Trk-fused gene plays a critical role in diet-induced adipose tissue expansion and is also involved in thyroid hormone action.

Mutations in the Trk-fused gene (TFG) cause hereditary motor and sensory neuropathy with proximal dominant involvement, which reportedly has high co-incidences with diabetes and dyslipidemia, suggesting critical roles of the TFG in metabolism as well. We found that TFG expression levels in white adipose tissues (WATs) were elevated in both genetically and diet-induced obese mice and that TFG deletion in preadipocytes from the stromal vascular fraction (SVF) markedly inhibited adipogenesis. To investigate its role in vivo, we generated tamoxifen-inducible adipocyte-specific TFG knockout (AiTFG KO) mice. While a marked down-regulation of the peroxisome proliferator-activated receptor gamma target, de novo lipogenesis (DNL), and mitochondria-related gene expressions were observed in subcutaneous WAT (scWAT) from AiTFG KO mice, these effects were blunted in SVF-derived adipocytes when the TFG was deleted after differentiation into adipocytes, implying cell nonautonomous effects. Intriguingly, expressions of thyroid hormone receptors, as well as carbohydrate responsive element-binding protein ß, which mediates the metabolic actions of thyroid hormone, were drastically down-regulated in scWAT from AiTFG KO mice. Reduced DNL and thermogenic gene expressions in AiTFG KO mice might be attributable to impaired thyroid hormone action in vivo. Finally, when adipocyte TFG was deleted in either the early or the late phase of high-fat diet feeding, the former brought about an impaired expansion of epididymal WAT, whereas the latter caused prominent adipocyte cell death. TFG deletion in adipocytes markedly exacerbated hepatic steatosis in both experimental settings. Collectively, these observations indicate that the TFG plays essential roles in maintaining normal adipocyte functions, including an enlargement of adipose tissue, thyroid hormone function, and thermogenic gene expressions, and in preserving hypertrophic adipocytes.

Identification of TFG- and Autophagy-Regulated Proteins and Glycerophospholipids in B Cells.

Autophagy supervises the proteostasis and survival of B lymphocytic cells. Trk-fused gene (TFG) promotes autophagosome-lysosome flux in murine CH12 B cells, as well as their survival. Hence, quantitative proteomics of CH12tfgKO and WT B cells in combination with lysosomal inhibition should identify proteins that are prone to lysosomal degradation and contribute to autophagy and B cell survival. Lysosome inhibition via NH4Cl unexpectedly reduced a number of proteins but increased a large cluster of translational, ribosomal, and mitochondrial proteins, independent of TFG. Hence, we propose a role for lysosomes in ribophagy in B cells. TFG-regulated proteins include CD74, BCL10, or the immunoglobulin JCHAIN. Gene ontology (GO) analysis reveals that proteins regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane-bound organelles. Likewise, TFG regulates the abundance of metabolic enzymes, such as ALDOC and the fatty acid-activating enzyme ACOT9. To test consequently for a function of TFG in lipid metabolism, we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol is more abundant in CH12tfgKO B cells. Several glycerophospholipid species with similar acyl side chains, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, show a dysequilibrium. We suggest a role for TFG in lipid homeostasis, mitochondrial functions, translation, and metabolism in B cells.

The Early Secretory Pathway Is Crucial for Multiple Aspects of the Hepatitis C Virus Life Cycle.

