muscles was assessed using the Bland-Altman analysis. Data are expressed as mean ± standard deviation. RESULTS: Nineteen patients were included in the analysis. Time to first PTC was significantly faster at the ADM muscle than the AP muscle (24.4 ± 11.4 min vs 32.4 ± 13.1 min, p = 0.006). PTC-TOF time was significantly longer with the ADM muscle than the AP muscle (19.4 ± 7.3 min vs 12.4 ± 10.6 min, p = 0.019). There were no significant differences in time to TOFC2 and sugammadex-facilitated recovery between the two muscles. Bland-Altman analyses showed acceptable ranges of bias and limits of agreement of the two muscles. CONCLUSIONS: The ADM muscle showed a good agreement with the AP muscle during rocuronium-induced neuromuscular block but faster recovery of PTC response when using EMG. TRIAL REGISTRATION: UMIN-CTR (Registration No. UMIN000044904 ). Registered 19 July 2021 -Retrospectively registered, https://center6.umin.ac.jp/cgi-bin/ctr_e/ctr_view.cgi?recptno=R000051290 .

A Comparison Between the Adductor Pollicis Muscle Using TOF-Watch SX and the Abductor Digiti Minimi Muscle Using TetraGraph in Rocuronium-Induced Neuromuscular Block: A Prospective Observational Study.

BACKGROUND: The commonly used acceleromyography (AMG)-based neuromuscular monitor TOF-Watch SX is no longer manufactured. Recently, a new portable electromyography (EMG)-based neuromuscular monitor TetraGraph was introduced in clinical anesthesia. The aim of the study was to compare the responses obtained simultaneously from the abductor digiti minimi (ADM) muscle with TetraGraph and the adductor pollicis (AP) muscle with TOF-Watch SX during rocuronium-induced neuromuscular block. METHODS: Patients undergoing orthopedic surgery with general anesthesia were enrolled in this prospective, observational study. During total intravenous general anesthesia, train-of-four (TOF) responses following 0.9-mg·kg -1 rocuronium administration were monitored at the AP muscle with TOF-Watch SX and the ADM muscle with TetraGraph on the opposite arms. Sugammadex 2 mg·kg -1 was administered when both devices showed TOF counts (TOFCs) = 2. The primary outcome was time from rocuronium administration to first appearance of posttetanic count (PTC) response (first PTC). The secondary outcomes were baseline TOF ratios (TOFRs), onset time, time to first reappearance of TOFC = 1 (time to TOFC1), time to first reappearance of TOFC = 2 (time to TOFC2), and time from sugammadex administration to TOFR ≥0.9 with TetraGraph or to normalized TOFR ≥0.9 with TOF-Watch SX (recovery time). We used paired t test and Wilcoxon signed-rank test to analyze parametric and nonparametric data, respectively. P <.05 defined statistical significance. RESULTS: A total of 20 patients were analyzed. The baseline TOFRs were significantly higher with TOF-Watch SX than with TetraGraph (105 [96-110] vs 100 [98-101]; P = .0002). The time to first PTC (minutes) (31.7 ± 9.6 vs 41.1 ± 12.3; P < .001), time to TOFC1 (minutes) (48.0 ± 12.7 vs 58.8 ± 19.2; P < .001), time to TOFC2 (minutes) (56.2 ± 15.7 vs 74.2 ± 23.7; P < .001), and recovery time (seconds) (61.5 [32-148] vs 75.5 [94-102]); P = .043) were significantly faster with TOF-Watch SX than with TetraGraph. There were no significant differences in onset time. CONCLUSIONS: TOF-Watch SX overestimated recovery from rocuronium-induced neuromuscular block compared with TetraGraph.

Cellular variability of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrate that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two ß-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by a failure of mRNA degradation after successful translation termination at the PTC.

Quantitative Kinetic Analyses of Histone Turnover Using Imaging and Flow Cytometry.

Dynamic histone changes occur as a central part of chromatin regulation. Deposition of histone variants and post-translational modifications of histones are strongly associated with properties of chromatin status. Characterizing the kinetics of histone variants allows important insights into transcription regulation, chromatin maintenance and other chromatin properties. Here we provide a protocol of quantitative and sensitive approaches to test the timing of incorporation and dissociation of histones using a two-color SNAP-labeling system, labelling pre-existing and newly-incorporated histones distinctly. Together with cell cycle synchronization methods and cell cycle markers, this approach enables a pulse-chase analysis to determine the turnover of histone variants during the cell cycle, detected using imaging or flow cytometry methods at single cell resolution. As well as testing global histone turnover, cell cycle-dependent cellular localization of histone variants can be also addressed using imaging approaches.

