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The variability of proteins at the sequence level creates an enormous potential for proteome complexity. Exploring the depths and limits of this complexity is an ongoing goal in biology. Here, we systematically survey human and plant high-throughput bottom-up native proteomics data for protein truncation variants, where substantial regions of the full-length protein are missing from an observed protein product. In humans, Arabidopsis, and the green alga Chlamydomonas, approximately one percent of observed proteins show a short form, which we can assign by comparison to RNA isoforms as either likely deriving from transcript-directed processes or limited proteolysis. While some detected protein fragments align with known splice forms and protein cleavage events, multiple examples are previously undescribed, such as our observation of fibrocystin proteolysis and nuclear translocation in a green alga. We find that truncations occur almost entirely between structured protein domains, even when short forms are derived from transcript variants. Intriguingly, multiple endogenous protein truncations of phase-separating translational proteins resemble cleaved proteoforms produced by enteroviruses during infection. Some truncated proteins are also observed in both humans and plants, suggesting that they date to the last eukaryotic common ancestor. Finally, we describe novel proteoform-specific protein complexes, where the loss of a domain may accompany complex formation., (© 2024. The Author(s).)

Background: A buried penis (BP) is rare in which the penile body is retracted into the prepubic adipose tissue. This research focuses on differences in smooth muscle myosin heavy chain (SMMHC) isoform expressions in the dartos fascia., Methods: A total of 82 children, 41 of whom had BPs, who applied for circumcision between May and November 2021, were included in the study. The cases were divided into four groups aged ≥6 years (NP6, n = 18) and aged ≤3 years (NP3, n = 17) with normal penile appearance, aged ≥6 years (BP6, n = 23) and aged ≤3 years (BP,n = 24) with a BP. SMMHC isoforms mRNA gene expression analyses were performed by quantitative PCR technique in dartos fascia obtained from foreskin removed by circumcision., Results: Compared to the NP3 group, the SM1 mRNA expressed in the BP6 group was statistically significantly higher (p < 0.005). SM2 mRNA levels expressed in dartos fascia were considerably higher in NP6 and NP3 groups compared to BP6 and BP3 groups (p < 0.001). The SM2/SM1 ratio was 0.85 in the BP6 group and 1.46 in the NP6 group, which was statistically significant (p = 0.006) and increased from 0.87 in the BP3 group to 2.21 in the NP3 group (p < 0.001)., Conclusion: In a buried penis, there is a difference in the expression of SMMHC isoforms. SM1 is highly expressed, while SM2 decreases, increasing the SM2/SM1 ratio. This causes increased contractility in the smooth muscle, leading to retraction of the penile body. The dartos fascia surrounding it resembles aberrant muscle tissue in boys with a BP., Level of Evidence: Level III., Type of Study: Case-control study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Diversity, a hallmark of G protein-coupled receptor (GPCR) signaling, partly stems from alternative splicing of a single gene generating more than one isoform for a receptor. Additionally, receptor responses to ligands can be attenuated by desensitization upon prolonged or repeated ligand exposure. Both phenomena have been demonstrated and exemplified by the deuterostome tachykinin signaling system, although the role of phosphorylation in desensitization remains a subject of debate. Here, we describe the signaling system for tachykinin-related peptides (TKRPs) in a protostome, mollusk Aplysia. We cloned the Aplysia TKRP precursor, which encodes three TKRPs (apTKRP-1, apTKRP-2a, and apTKRP-2b) containing the FXGXR-amide motif. In situ hybridization and immunohistochemistry showed predominant expression of TKRP mRNA and peptide in the cerebral ganglia. TKRPs and their posttranslational modifications were observed in extracts of central nervous system ganglia using mass spectrometry. We identified two Aplysia TKRP receptors (apTKRPRs), named apTKRPR-A and apTKRPR-B. These receptors are two isoforms generated through alternative splicing of the same gene and differ only in their intracellular C termini. Structure-activity relationship analysis of apTKRP-2b revealed that both C-terminal amidation and conserved residues of the ligand are critical for receptor activation. C-terminal truncates and mutants of apTKRPRs suggested that there is a C-terminal phosphorylation-independent desensitization for both receptors. Moreover, apTKRPR-B also exhibits phosphorylation-dependent desensitization through the phosphorylation of C-terminal Ser/Thr residues. This comprehensive characterization of the Aplysia TKRP signaling system underscores the evolutionary conservation of the TKRP and TK signaling systems, while highlighting the intricacies of receptor regulation through alternative splicing and differential desensitization mechanisms., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in a variety of human cancers. Two overlapping NEAT1 isoforms, NEAT1&#95;1 and NEAT1&#95;2, are produced through mutually exclusive alternative 3' end formation. Previous studies extensively investigated NEAT1 dysregulation in tumors, but often failed to achieve distinct quantification of the two NEAT1 isoforms. Moreover, molecular mechanisms governing the biogenesis of NEAT1 isoforms and the functional impacts of their dysregulation in tumorigenesis remain poorly understood. In this study, we employed an isoform-specific quantification assay and found differential dysregulation of NEAT1 isoforms in patient-derived glioblastoma multiforme cells. We further showed usage of the NEAT1 proximal polyadenylation site (PAS) is a critical mechanism that controls glioma NEAT1 isoform production. CRISPR-Cas9-mediated PAS deletion reduced NEAT1&#95;1 and reciprocally increased NEAT1&#95;2, which enhanced nuclear paraspeckle formation in human glioma cells. Moreover, the utilization of the NEAT1 PAS is facilitated by the RNA-binding protein quaking (QKI), which binds to the proximal QKI recognition elements. Functionally, we identified transcriptomic changes and altered biological pathways caused by NEAT1 isoform imbalance in glioma cells, including the pathway for the regulation of cell migration. Finally, we demonstrated the forced increase of NEAT1&#95;2 upon NEAT1 PAS deletion is responsible for driving glioma cell migration and promoting the expression of genes implicated in the regulation of cell migration. Together, our studies uncovered a novel mechanism that regulates NEAT1 isoforms and their functional impacts on the glioma transcriptome, which affects pathological pathways of glioma, represented by migration., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Skeletal muscle tissue increases or decreases its volume by synthesizing or degrading myofibrillar proteins. The ubiquitin-proteasome system plays a pivotal role during muscle atrophy, where muscle ring finger proteins (Murf) function as E3 ubiquitin ligases responsible for identifying and targeting substrates for degradation. Our previous study demonstrated that overexpression of Ozz, an E3 specific to embryonic myosin heavy chain (Myh3), precisely reduced the Myh3 replacement rate in the thick filaments of myotubes (E. Ichimura et al., Physiol Rep. 9:e15003, 2021). These findings strongly suggest that E3 plays a critical role in regulating myosin replacement. Here, we hypothesized that the Murf isoforms, which recognize Myhs as substrates, reduced the myosin replacement rates through the enhanced Myh degradation by Murfs. First, fluorescence recovery after a photobleaching experiment was conducted to assess whether Murf isoforms affected the GFP-Myh3 replacement. In contrast to Murf2 or Murf3 overexpression, Murf1 overexpression selectively facilitated the GFP-Myh3 myosin replacement. Next, to examine the effects of Murf1 overexpression on the replacement of myosin isoforms, Cherry-Murf1 was coexpressed with GFP-Myh1, GFP-Myh4, or GFP-Myh7 in myotubes. Intriguingly, Murf1 overexpression enhanced the myosin replacement of GFP-Myh4 but did not affect those of GFP-Myh1 or GFP-Myh7. Surprisingly, overexpression of Murf1 did not enhance the ubiquitination of proteins. These results indicate that Murf1 selectively regulated myosin replacement in a Myh isoform-dependent fashion, independent of enhanced ubiquitination. This suggests that Murf1 may have a role beyond functioning as a ubiquitin ligase E3 in thick filament myosin replacement., (© 2024. The Society for In Vitro Biology.)