Although most of the early events of the hepatitis C virus (HCV) life cycle are well characterized, our understanding of HCV egress is still unclear. Some reports implicate the conventional endoplasmic reticulum (ER)-Golgi route, while some propose noncanonical secretory routes. Initially, the envelopment of HCV nucleocapsid occurs by budding into the ER lumen. Subsequently, the HCV particle exit from the ER is assumed to be mediated by coat protein complex II (COPII) vesicles. COPII vesicle biogenesis also involves the recruitment of cargo to the site of vesicle biogenesis via interaction with COPII inner coat proteins. We investigated the modulation and the specific role of the individual components of the early secretory pathway in HCV egress. We observed that HCV inhibits cellular protein secretion and triggers the reorganization of the ER exit sites and ER-Golgi intermediate compartments (ERGIC). Gene-specific knockdown of the components of this pathway such as SEC16A, TFG, ERGIC-53, and COPII coat proteins demonstrated the functional significance of these components and the distinct role played by these proteins in various aspects of the HCV life cycle. SEC16A is essential for multiple steps in the HCV life cycle, whereas TFG is specifically involved in HCV egress and ERGIC-53 is crucial for HCV entry. Overall, our study establishes that the components of the early secretory pathway are essential for HCV propagation and emphasize the importance of the ER-Golgi secretory route in this process. Surprisingly, these components are also required for the early stages of the HCV life cycle due to their role in overall intracellular trafficking and homeostasis of the cellular endomembrane system. IMPORTANCE The virus life cycle involves entry into the host, replication of the genome, assembly of infectious progeny, and their subsequent release. Different aspects of the HCV life cycle, including entry, genome replication, and assembly, are well characterized; however, our understanding of the HCV release is still not clear and subject to debate due to varied findings. Here, we attempted to address this controversy and enhance our understanding of HCV egress by evaluating the role of the different components of the early secretory pathway in the HCV life cycle. To our surprise, we found that the components of the early secretory pathway are not only essential for HCV release but also contribute to many other earlier events of the HCV life cycle. This study emphasizes the importance of the early secretory pathway for the establishment of productive HCV infection in hepatocytes.

A novel TFG variant of uncertain significance in amyotrophic lateral sclerosis: A case report and review of literature.

Introduction: Amyotrophic lateral sclerosis is a neurodegenerative disease with wide variation of genetics associated with it. Among the different genes described, mutation in TFG is a rare finding in amyotrophic lateral sclerosis. Case presentation: A 35 years old right-handed male presenting with ipsilateral weakness was diagnosed with amyotrophic lateral sclerosis. He was found to have missense variant of TFG with uncertain significance on exome sequencing. Clinical discussion: The genetics involved in amyotrophic lateral sclerosis is ever-evolving. The identification of new TFG variant in this disease adds another evidence to the role of TFG in neurodegenerative disease. Conclusions: The finding of TFG variant of uncertain significance is a rare finding in amyotrophic lateral sclerosis. And with the identification of new TFG variant, it leads to further understanding of spectrum of TFG and its pathophysiology in amyotrophic lateral sclerosis.

Changes in TFG gene expression in bovine leucocytes transformed by Theileria annulata.

Theileria annulata schizont-infected host cells in culture in vitro show unlimited proliferation similar to tumor cells; thus far, T. annulata and T. parva are the only eukaryotes that have been found to transform mammalian cells (immortalized). The transformation of these cells is reversible; when the parasite is eliminated in transformed cells by buparvaquone (BW720c), the host cells show normal growth and apoptosis. TFG is a tropomyosin-receptor kinase fused gene that is conserved among many species and is an important proto-oncogene. In this study, the bovine TFG gene was amplified by PCR from the cDNA of T. annulata schizont-transformed cells, cloned into the pGEX-4T-1 vector and expressed in Escherichia coli BL21 (DE3). After purification, the fusion protein was injected into rabbits to produce polyclonal antibodies. Using T. annulata-transformed cells together with BW720c treatment to kill the parasite, we aimed to identify changes in TFG gene expression by real-time PCR and Western blotting. The results showed that the bovine TFG gene was ~582 bp in size; SDS-PAGE analysis showed that the fusion protein was expressed in BL21 (DE3) cells with a molecular mass of 48 kD, and Western blotting indicated that the polyclonal antibodies could react with bovine TFG proteins from T. annulata-transformed cells and showed high specificity. Compared with that in the control group, the transcription level of the host TFG gene decreased significantly in the BW720c test group, and the expression of host tumor-related TFG protein decreased sharply after 72 h of drug treatment, suggesting that the TFG protein expression in transformed cells was directly related to T. annulata. This finding laid a foundation for further study on the interaction between T. annulata and host cells.

TFG mutation induces haploinsufficiency and drives axonal Charcot-Marie-Tooth disease by causing neurite degeneration.

