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The formation of self-perpetuating protein aggregates such as amyloids is associated with various diseases and provides a basis for transmissible (infectious or heritable) protein isoforms (prions). Many human proteins involved in the regulation of transcription contain potentially amyloidogenic regions. Here, it is shown that short N-terminal isoforms of the human protein PHC3, a component of the chromatin-modifying complex PRC1 (Polycomb repressive complex 1), can form prion-like aggregates in yeast assays, exhibit amyloid properties in the E. coli-based C-DAG assay, and produce detergent-resistant aggregates when ectopically expressed in cultured human cells. Moreover, aggregates of short isoforms can sequester the full-length PHC3 protein, causing its accumulation in the cytosol instead of the nucleus. The introduction of an aggregating short PHC3 isoform alters the transcriptional profile of cultured human cells. It is proposed that the aggregation of short isoforms is involved in the feedback downregulation of PRC1 activity, leading to more open chromatin configuration.

Germline variants in the NSD1 gene are responsible for Sotos syndrome, while somatic variants promote neoplastic cell transformation. Our previous studies revealed three alternative RNA isoforms of NSD1 present in fibroblast cell lines (FBs): the canonical full transcript and 2 alternative transcripts, termed AT2 (NSD1 Δ5Δ7) and AT3 (NSD1 Δ19–23 at the 5′ end). The precise molecular pathways affected by each specific isoform of NSD1 are uncharacterized to date. To elucidate the role of these isoforms, their expression was suppressed by siRNA knockdown in FBs and protein expression and transcriptome data was explored. We demonstrate that one gene target of NSD1 isoform AT2 is ARP3 actin-related protein 3 homolog B (ACTR3B). We show that loss of both canonical NSD1 and AT2 isoforms impaired the ability of fibroblasts to regulate the actin cytoskeleton, and we observed that this caused selective loss of stress fibers. Our findings provide novel insights into NSD1 function by distinguishing isoform function and demonstrating an essential role of NSD1 in regulating the actin cytoskeleton and stress fiber formation in fibroblasts.

VM202, a plasmid DNA that expresses two isoforms of hepatocyte growth factor, may elicit angiogenic effects that could benefit patients with critical limb ischemia (CLI). In a phase 2, double-blind trial in 52 CLI patients, we examined the safety and potential efficacy of intramuscular injections of low-dose ( n =21) or high-dose ( n =20) VM202 or placebo ( n =11) in the affected limb (days 0, 14, 28 and 42). Adverse events and serious adverse events were similar among the groups; no malignancy or proliferative retinopathy was seen. In exploratory efficacy analyses, we found no differences in ankle or toe-brachial index, VAS, VascuQuol or amputation rate among the groups. Complete ulcer healing was significantly better in high-dose (8/13 ulcers; P <0.01) versus placebo (1/9) patients. Clinically meaningful reductions (>50%) in ulcer area occurred in high-dose (9/13 ulcers) and low-dose (19/27) groups versus placebo (1/9; P <0.05 and P <0.005, respectively). At 12 months, significant differences were seen in TcPO 2 between the high-dose and placebo groups (47.5±17.8 versus 36.6±24.0 mm Hg, respectively; P <0.05) and in the change from baseline among the groups ( P <0.05). These data suggest that VM202 is safe and may provide therapeutic bioactivity in CLI patients.