Leber congenital amaurosis (LCA) and early-onset retinal degeneration (EORD) are inherited retinal diseases (IRD) characterized by early-onset vision impairment. Herein, we studied 15 Saudi families by whole exome sequencing (WES) and run-of-homozygosity (ROH) detection via AutoMap in 12/15 consanguineous families. This revealed (likely) pathogenic variants in 11/15 families (73%). A potential founder variant was found in RPGRIP1. Homozygous pathogenic variants were identified in known IRD genes (ATF6, CRB1, CABP4, RDH12, RIMS2, RPGRIP1, SPATA7). We established genotype-driven clinical reclassifications for ATF6, CABP4, and RIMS2. Specifically, we observed isolated IRD in the individual with the novel RIMS2 variant, and we found a retina-enriched RIMS2 isoform conserved but not annotated in mouse. The latter illustrates potential different phenotypic consequences of pathogenic variants depending on the particular tissue/cell-type specific isoforms they affect. Lastly, a compound heterozygous genotype in GUCY2D in one non-consanguineous family was demonstrated, and homozygous variants in novel candidate genes ATG2B and RUFY3 were found in the two remaining consanguineous families. Reporting these genes will allow to validate them in other IRD cohorts. Finally, the missing heritability of the two unsolved IRD cases may be attributed to variants in non-coding regions or structural variants that remained undetected, warranting future WGS studies., (© 2024 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