AIMS: TFG-related axonal Charcot-Marie-Tooth (CMT) disease is a late-onset, autosomal dominant, hereditary motor, and sensory neuropathy characterized by slowly progressive weakness and atrophy of the distal muscles. The objective of this study was to determine the common pathogenic mechanism of TFG-related CMT type 2 (CMT2) caused by different mutations and establish a direct association between TFG haploinsufficiency and neurodegeneration. METHODS: Three individuals carrying the TFG p.G269V mutation but with varying disease durations were studied. The effect of the p.G269V mutation was confirmed by analyzing protein samples extracted from the blood of two individuals. The functional consequences of both CMT2 mutant gene products were evaluated in vitro. The effect of TFG deficiency in the nervous system was examined using zebrafish models and cultured mouse neurons. RESULTS: Overexpression of p.G269V TFG failed to enhance soluble TFG levels by generating insoluble TFG aggregates. TFG deficiency disrupted neurite outgrowth and induced neuronal apoptosis both in vivo and in vitro and further impaired locomotor capacity in zebrafish, which was consistent with the phenotype in patients. Wnt signaling was activated as a protective factor in response to TFG deficiency. CONCLUSION: CMT2-related TFG mutation induces TFG haploinsufficiency within cells and drives disease by causing progressive neurite degeneration.

A Novel TFG Mutation in a Korean Family with α-Synucleinopathy and Amyotrophic Lateral Sclerosis.

BACKGROUND: Tropomyosin-receptor kinase fused gene (TFG) functions as a regulator of intracellular protein packaging and trafficking at the endoplasmic reticulum exit sites. TFG has recently been proposed as a cause of multisystem proteinopathy. OBJECTIVES: Here, we describe a Korean family presenting with Parkinson's disease or amyotrophic lateral sclerosis caused by a novel variant of TFG (c.1148 G > A, p.Arg383His). METHODS: We collected clinical, genetic, dopamine transporter imaging, nerve conduction, and electromyography data from the seven subjects. To verify the pathogenicity of the R383H variant, we studied cell viability and the abnormal aggregation of α-synuclein and TAR DNA-binding protein 43 (TDP-43) in HeLa cells expressing R383H-TFG. RESULTS: The clinical phenotypes of the R383H-TFG mutation varied; of the five family members, one had Parkinson's disease, three had subclinical parkinsonism, and one (the proband) had amyotrophic lateral sclerosis. The individual with multiple system atrophy was the proband's paternal cousin, but the TFG genotype was not confirmed due to unavailability of samples. Our in vitro studies showed that R383H-TFG overexpression impaired cell viability. In cells co-expressing R383H-TFG and α-synuclein, insoluble α-synuclein aggregates increased in concentration and were secreted from the cells and co-localized with R383H-TFG. The levels of cytoplasmic insoluble aggregates of TDP-43 increased in HeLa cells expressing R383H-TFG and co-localized with R383H-TFG. CONCLUSIONS: Clinical and in vitro studies have supported the pathogenic role of the novel TFG mutation in α-synucleinopathy and TDP-43 proteinopathy. These findings expand the phenotypic spectrum of TFG and suggest a pivotal role of endoplasmic reticulum dysfunction during neurodegeneration. © 2021 International Parkinson and Movement Disorder Society.

Development of small-molecule tropomyosin receptor kinase (TRK) inhibitors for NTRK fusion cancers.

Tropomyosin receptor kinase A, B and C (TRKA, TRKB and TRKC), which are well-known members of the cell surface receptor tyrosine kinase (RTK) family, are encoded by the neurotrophic receptor tyrosine kinase 1, 2 and 3 (NTRK1, NTRK2 and NTRK3) genes, respectively. TRKs can regulate cell proliferation, differentiation and even apoptosis through the RAS/MAPKs, PI3K/AKT and PLCγ pathways. Gene fusions involving NTRK act as oncogenic drivers of a broad diversity of adult and pediatric tumors, and TRKs have become promising antitumor targets. Therefore, achieving a comprehensive understanding of TRKs and relevant TRK inhibitors should be urgently pursued for the further development of novel TRK inhibitors for potential clinical applications. This review focuses on summarizing the biological functions of TRKs and NTRK fusion proteins, the development of small-molecule TRK inhibitors with different chemotypes and their activity and selectivity, and the potential therapeutic applications of these inhibitors for future cancer drug discovery efforts.