Imaging of DNA and RNA in Living Eukaryotic Cells to Reveal Spatiotemporal Dynamics of Gene Expression.

This review focuses on imaging DNA and single RNA molecules in living cells to define eukaryotic functional organization and dynamic processes. The latest advances in technologies to visualize individual DNA loci and RNAs in real time are discussed. Single-molecule fluorescence microscopy provides the spatial and temporal resolution to reveal mechanisms regulating fundamental cell functions. Novel insights into the regulation of nuclear architecture, transcription, posttranscriptional RNA processing, and RNA localization provided by multicolor fluorescence microscopy are reviewed. A perspective on the future use of live imaging technologies and overcoming their current limitations is provided.

A Cellular Stress Response Induced by the CRISPR-dCas9 Activation System Is Not Heritable Through Cell Divisions.

The CRISPR-Cas9 system can be modified to perform "epigenetic editing" by utilizing the catalytically inactive (dead) Cas9 (dCas9) to recruit regulatory proteins to specific genomic locations. In prior studies, epigenetic editing with multimers of the transactivator VP16 and guide RNAs (gRNAs) was found to cause adverse cellular responses. These side effects may confound studies inducing new cellular properties, especially if the cellular responses are maintained through cell divisions-an epigenetic regulatory property. Here, we show how distinct components of this CRISPR-dCas9 activation system, particularly dCas9 with untargeted gRNAs, upregulate genes associated with transcriptional stress, defense response, and regulation of cell death. Our results highlight a previously undetected acute stress response to CRISPR-dCas9 components in human cells, which is transient and not maintained through cell divisions.

Retargeting of macroH2A following mitosis to cytogenetic-scale heterochromatic domains.

The heritability of chromatin states through cell division is a potential contributor to the epigenetic maintenance of cellular memory of prior states. The macroH2A histone variant has properties of a regulator of epigenetic cell memory, including roles controlling gene silencing and cell differentiation. Its mechanisms of regional genomic targeting and maintenance through cell division are unknown. Here, we combined in vivo imaging with biochemical and genomic approaches to show that human macroH2A is incorporated into chromatin in the G1 phase of the cell cycle following DNA replication. The newly incorporated macroH2A retargets the same large heterochromatic domains where macroH2A was already enriched in the previous cell cycle. It remains heterotypic, targeting individual nucleosomes that do not already contain a macroH2A molecule. The pattern observed resembles that of a new deposition of centromeric histone variants during the cell cycle, indicating mechanistic similarities for macrodomain-scale regulation of epigenetic properties of the cell.

DNMT3A and TET2 in the Pre-Leukemic Phase of Hematopoietic Disorders.

In recent years, advances in next-generation sequencing (NGS) technology have provided the opportunity to detect putative genetic drivers of disease, particularly cancers, with very high sensitivity. This knowledge has substantially improved our understanding of tumor pathogenesis. In hematological malignancies such as acute myeloid leukemia and myelodysplastic syndromes, pioneering work combining multi-parameter flow cytometry and targeted resequencing in leukemia have clearly shown that different classes of mutations appear to be acquired in particular sequences along the hematopoietic differentiation hierarchy. Moreover, as these mutations can be found in "normal" cells recovered during remission and can be detected at relapse, there is strong evidence for the existence of "pre-leukemic" stem cells (pre-LSC). These cells, while phenotypically normal by flow cytometry, morphology, and functional studies, are speculated to be molecularly poised to transform owing to a limited number of predisposing mutations. Identifying these "pre-leukemic" mutations and how they propagate a pre-malignant state has important implications for understanding the etiology of these disorders and for the development of novel therapeutics. NGS studies have found a substantial enrichment for mutations in epigenetic/chromatin remodeling regulators in pre-LSC, and elegant genetic models have confirmed that these mutations can predispose to a variety of hematological malignancies. In this review, we will discuss the current understanding of pre-leukemic biology in myeloid malignancies, and how mutations in two key epigenetic regulators, DNMT3A and TET2, may contribute to disease pathogenesis.

RNA:DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships.