Renal development is a complex process in which two major processes, tubular branching and nephron development, regulate each other reciprocally. Our previous findings have indicated that collagen XVIII (ColXVIII), an extracellular matrix protein, affects the renal branching morphogenesis. We investigate here the role of ColXVIII in nephron formation and the behavior of nephron progenitor cells (NPCs) using isoform-specific ColXVIII knockout mice. The results show that the short ColXVIII isoform predominates in the early epithelialized nephron structures whereas the two longer isoforms are expressed only in the later phases of glomerular formation. Meanwhile, electron microscopy showed that the ColXVIII mutant embryonic kidneys have ultrastructural defects at least from embryonic day 16.5 onwards. Similar structural defects had previously been observed in adult ColXVIII-deficient mice, indicating a congenital origin. The lack of ColXVIII led to a reduced NPC population in which changes in NPC proliferation and maintenance and in macrophage influx were perceived to play a role. The changes in NPC behavior in turn led to notably reduced overall nephron formation. In conclusion, the results show that ColXVIII has multiple roles in renal development, both in ureteric branching and in NPC behavior., Competing Interests: Declaration of competing interest There are no competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ferroportin (FPN) is a transmembrane protein and is the only known iron exporter that helps in maintaining iron homeostasis in vertebrates. To maintain stable iron equilibrium in the body, ferroportin works in conjunction with a peptide called hepcidin. In this study, we have identified an alternatively spliced novel isoform of the human SLC40A1 gene, which encodes for the FPN protein and is found to be expressed in different tissues. The novel transcript has an alternate last exon and encodes 31-amino acid long peptide sequence that replaces 104 amino acids at C-terminal in the novel transcript. Molecular modelling and molecular dynamics (MD) simulation studies revealed key structural features of the novel isoform (FPN-N). FPN-N was predicted to have 12 transmembrane domains similar to the reported isoform (FPN), despite being much smaller in size. FPN-N was found to interact with hepcidin, a key regulator of ferroportin activity. Also, the iron-binding sites were retained in the novel isoform as revealed by the MD simulation of FPN-N in bilipid membrane. The novel isoform identified in this study may play important role in iron homeostasis. However, further studies are required to characterize the FPN-N isoform and decipher its role inside the cell., (© 2024 International Union of Biochemistry and Molecular Biology.)