TFG is required for autophagy flux and to prevent endoplasmic reticulum stress in CH12 B lymphoma cells.

Plasma cells depend on quality control of newly synthesized antibodies in the endoplasmic reticulum (ER) via macroautophagy/autophagy and proteasomal degradation. The cytosolic adaptor protein TFG (Trk-fused gene) regulates ER-Golgi transport, the secretory pathway and proteasome activity in non-immune cells. We show here that TFG is upregulated during lipopolysaccharide- and CpG-induced differentiation of B1 and B2 B cells into plasmablasts, with the highest expression of TFG in mature plasma cells. CRISPR-CAS9-mediated gene disruption of tfg in the B lymphoma cell line CH12 revealed increased apoptosis, which was reverted by BCL2 but even more by ectopic TFG expression. Loss of TFG disrupted ER structure, leading to an expanded ER and increased expression of ER stress genes. When compared to wild-type CH12 cells, tfg KO CH12 cells were more sensitive toward ER stress induced by tunicamycin, monensin and proteasome inhibition or by expression of an ER-bound immunoglobulin (Ig) µ heavy (µH) chain. CH12 tfg KO B cells displayed more total LC3, lower LC3-II turnover and increased numbers and size of autophagosomes. Tandem-fluorescent-LC3 revealed less accumulation of GFP-LC3 in starved and chloroquine-treated CH12 tfg KO B cells. The GFP:RFP ratio of tandem-fluorescent-LC3 was higher in tunicamycin-treated CH12 tfg KO B cells, suggesting less autophagy flux during induced ER stress. Based on these data, we suggest that TFG controls autophagy flux in CH12 B cells and propose that TFG is a survival factor that alleviates ER stress through the support of autophagy flux in activated B cells and mature plasma cells.Abbreviations: Ab, antibody; Ag, antigen; ASC, antibody-secreting cells; ATG, autophagy-related; BCR, B cell receptor; COPII, coat protein complex II; CpG, non-methylated CpG oligonucleotide; ER, endoplasmic reticulum; ERAD, ER-associated degradation; FO, follicular; GFP, green fluorescent protein; HC, heavy chain; Ig, immunoglobulin; IRES, internal ribosomal entry site; LC, light chain; MZ, marginal zone; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B cells; TLR, toll-like receptor; UPR, unfolded protein response.

Identification of a novel fusion gene, TRIM52-RACK1, in oral squamous cell carcinoma.

Gene fusion is caused by the linkage of previously separate genes or sequences. Recently, an increasing number of novel fusion genes have been identified and associated with tumor progression, and several of them have been suggested as promising targets for tumor therapy. However, there are hardly any studies reporting the association of fusion genes with the progression of oral squamous cell carcinoma (OSCC). In this study, we identified a total of 11 fused genes in OSCC cells. We further analyzed the structure of one fused gene, TRIM52-RACK1, and detected its function in tumor progression in vitro. We found that TRIM52-RACK1 was caused by a deletion of 181,257,187-181,247,386 at 5q35.3 and it promoted OSCC cell proliferation, migration, and invasion. Therefore, TRIM52-RACK1 can be a promising target for tumor therapy in OSCC.

[Analysis of copy number variation of CYP21A2 gene and the type of CYP21A1P/CYP21A2 fused gene in patients with 21-hydroxylase deficiency].