BACKGROUND: RNA:DNA hybrids represent a non-canonical nucleic acid structure that has been associated with a range of human diseases and potential transcriptional regulatory functions. Mapping of RNA:DNA hybrids in human cells reveals them to have a number of characteristics that give insights into their functions. RESULTS: We find RNA:DNA hybrids to occupy millions of base pairs in the human genome. A directional sequencing approach shows the RNA component of the RNA:DNA hybrid to be purine-rich, indicating a thermodynamic contribution to their in vivo stability. The RNA:DNA hybrids are enriched at loci with decreased DNA methylation and increased DNase hypersensitivity, and within larger domains with characteristics of heterochromatin formation, indicating potential transcriptional regulatory properties. Mass spectrometry studies of chromatin at RNA:DNA hybrids shows the presence of the ILF2 and ILF3 transcription factors, supporting a model of certain transcription factors binding preferentially to the RNA:DNA conformation. CONCLUSIONS: Overall, there is little to indicate a dependence for RNA:DNA hybrids forming co-transcriptionally, with results from the ribosomal DNA repeat unit instead supporting the intriguing model of RNA generating these structures in trans. The results of the study indicate heterogeneous functions of these genomic elements and new insights into their formation and stability in vivo.

Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences.

Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts.

Temporal and spatial characterization of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism responsible for "surveying" mRNAs during translation and degrading those that harbor a premature termination codon (PTC). Currently the intracellular spatial location of NMD and the kinetics of its decay step in mammalian cells are under debate. To address these issues, we used single-RNA fluorescent in situ hybridization (FISH) and measured the NMD of PTC-containing ß-globin mRNA in intact single cells after the induction of ß-globin gene transcription. This approach preserves temporal and spatial information of the NMD process, both of which would be lost in an ensemble study. We determined that decay of the majority of PTC-containing ß-globin mRNA occurs soon after its export into the cytoplasm, with a half-life of <1 min; the remainder is degraded with a half-life of >12 h, similar to the half-life of normal PTC-free ß-globin mRNA, indicating that it had evaded NMD. Importantly, NMD does not occur within the nucleoplasm, thus countering the long-debated idea of nuclear degradation of PTC-containing transcripts. We provide a spatial and temporal model for the biphasic decay of NMD targets.

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.

Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin beta.

Mammalian mRNAs lose and acquire proteins throughout their life span while undergoing processing, transport, translation, and decay. How translation affects messenger RNA (mRNA)-protein interactions is largely unknown. The pioneer round of translation uses newly synthesized mRNA that is bound by cap-binding protein 80 (CBP80)-CBP20 (also known as the cap-binding complex [CBC]) at the cap, poly(A)-binding protein N1 (PABPN1) and PABPC1 at the poly(A) tail, and, provided biogenesis involves pre-mRNA splicing, exon junction complexes (EJCs) at exon-exon junctions. Subsequent rounds of translation engage mRNA that is bound by eukaryotic translation initiation factor 4E (eIF4E) at the cap and PABPC1 at the poly(A) tail, but that lacks detectable EJCs and PABPN1. Using the level of intracellular iron to regulate the translation of specific mRNAs, we show that translation promotes not only removal of EJC constituents, including the eIF4AIII anchor, but also replacement of PABPN1 by PABPC1. Remarkably, translation does not affect replacement of CBC by eIF4E. Instead, replacement of CBC by eIF4E is promoted by importin beta (IMPbeta): Inhibiting the binding of IMPbeta to the complex of CBC-IMPalpha at an mRNA cap using the IMPalpha IBB (IMPbeta-binding) domain or a RAN variant increases the amount of CBC-bound mRNA and decreases the amount of eIF4E-bound mRNA. Our studies uncover a previously unappreciated role for IMPbeta and a novel paradigm for how newly synthesized messenger ribonucleoproteins (mRNPs) are matured.

Efficiency of the pioneer round of translation affects the cellular site of nonsense-mediated mRNA decay.

In mammalian cells, nonsense-mediated mRNA decay (NMD) is a consequence of nonsense codon recognition during a pioneer round of translation. This round can occur largely before or largely after the release of newly synthesized mRNA from nuclei, depending on the mRNA, and likely utilizes cytoplasmic ribosomes. We show that increasing the cellular concentration of the splicing factor SF2/ASF augments the efficiency of NMD and ultimately shifts NMD that takes place after mRNA export to the cytoplasm to NMD that occurs before mRNA release from nuclei. These changes are accompanied by an increased association of pioneer translation initiation complexes with SF2/ASF, translationally active ribosomes, and the translational activator TAP. Increased TAP binding correlates with increased SF2/ASF binding, but not increased REF/Aly or Y14 binding. Our results uncover an additional role for SF2/ASF and indicate that the efficiency of the pioneer round of translation influences the efficiency of subsequent rounds of translation.

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.