HSPB8 is a heat shock protein belonging to a family of ATP-independent stress proteins called HSPB which are present far and wide in the cells of various organisms. They are committed to protein quality control (PQC) and strive to avert protein aggregation and to procreate a pool of non-native proteins that can be swiftly folded. Their fundamental expression or stress inducibility is regulated by various cis-elements localized in the HSPB regulatory regions. In the current study we have predicted and confirmed two alternatively spliced novel transcripts of HSPB8 gene in liver, brain, and heart. These spliced variants have smaller sizes owing to smaller N terminal regions and showed remarkable changes in their cellular localization. Novel isoform (HSPB8-N1) was predicted to be majorly localized to nuclear region while the reported isoform (HSPB8) and one of the novel isoforms (HSPB8-N2) were predicted to be cytoplasmic in nature. There were many changes observed in the phosphorylation sites of the novel isoforms as well. The newly reported isoforms lack several structural motifs that are essential for various functional endeavors of the HSPB8 protein. In silico analysis of the conceptually translated protein was carried out using various bioinformatics tools to gain an understanding of their properties in order to explore their possible potential in therapeutics., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The transcriptional coactivator PGC-1α has been implicated in the regulation of multiple metabolic processes. However, the previously reported metabolic phenotypes of mice deficient in PGC-1α have been inconsistent. PGC-1α exists as multiple isoforms, including variants transcribed from an alternative first exon. We show here that alternative PGC-1α variants are the main entity that increases PGC-1α during exercise. These variants, unlike the canonical isoform of PGC-1α, are robustly upregulated in human skeletal muscle after exercise. Furthermore, the extent of this upregulation correlates with oxygen consumption. Mice lacking these variants manifest impaired energy expenditure during exercise, leading to the development of obesity and hyperinsulinemia. The alternative variants are also upregulated in brown adipose tissue in response to cold exposure, and mice lacking these variants are intolerant of a cold environment. Our findings thus indicate that an increase in PGC-1α expression, attributable mostly to upregulation of alternative variants, is pivotal for adaptive enhancement of energy expenditure and heat production and thereby essential for the regulation of whole-body energy metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Galectin 8 belongs to the tandem repeat subclass of the galectin superfamily. It possesses two homologous carbohydrate recognition domains linked by a short peptide and preferentially binds to β-galactoside-containing glycol-conjugates in a calcium-independent manner. This study identified Galectin-8-like isoform X1 (PhGal8X1) from red-lip mullet (Planiliza haematocheilus) and investigated its role in regulating fish immunity. The open reading frame of PhGal8X1 was 918bp, encoding a soluble protein of 305 amino acids. The protein had a theoretical isoelectric (pI) point of 7.7 and an estimated molecular weight of 34.078 kDa. PhGal8X1 was expressed in various tissues of the fish, with prominent levels in the brain, stomach, and intestine. PhGal8X1 expression was significantly (p < 0.05) induced in the blood and spleen upon challenge with different immune stimuli, including polyinosinic:polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. The recombinant PhGal8X1 protein demonstrated agglutination activity towards various bacterial pathogens at a minimum effective concentration of 50 μg/mL or 100 μg/mL. Subcellular localization observations revealed that PhGal8X1 was primarily localized in the cytoplasm. PhGal8X1 overexpression in fathead minnow cells significantly (p < 0.05) inhibited viral hemorrhagic septicemia virus (VHSV) replication. The expression levels of four proinflammatory cytokines and two chemokines were significantly (p < 0.05) upregulated in PhGal8X1 overexpressing cells in response to VHSV infection. Furthermore, overexpression of PhGal8X1 exhibited protective effects against oxidative stress induced by H 2 O 2 through the upregulation of antioxidant enzymes. Taken together, these findings provide compelling evidence that PhGal8X1 plays a crucial role in enhancing innate immunity and promoting cell survival through effective regulation of antibacterial, antiviral, and antioxidant defense mechanisms in red-lip mullet., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Betalains are coloring pigments produced in some families of the order Caryophyllales, where they replace anthocyanins as coloring pigments. While the betalain pathway itself is well studied, the tissue-specific regulation of the pathway remains mostly unknown. We enhance the high-quality Amaranthus hypochondriacus reference genome and produce a substantially more complete genome annotation, incorporating isoform details. We annotate betalain and anthocyanin pathway genes along with their regulators in amaranth and map the genetic control and tissue-specific regulation of the betalain pathway. Our improved genome annotation allowed us to identify causal mutations that lead to a knock-out of red betacyanins in natural accessions of amaranth. We reveal the tissue-specific regulation of flower color via a previously uncharacterized MYB transcription factor, AhMYB2. Downregulation of AhMYB2 in the flower leads to reduced expression of key betalain enzyme genes and loss of red flower color. Our improved amaranth reference genome represents the most complete genome of amaranth to date and is a valuable resource for betalain and amaranth research. High similarity of the flower betalain regulator AhMYB2 to anthocyanin regulators and a partially conserved interaction motif support the co-option of anthocyanin regulators for the betalain pathway as a possible reason for the mutual exclusiveness of the two pigments., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Background: Versican is a large extracellular matrix (ECM) proteoglycan with four isoforms V0-3. Elevated V0/V1 levels in breast cancer and glioma regulate cell migration and proliferation, but the role of versican in pancreatic ductal adenocarcinoma (PDAC) remains unclear., Methods: In this study, we evaluated the expression levels of versican isoforms, as well as their cellular source and interacting partners, in vivo, in human and mouse primary and metastatic PDAC tumours and in vitro, in pancreatic tumour cells and fibroblasts using immunostaining, confocal microscopy and qPCR techniques. We also investigated the effect of versican expression on fibroblast proliferation and migration using genetic and pharmacological approaches., Results: We found that versican V0/V1 is highly expressed by cancer-associated fibroblasts (CAFs) in mouse and human primary and metastatic PDAC tumours. Our data also show that exposing fibroblasts to tumour-conditioned media upregulates V0 and V1 expressions, while Verbascoside (a CD44 inhibitor) downregulates V0/V1 expression. Importantly, V0/V1 knockdown significantly inhibits fibroblast proliferation. Mechanistically, we found that inhibiting hyaluronan synthesis does not affect versican co-localisation with CD44 in fibroblasts., Conclusion: CAFs express high levels of versican V0/V1 in primary and liver metastatic PDAC tumours and versican V0/V1 supports fibroblast proliferation., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