Objective: To analyze the copy number variation of CYP21A2 gene in 21-hydroxylase deficiency (21-OHD) patients, and identify the three copy repetition, single copy deletion of CYP21A2 gene and the type and proportion of CYP21A1P/CYP21A2 fused gene in 21-OHD patients. Methods: A total of 424 patients (140 males and 284 females) with 21-OHD who visited Peking Union Medical College Hospital from January 2015 to January 2018 were enrolled and the average age was (17.1±12.4) years. All clinical and biochemical data were collected. DNAs were extracted from peripheral blood leukocytes, and CYP21A2 gene mutation and copy number variation were detected by Sanger sequencing and multiple ligation probe amplification (MLPA). Results: Of 424 21-OHD patients, 287 (67.7%) had two copies of CYP21A2 gene, 137 (32.3%) had copy number variation, of which 1 patients (0.2%) had 3 copies of CYP21A2 gene and 136 (32.1%) were carriers of large deletion/rearrangement mutation of CYP21A2 gene. Three pathogenic mutations including a truncated Q319X protein mutation were detected in the patient with 3 copies of CYP21A2 gene. Of 136 patients with large deletion/rearrangement mutation of CYP21A2 gene, 82 (60.3%) carried fused CYP21A1P/CYP21A2 gene, and the remaining 54 harbored the one allele deletion of CYP21A2. The most common types of fused CYP21A1P/CYP21A2 gene were CH-5, CH-1 and CH-2, with the frequency being 31.7% (26 cases), 26.8% (22 cases) and 19.5% (16 cases), respectively, and followed by CH-4 and CH-7, with the incidence being 8.5% (7 cases) and 4.9% (4 cases), respectively. In addition, two cases of CH-3, CH-6 and CH-8 and one case of CH-9 were detected. Conclusions: This is the first study to detect the occurrence of CYP21A2 gene copy number variation and fused CYP21A1P/CYP21A2 gene in a large cohort of 21-OHD patients. The number of CYP21A2 gene copies in 21-OHD patients includes 2 copies, 1 copy deletion and 3 copies duplication. One copy deletion of CYP21A2 includes one allele deletion of CYP21A2 gene and fused CYP21A1P/CYP21A2 gene. In patients with 3 copies of CYP21A2 gene, pathogenic mutations should be verified in all 3 copies of CYP21A2 gene to make the precise diagnosis. Therefore, the accurate molecular diagnosis of 21-OHD patients should take both genotype and copy number variation of CYP21A2 into account.

Muscle pathology of hereditary motor and sensory neuropathy with proximal dominant involvement with TFG mutation.

BACKGROUND: Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is characterized by adult onset, a slowly progressive course and autosomal dominant inheritance. It remains unclear whether myopathic changes occur histopathologically. METHODS: We encountered 2 patients in a family with a heterozygous p.P285L mutation in TRK-fused gene (TFG), which is known to cause HMSN-P. The affected individuals developed proximal-dominant muscle weakness in their 40s, which slowly progressed to a motor neuron disease-like phenotype. RESULTS: Muscle biopsy showed myopathic pathology including fiber size variability, increased internal nuclei, fiber splitting, and core-like structures, associated with neurogenic changes: large groups of atrophic fibers and fiber type-grouping. Immunohistochemistry revealed sarcoplasmic aggregates of TFG, TDP-43, and p62 without congophilic material. CONCLUSIONS: The present study demonstrates myopathic changes in HMSN-P. Although the mechanisms underlying the skeletal muscle involvement remain to be elucidated, immunohistochemistry suggests that abnormal protein aggregation may be involved in the myopathic pathology.

Continuum of phenotypes in hereditary motor and sensory neuropathy with proximal predominance and Charcot-Marie-Tooth patients with TFG mutation.

Charcot-Marie-Tooth (CMT) is a common neuropathy, and hereditary motor and sensory neuropathy with proximal predominance (HMSN-P) is a recently described rare neuromuscular disease. Although many genes have been implicated for CMT, TFG is the only known HMSN-P-causing gene. Within the framework of diagnostic criteria, clinical variation is evident among CMT-diagnosed and also HMSN-P-diagnosed individuals. Mutations that cause p.(Pro285Leu) and p.(Gly269Val) in TFG were earlier reported as cause of HMSN-P in two Iranian pedigrees. Here, we report the identification of p.(Gly269Val) in TFG as cause of CMT in a large Iranian pedigree. The clinical features of patients of the three pedigrees are presented and critically compared. Similarities between the two HMSN-P-diagnosed pedigrees with different TFG mutations, and differences between the two differentially diagnosed pedigrees with the same p.(Gly269Val) mutation were evident. The clinical features of the HMSN-P pedigree with the p.(Pro285Leu) and the CMT pedigree with the p.(Gly269Val) mutation were clearly congruent with the respective diagnoses, whereas the features of the HMSN-P-diagnosed pedigree with the p.(Gly269Val) were intermediate between the other two pedigrees. It is therefore suggested that the clinical features of the three Iranian pedigrees with TFG mutations and diagnosed with HMSN-P or CMT represent a continuum.