Background Transcriptome reconstruction, defined as the identification of all protein isoforms that may be expressed by a gene, is a notably difficult computational task. With real data, the best methods based on RNA-seq data identify barely 21 % of the expressed transcripts. While waiting for algorithms and sequencing techniques to improve — as has been strongly suggested in the literature — it is important to evaluate assisted transcriptome prediction ; this is the question of how alternative transcription in one species performs as a predictor of protein isoforms in another relatively close species. Most evidence-based gene predictors use transcripts from other species to annotate a genome, but the predictive power of procedures that use exclusively transcripts from external species has never been quantified. The cornerstone of such an evaluation is the correct identification of pairs of transcripts with the same splicing patterns, called splicing orthologs . Results We propose a rigorous procedural definition of splicing orthologs, based on the identification of all ortholog pairs of splicing sites in the nucleotide sequences, and alignments at the protein level. Using our definition, we compared 24 382 human transcripts and 17 909 mouse transcripts from the highly curated CCDS database, and identified 11 122 splicing orthologs. In prediction mode, we show that human transcripts can be used to infer over 62 % of mouse protein isoforms. When restricting the predictions to transcripts known eight years ago, the percentage grows to 74 %. Using CCDS timestamped releases, we also analyze the evolution of the number of splicing orthologs over the last decade. Conclusions Alternative splicing is now recognized to play a major role in the protein diversity of eukaryotic organisms, but definitions of spliced isoform orthologs are still approximate. Here we propose a definition adapted to the subtle variations of conserved alternative splicing sites, and use it to validate numerous accurate orthologous isoform predictions.

The Connexin43 transmembrane protein (Cx43), encoded by the GJA1 gene, is a member of a multigenic family of proteins that oligomerize to form hemichannels and intercellular channels, allowing gap junctional intercellular communication between adjacent cells or communication between the intracellular and extracellular compartments. Cx43 has long been shown to play a significant but complex role in cancer development, acting as a tumor suppressor and/or tumor promoter. The effects of Cx43 are associated with both channel-dependent and -independent functionalities and differ depending on the expression level, subcellular location and the considered stage of cancer progression. Recently, six isoforms of Cx43 have been described and one of them, called GJA1-20k, has also been found to be expressed in cancer cells. This isoform is generated by alternative translation and corresponds to the end part of the fourth transmembrane domain and the entire carboxyl-terminal (CT) domain. Initial studies in the cardiac model implicated GJA1-20k in the trafficking of full-length Cx43 to the plasma membrane, in cytoskeletal dynamics and in mitochondrial fission and subcellular distribution. As these processes are associated with cancer progression, a potential link between Cx43 functions, mitochondrial activity and GJA1-20k expression can be postulated in this context. This review synthetizes the current knowledge on GJA1-20k and its potential involvement in processes related to epithelial-to-mesenchymal transition (EMT) and the proliferation, dissemination and quiescence of cancer cells. Particular emphasis is placed on the putative roles of GJA1-20k in full-length Cx43 exportation to the plasma membrane, mitochondrial activity and functions originally attributed to the CT domain.

This study aimed to evaluate the safety and preliminary efficacy of intramuscular injections of plasmid DNA (VM202) expressing two isoforms of hepatocyte growth factor (HGF) in subjects with painful diabetic peripheral neuropathy (PDPN). Twelve patients in three cohorts (4, 8, and 16 mg) received two sets of VM202 injections separated by two weeks. Safety and tolerability were evaluated and the visual analog scale (VAS), the short form McGill questionnaire (SF-MPQ), and the brief pain inventory for patients with diabetic peripheral neuropathy (BPI-DPN) measured pain level throughout 12 months after treatment. No serious adverse events (AEs) were observed. The mean VAS was reduced from baseline by 47.2% (P = 0.002) at 6 months and by 44.1% (P = 0.005) at 12 months after treatment. The VAS scores for the 4, 8, and 16 mg dose cohorts at 6 months follow-up decreased in a dose–responsive manner, by 21% (P = 0.971), 53% (P = 0.014), and 62% (P = 0.001), respectively. The results with the BPI-DPN and SF-MPQ showed patterns similar to the VAS scores. In conclusion, VM202 treatment appeared to be safe, well tolerated, and sufficient to provide long term symptomatic relief and improvement in the quality of life in patients with PDPN.

The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13); high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13); or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % (mean (SD)). High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2)% (p<0.00001) and 45.2 (7.3)% (p<0.00001). Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8)% (p<0.00001) and 46.1 (10.4)% (p<0.00001). In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4)% (p=0.029); low-dose Epoetin beta: 73.1 (17.8)% (p=0.039)). In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal.