The majority of cancer deaths are caused by solid tumors, where the four most prevalent cancers (breast, lung, colorectal and prostate) account for more than 60% of all cases (1). Tumor cell heterogeneity driven by variable cancer microenvironments, such as hypoxia, is a key determinant of therapeutic outcome. We developed a novel culture protocol, termed the Long-Term Hypoxia (LTHY) time course, to recapitulate the gradual development of severe hypoxia seen in vivo to mimic conditions observed in primary tumors. Cells subjected to LTHY underwent a non-canonical epithelial to mesenchymal transition (EMT) based on miRNA and mRNA signatures as well as displayed EMT-like morphological changes. Concomitant to this, we report production of a novel truncated isoform of WT1 transcription factor (tWt1), a non-canonical EMT driver, with expression driven by a yet undescribed intronic promoter through hypoxia-responsive elements (HREs). We further demonstrated that tWt1 initiates translation from an intron-derived start codon, retains proper subcellular localization and DNA binding. A similar tWt1 is also expressed in LTHY-cultured human cancer cell lines as well as primary cancers and predicts long-term patient survival. Our study not only demonstrates the importance of culture conditions that better mimic those observed in primary cancers, especially with regards to hypoxia, but also identifies a novel isoform of WT1 which correlates with poor long-term survival in ovarian cancer., (© 2024. The Author(s).)

Purpose: The contribution of androgen receptors (AR) on bladder cancer has been demonstrated in pre-clinical studies, however in clinical studies, only the canonical AR (AR-FL) protein was measured by immunohistochemistry and conflicting results were obtained. To get better insight into the alterations of AR signalling, we used western blotting (WB) method and simultaneously measured both mRNA and protein levels of AR-FL and AR-V7., Methods: 23 naive non-muscle invasive bladder cancer patients and 12 healthy individuals were included. AR-FL protein, AR-FL mRNA, AR-V7 protein and AR-V7 mRNA levels were quantitatively measured by WB and qRT-PCR., Results: While AR-FL protein and AR-V7 mRNA were significantly higher in bladder cancer, AR-FL mRNA and AR-V7 protein were lower. AR-V7 mRNA level was higher in patients with tumour size over 3 cm and AR-FL protein was higher in single tumours (p < 0,005). The small sampling size and the inclusion of only male participants were the main limitations., Conclusions: The increase of AR-FL protein in bladder cancer supports the contribution of the AR pathway in bladder cancer. The presence of high AR-FL protein despite low mRNA levels may be due to a disruption in post-transcriptional regulatory mechanisms. AR-V7 was demonstrated for the first time in bladder tissue and found significantly different in bladder cancer tissues. Our study reached new and valuable findings and will shed light on the studies that aim to clarify the role of the AR pathway in bladder cancer., (© 2024. The Author(s).)

Lipoprotein(a) (Lp(a)) is a low-density lipoprotein (LDL)-like particle in which the apolipoprotein B component is covalently linked to apolipoprotein(a) (apo(a)). Lp(a) is a well-established independent risk factor for cardiovascular diseases. Plasma Lp(a) concentrations vary enormously between individuals and ethnic groups. Several nucleotide polymorphisms in the SLC22A3 gene associate with Lp(a) concentration in people of different ethnicities. We investigated the association of a Polynesian-specific (Māori and Pacific peoples) SLC22A3 gene coding variant p.Thr44Met) with the plasma concentration of Lp(a) in a cohort of 302 healthy Polynesian males. An apo(a)-size independent assay assessed plasma Lp(a) concentrations; all other lipid and apolipoprotein concentrations were measured using standard laboratory techniques. Quantitative real-time polymerase chain reaction was used to determine apo(a) isoforms. The range of metabolic (HbA1c, blood pressure, and blood lipids) and blood lipid variables were similar between the non-carriers and carriers in age, ethnicity and BMI adjusted models. However, rs8187715 SLC22A3 variant was significantly associated with lower Lp(a) concentrations. Median Lp(a) concentration was 10.60 nmol/L (IQR: 5.40-41.00) in non-carrier group, and was 7.60 nmol/L (IQR: 5.50-12.10) in variant carrier group (P<0.05). Lp(a) concentration inversely correlated with apo(a) isoform size. After correction for apo(a) isoform size, metabolic parameters and ethnicity, the association between the SLC22A3 variant and plasma Lp(a) concentration remained. The present study is the first to identify the association of this gene variant and low plasma Lp(a) concentrations. This provides evidence for better guidance on ethnic specific cut-offs when defining 'elevated' and 'normal' plasma Lp(a) concentrations in clinical applications., (© 2024 The Author(s).)