TRK-fused Gene Protein Localization Is Prominent in Serotonergic and Noradrenergic Cell Groups, and Some Lower Motor Neurons in the Corticospinal Tract of the Rat Brainstem.

The TRK-fused gene (TFG) is reported to be involved in the regulation of cell size, apoptosis, cell growth, ER-Golgi protein secretion, NF-κß pathway signaling, the ubiquitin-proteasome system, and pancreatic ß-cell mass and function. TFG mutations were reported in some neurodegenerative diseases affecting sensory and motor functions. However, the function of TFG in the nervous system and how TFG mutations lead to neurodegeneration remain unclear. In this study, we employed double immunohistochemistry to investigate the details of TFG localization patterns in monoaminergic and cholinergic neurons in the brainstem. Intense TFG immunoreactivity was observed in the dorsal raphe nucleus, the locus coeruleus, and the ventral horn of the spinal cord. TFG immunoreactivity was observed in some serotonergic neurons in all B1-B9 cell groups, and some noradrenergic neurons in all A1-A7 cell groups in the rat brainstem, while no immunoreactivity was observed in the dopaminergic neurons in A8-A10 cell groups. TFG immunoreactivity was observed in all ChAT-positive motor nuclei in the lower corticospinal tract of the rat brainstem.

Pathogenic TFG Mutations Underlying Hereditary Spastic Paraplegia Impair Secretory Protein Trafficking and Axon Fasciculation.

Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated in both diseases, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the TFG coiled-coil domain, which underlies early-onset forms of HSP. We find that the TFG (p.R106C) mutation alters compaction of TFG ring complexes, which play a critical role in the export of cargoes from the endoplasmic reticulum (ER). Using CRISPR-mediated genome editing, we engineered human stem cells that express the mutant form of TFG at endogenous levels and identified specific defects in secretion from the ER and axon fasciculation following neuronal differentiation. Together, our data highlight a key role for TFG-mediated protein transport in the pathogenesis of HSP.

The natural history of hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) in 97 Japanese patients.

Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is a motor and sensory neuronopathy with autosomal dominant inheritance, adult onset, slowly progressive course, and is associated with TRK-fused gene (TFG) mutation. At advanced stages, respiratory failure and dysphagia becomes life-threatoning, and patients typically die by their 70s. Although there is currently no evidence for effective treatment, a therapy may be found by elucidation of the function of TFG. Recently its pathomechanism has been proposed to be associated with abnormalities in protein transfer from the endoplasmic reticulum. Such pathomechanisms might involve a similar process in amyotrophic lateral sclerosis; thus, its pathomechanisms and treatment strategy might make it a good model for neurodegenerative disorders. It is of great value to clarify the natural history of HMSN-P, in oder to judge the treatment effect. By evaluating 97 patients (79 out of 97 were examined and all confirmed with p.Pro 285 Leu mutation) in this study, it was confirmed that this disease follows a uniform course in the earlier stages, and there are individual differences in the onset between 20 and 30 years. Such uniformity might be due to the proposed single gene abnormality. At advanced stages, there are larger individual differences in the progression, but the reasons for these are unknown. Longer survival might be achieved with a better care for respiratory failure and dysphagia if such cares were undertaken at appropriate times.

Discriminating RNA variants with single-molecule allele-specific FISH.