In the absence of DHX9, circular RNAs accumulate and transcription and translation are dysregulated—effects that are exacerbated by concomitant depletion of the RNA-editing enzyme ADAR. DHX9 suppresses Alu-derived defects In the human genome, there are more than a million copies of the Alu transposable element. Movement of Alu elements is a common source of mutations, but as insertions usually occur in non-coding regions, they are often without discernible effect. Alu elements located near one another in an inverted orientation will form secondary structures that may affect various nuclear processes. Asifa Akhtar and colleagues find that the RNA helicase, DHX9, binds transcribed ‘IRAlus’ (inverted repeat Alu elements). In the absence of DHX9, circular RNAs accumulate, and transcription and translation are dysregulated. These effects are further exacerbated by co-depletion of DHX9 and ADAR p150, an interferon-inducible RNA modification enzyme. The authors conclude that these proteins protect against transposon insertion, which can have deleterious effects on gene expression. Transposable elements are viewed as ‘selfish genetic elements’, yet they contribute to gene regulation and genome evolution in diverse ways 1 . More than half of the human genome consists of transposable elements 2 . Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome 2 . Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability 3 . Alu elements are the main targets of the RNA-editing enzyme ADAR 4 and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC 5 , but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant 6 nuclear RNA helicase 7 , binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression.

T cells recognize antigens by using T cell receptors (TCRs) encoded by gene segments, called variable (V), diversity (D), and joining (J), that undergo somatic recombination to create diverse binding specificities. Four TCR chains (α, β, γ, and δ) have been identified to date, and, as T cells develop in the thymus, they express exclusively either an αβTCR or a γδTCR heterodimer. Here, we show that marsupials have an additional TCR (TCRμ) that has V, D, and J that are either somatically recombined, as in conventional TCRs, or are already prejoined in the germ-line DNA in a manner consistent with their creation by retrotransposition. TCRμ does not have a known homolog in eutherian mammals but has features analogous to a recently described TCRδ isoform in sharks. TCRμ is expressed in at least two mRNA isoforms that appear capable of encoding a full-length protein, both of which are transcribed in the thymus and spleen. One contains two variable domains: a somatically recombined V and a prejoined V. This appears to be the dominant isoform in peripheral lymphoid tissue. The other isoform contains only the prejoined V and is structurally more similar to conventional TCR chains, however invariant. Unlike other TCRs, TCRμ uses prejoined gene segments and is likely present in all marsupials. Its similarity to a TCR isoform in sharks suggests that it, or something similar, may be present in other vertebrate lineages and, therefore, may represent an ancient receptor system.

The EGF-family of tyrosine-kinase receptors activates cytoplasmic pathways involved in cell proliferation, migration and differentiation in response to specific extracellular ligands. Beside these canonical pathways, the nuclear localization of the ErbB receptors in primary tumours and cancer cell lines led to investigate their role as transcriptional regulators of cancer genes. The nuclear localization of ErbB3 has been reported in various cancer tissues and cell lines but the nuclear functions and the putative correlation with tumour progression and resistance to therapy remain unclear. We first assessed ErbB3 expression in normal and tumour prostate tissues. The nuclear staining was mainly due to an isoform matching the C-terminus domain of the full length ErbB3185kDa receptor. Nuclear staining was also restricted to cancer cells and was increased in advanced castration-resistant prostate cancer when compared to localized tumours, suggesting it could be involved in the progression of prostate cancer up to the terminal castration-resistant stage. ChIP-on-chip experiments were performed on immortalized and tumour cell lines selected upon characterization of endogenous nuclear expression of an ErbB380kDa isoform. Among the 1840 target promoters identified, 26 were selected before ErbB380kDa-dependent gene expression was evaluated by real-time quantitative RT-PCR, providing evidence that ErbB380kDa exerted transcriptional control on those genes. Some targets are already known to be involved in prostate cancer progression even though no link was previously established with ErbB3 membrane and/or nuclear signalling. Many others, not yet associated with prostate cancer, could provide new therapeutic possibilities for patients expressing ErbB380kDa. Detecting ErbB380kDa could thus constitute a useful marker of prognosis and response to therapy.