The different susceptibility to HIV-1 infection in U937 cells-permissive (Plus) or nonpermissive (Minus)-is linked to the expression in Minus cells of interferon (IFN)-γ inducible antiviral factors such as tripartite motif-containing protein 22 (TRIM22) and class II transactivator (CIITA). CIITA interacts with Cyclin T1, a key component of the Positive-Transcription Elongation Factor b (P-TEFb) complex needed for the efficient transcription of HIV-1 upon interaction with the viral transactivator Tat. TRIM22 interacts with CIITA, recruiting it into nuclear bodies together with Cyclin T1. A 50 kDa Cyclin T1 was found only in Minus cells, alongside the canonical 80 kDa protein. The expression of this truncated form remained unaffected by proteasome inhibitors but was reduced by IFNγ treatment. Unlike the nuclear full-length protein, truncated Cyclin T1 was also present in the cytoplasm, and this subcellular localization correlated with its capacity to inhibit Tat-mediated HIV-1 transcription. The 50 kDa Cyclin T1 in Minus cells likely contributes to their non-permissive phenotype by acting as a dominant negative factor, disrupting P-TEFb complex formation and function. Its reduction upon IFNγ treatment suggests a regulatory loop by which its inhibitory role on HIV-1 replication is then exerted by the IFNγ-induced CIITA, which binds to the canonical Cyclin T1, displacing it from the P-TEFb complex.

Disrupted alternative splicing plays a determinative role in neurological diseases, either as a direct cause or as a driver in disease susceptibility. Transcriptomic profiling of aged human postmortem brain samples has uncovered hundreds of aberrant mRNA splicing events in Alzheimer's disease (AD) brains, associating dysregulated RNA splicing with disease. We previously identified a complex array of alternative splicing combinations across apolipoprotein E receptor 2 (APOER2), a transmembrane receptor that interacts with both the neuroprotective ligand Reelin and the AD-associated risk factor, APOE. Many of the human APOER2 isoforms, predominantly featuring cassette splicing events within functionally important domains, are critical for the receptor's function and ligand interaction. However, a comprehensive repertoire and the functional implications of APOER2 isoforms under both physiological and AD conditions are not fully understood. Here, we present an in-depth analysis of the splicing landscape of human APOER2 isoforms in normal and AD states. Using single-molecule, long-read sequencing, we profiled the entire APOER2 transcript from the parietal cortex and hippocampus of Braak stage IV AD brain tissues along with age-matched controls and investigated several functional properties of APOER2 isoforms. Our findings reveal diverse patterns of cassette exon skipping for APOER2 isoforms, with some showing region-specific expression and others unique to AD-affected brains. Notably, exon 15 of APOER2, which encodes the glycosylation domain, showed less inclusion in AD compared to control in the parietal cortex of females with an APOE ɛ3/ɛ3 genotype. Also, some of these APOER2 isoforms demonstrated changes in cell surface expression, APOE-mediated receptor processing, and synaptic number. These variations are likely critical in inducing synaptic alterations and may contribute to the neuronal dysfunction underlying AD pathogenesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gallo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Eukaryotic release factor eRF1, encoded by the ETF1 gene, recognizes stop codons and induces peptide release during translation termination. ETF1 produces several different transcripts as a result of alternative splicing, from which two eRF1 isoforms can be formed. Isoform 1 codes well-studied canonical eRF1, and isoform 2 is 33 amino acid residues shorter than isoform 1 and completely unstudied. Using a reconstituted mammalian in vitro translation system, we showed that the isoform 2 of human eRF1 is also involved in translation. We showed that eRF1iso2 can interact with the ribosomal subunits and pre-termination complex. However, its codon recognition and peptide release activities have decreased. Additionally, eRF1 isoform 2 exhibits unipotency to UGA. We found that eRF1 isoform 2 interacts with eRF3a but stimulated its GTPase activity significantly worse than the main isoform eRF1. Additionally, we studied the eRF1 isoform 2 effect on stop codon readthrough and translation in a cell-free translation system. We observed that eRF1 isoform 2 suppressed stop codon readthrough of the uORFs and decreased the efficiency of translation of long coding sequences. Based on these data, we assumed that human eRF1 isoform 2 can be involved in the regulation of translation termination. Moreover, our data support previously stated hypotheses that the GTS loop is important for the multipotency of eRF1 to all stop codons. Whereas helix α1 of the N-domain eRF1 is proposed to be involved in conformational rearrangements of eRF1 in the A-site of the ribosome that occur after GTP hydrolysis by eRF3, which ensure hydrolysis of peptidyl-tRNA at the P site of the ribosome.