DNA mutations of various types often affect the cellular localization and function of gene products. The role of mutant transcripts in the pathogenesis of human disease is increasingly recognized. Among the pathogenic RNA variants are transcripts with single nucleotide substitutions, small insertions or deletions, aberrantly or alternatively spliced transcripts and RNAs derived from fused genes. To discriminate among transcripts, particularly those of low abundance, showing small or large sequence differences, a highly sensitive and specific RNA imaging method is required. The method that fulfills these criteria is single-molecule fluorescence in situ hybridization (smFISH) combined with probes discriminating among RNA variants. With this method, RNA transcripts produced from individual alleles can be imaged, and differences in their transcription, processing, cellular localization and decay can be revealed. In addition to its applications for studying physiological processes involving RNA variants, smFISH offers several advantages for disease related mutation research. Further development of allele-specific microscopic methods may broaden group of RNA variants analyzed, including RNAs with expanded repeat tract, different variants of 3'UTR, RNAs differing in length of polyA tract or transcripts produced from alternative start codons. Moreover, first attempts for allele-specific RNA live imaging were made adding time-lapse analysis. In this review, we discuss important aspects of the variant-specific smFISH methodology and present examples of its applications in deciphering RNA-mediated pathogenic mechanisms in a variety of human diseases, including cancer, neurological, immunological and cardiovascular diseases.

TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER-Golgi intermediate compartments.

The conserved coat protein complex II (COPII) mediates the initial steps of secretory protein trafficking by assembling onto subdomains of the endoplasmic reticulum (ER) in two layers to generate cargo-laden transport carriers that ultimately fuse with an adjacent ER-Golgi intermediate compartment (ERGIC). Here, we demonstrate that Trk-fused gene (TFG) binds directly to the inner layer of the COPII coat. Specifically, the TFG C terminus interacts with Sec23 through a shared interface with the outer COPII coat and the cargo receptor Tango1/cTAGE5. Our findings indicate that TFG binding to Sec23 outcompetes these other associations in a concentration-dependent manner and ultimately promotes outer coat dissociation. Additionally, we demonstrate that TFG tethers vesicles harboring the inner COPII coat, which contributes to their clustering between the ER and ERGIC in cells. Together, our studies define a mechanism by which COPII transport carriers are retained locally at the ER/ERGIC interface after outer coat disassembly, which is a prerequisite for fusion with ERGIC membranes.

The calcium-binding protein ALG-2 promotes endoplasmic reticulum exit site localization and polymerization of Trk-fused gene (TFG) protein.

Apoptosis-linked gene 2 (ALG-2), which is a gene product of PDCD6, is a 22-kDa Ca2+ -binding protein. Accumulating evidence points to a role for ALG-2 as a Ca2+ -responsive adaptor protein. On binding to Ca2+ , ALG-2 undergoes a conformational change that facilitates its interaction with various proteins. It also forms a homodimer and heterodimer with peflin, a paralog of ALG-2. However, the differences in cellular roles for the ALG-2 homodimer and ALG-2/peflin heterodimer are unclear. In the present study, we found that Trk-fused gene (TFG) protein interacted with the ALG-2 homodimer. Immunostaining analysis revealed that TFG and ALG-2 partially overlapped at endoplasmic reticulum exit sites (ERES), a platform for COPII-mediated protein transport from the endoplasmic reticulum. Time-lapse live-cell imaging demonstrated that both green fluorescent protein-fused TFG and mCherry-fused ALG-2 are recruited to ERES after thapsigargin treatment, which raises intracellular Ca2+ levels. Furthermore, overexpression of ALG-2 induced the accumulation of TFG at ERES. TFG has an ALG-2-binding motif and deletion of the motif decreased TFG binding to ALG-2 and shortened its half-life at ERES, suggesting a critical role for ALG-2 in retaining TFG at ERES. We also demonstrated, by in vitro cross-linking assays, that ALG-2 promoted the polymerization of TFG in a Ca2+ -dependent manner. Collectively, the results suggest that ALG-2 acts as a Ca2+ -sensitive adaptor to concentrate and polymerize TFG at ERES, supporting a potential role for ALG-2 in COPII-dependent trafficking from the endoplasmic reticulum.