The mangrove red snapper, Lutjanus argentimaculatus, is a marine food fish of economic and aquaculture importance. The application of genomic selection-based breeding programs for this species is limited by the absence of a reference genome and transcriptome profiles. The current study attempted to fill this void by generating genomic and transcriptomic resources for red snapper. Using PacBio long reads, and Arima Hi-C linked reads, a scaffold-level genome assembly was generated for L. argentimaculatus. The assembly is of 1.03 Gb comprising of 400 scaffolds with N50 of 33.8 Mb and was assessed to be 97.2% complete upon benchmarking with BUSCO. Full-length transcriptome generated with PacBio Iso-Sequencing strategy using six tissues (muscle, gills, liver, kidney, stomach, and gonad) contained 56,515 isoforms belonging to 18,108 unique genes with N50 length of 3,973 bp. The resources generated will have potential applications in the functional studies, conservation, broodstock management and selective breeding programmes of L. argentimaculatus., (© 2024. The Author(s).)

In the developing human brain, only 53 stochastically expressed clustered protocadherin (cPcdh) isoforms enable neurites from individual neurons to recognize and self-avoid while simultaneously maintaining contact with neurites from other neurons. Cell assays have demonstrated that self-recognition occurs only when all cPcdh isoforms perfectly match across the cell boundary, with a single mismatch in the cPcdh expression profile interfering with recognition. It remains unclear, however, how a single mismatched isoform between neighboring cells is sufficient to block erroneous recognitions. Using systematic cell aggregation experiments, we show that abolishing cPcdh interactions on the same membrane ( cis ) results in a complete loss of specific combinatorial binding between cells ( trans ). Our computer simulations demonstrate that the organization of cPcdh in linear array oligomers, composed of cis and trans interactions, enhances self-recognition by increasing the concentration and stability of cPcdh trans complexes between the homotypic membranes. Importantly, we show that the presence of mismatched isoforms between cells drastically diminishes the concentration and stability of the trans complexes. Overall, we provide an explanation for the role of the cPcdh assembly arrangements in neuronal self/non-self-discrimination underlying neuronal self-avoidance., Competing Interests: Competing interests statement:The authors declare no competing interest.

Regulation of gene expression is critical for fate commitment of stem and progenitor cells during tissue formation. In the context of mammalian brain development, a plethora of studies have described how changes in the expression of individual genes characterize cell types across ontogeny and phylogeny. However, little attention has been paid to the fact that different transcripts can arise from any given gene through alternative splicing (AS). Considered a key mechanism expanding transcriptome diversity during evolution, assessing the full potential of AS on isoform diversity and protein function has been notoriously difficult. Here, we capitalize on the use of a validated reporter mouse line to isolate neural stem cells, neurogenic progenitors and neurons during corticogenesis and combine the use of short- and long-read sequencing to reconstruct the full transcriptome diversity characterizing neurogenic commitment. Extending available transcriptional profiles of the mammalian brain by nearly 50,000 new isoforms, we found that neurogenic commitment is characterized by a progressive increase in exon inclusion resulting in the profound remodeling of the transcriptional profile of specific cortical cell types. Most importantly, we computationally infer the biological significance of AS on protein structure by using AlphaFold2, revealing how radical protein conformational changes can arise from subtle changes in isoforms sequence. Together, our study reveals that AS has a greater potential to impact protein diversity and function than previously thought, independently from changes in gene expression., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Voltage-gated K + (K V ) channels govern K +  ion flux across cell membranes in response to changes in membrane potential. They are formed by the assembly of four subunits, typically from the same family. Electrically silent K V channels (K V S), however, are unable to conduct currents on their own. It has been assumed that these K V S must obligatorily assemble with subunits from the K V 2 family into heterotetrameric channels, thereby giving rise to currents distinct from those of homomeric K V 2 channels. Herein, we show that K V S subunits indeed also modulate the activity, biophysical properties and surface expression of recombinant K V 7 isoforms in a subunit-specific manner. Employing co-immunoprecipitation, and proximity labelling, we unveil the spatial coexistence of K V S and K V 7 within a single protein complex. Electrophysiological experiments further indicate functional interaction and probably heterotetramer formation. Finally, single-cell transcriptomic analyses identify native cell types in which this K V S and K V 7 interaction may occur. Our findings demonstrate that K V cross-family interaction is much more versatile than previously thought-possibly serving nature to shape potassium conductance to the needs of individual cell types., (© 2024. The Author(s).)

Tau pathologies are detected in the brains of some of the most common neurodegenerative diseases including Alzheimer's disease (AD), Lewy body dementia (LBD), chronic traumatic encephalopathy (CTE), and frontotemporal dementia (FTD). Tau proteins are expressed in six isoforms with either three or four microtubule-binding repeats (3R tau or 4R tau) due to alternative RNA splicing. AD, LBD, and CTE brains contain pathological deposits of both 3R and 4R tau. FTD patients can exhibit either 4R tau pathologies in most cases or 3R tau pathologies less commonly in Pick's disease, which is a subfamily of FTD. Here, we report the isoform-specific roles of tau in FTD. The P301L mutation, linked to familial 4R tau FTD, induces mislocalization of 4R tau to dendritic spines in primary hippocampal cultures that were prepared from neonatal rat pups of both sexes. Contrastingly, the G272V mutation, linked to familial Pick's disease, induces phosphorylation-dependent mislocalization of 3R tau but not 4R tau proteins to dendritic spines. The overexpression of G272V 3R tau but not 4R tau proteins leads to the reduction of dendritic spine density and suppression of mEPSCs in 5-week-old primary rat hippocampal cultures. The decrease in mEPSC amplitude caused by G272V 3R tau is dynamin-dependent whereas that caused by P301L 4R tau is dynamin-independent, indicating that the two tau isoforms activate different signaling pathways responsible for excitatory synaptic dysfunction. Our 3R and 4R tau studies here will shed new light on diverse mechanisms underlying FTD, AD, LBD, and CTE., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

BRCA1 , a crucial tumor suppressor gene, has several splicing isoforms, including Δ9-11, Δ11, and Δ11q, which lack exon 11, coding for significant portions of the protein. These isoforms are naturally present in both normal and cancerous cells, exhibiting altered activity compared to the full-length BRCA1. Despite this, the impact on cancer risk of the germline intronic variants promoting the exclusive expression of these Δ11 isoforms remains uncertain. Consequently, they are classified as variants of uncertain significance (VUS), posing challenges for traditional genetic classification methods due to their rarity and complexity. Our research utilizes a yeast-based functional assay, previously validated for assessing missense BRCA1 variants, to compare the activity of the Δ11 splicing isoforms with known pathogenic missense variants. This approach allows us to elucidate the functional implications of these isoforms and determine whether their exclusive expression could contribute to increased cancer risk. By doing so, we aim to provide insights into the pathogenic potential of intronic VUS-generating BRCA1 splicing isoforms and improve the classification of BRCA1 variants.

In budding yeast, the integrity of both the nuclear and mitochondrial genomes relies on dual-targeted isoforms of the conserved Pif1 helicase, generated by alternative translation initiation (ATI) of PIF1 mRNA from two consecutive AUG codons flanking a mitochondrial targeting signal. Here, we demonstrate that ribosomal leaky scanning is the specific ATI mechanism that produces not only these, but also novel, previously uncharacterized Pif1 isoforms. Both in-frame, downstream AUGs as well as near-cognate start codons contribute to the generation of these alternative isoforms. This has crucial implications for the rational design of genuine separation-of-function alleles and provides an explanation for the suboptimal behaviour of the widely employed mitochondrial- (pif1-m1) and nuclear-deficient (pif1-m2) alleles, with mutations in the first or second AUG codon, respectively. We have taken advantage of this refined model to develop improved versions of these alleles, which will serve as valuable tools to elucidate novel functions of this helicase and to disambiguate previously described genetic interactions of PIF1 in the context of nuclear and mitochondrial genome stability., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The fly Drosophila yakuba has lost an ancestral component of the male courtship song: this is due to ontogenetic death of effector neurons in the ventral nerve cord, a result of the D. yakuba sex-determining gene dsx producing a male isoform, dsxM, with cell-death-promoting activity similar to that of the female isoform, dsxF, in D. melanogaster., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Alternative splicing is the process of generating different mRNAs from the same primary transcript, which contributes to increase the transcriptome and proteome diversity. Abnormal splicing has been associated with the development of several diseases including cancer. Given that mutations and abnormal levels of the RIPK2 transcript and RIP-2 protein are frequent in tumors, and that RIP-2 modulates immune and inflammatory responses, we investigated alternative splicing events that result in partial deletions of the kinase domain at the N-terminus of RIP-2. We also investigated the structure and expression of the RIPK2 truncated variants and isoforms in different environments. In addition, we searched data throughout Supraprimates evolution that could support the biological importance of RIPK2 alternatively spliced products. We observed that human variants and isoforms were differentially regulated following temperature stress, and that the truncated transcript was more expressed than the long transcript in tumor samples. The inverse was found for the longer protein isoform. The truncated variant was also detected in chimpanzee, gorilla, hare, pika, mouse, rat, and tree shrew. The fact that the same variant has been preserved in mammals with divergence times up to 70 million years raises the hypothesis that it may have a functional significance., Competing Interests: Conflicts of Interest The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Latrophilins (LPHNs), a group of the G-protein-coupled receptor to which a spider venom latrotoxin (LTX) is known to bind, remain largely uncharacterized in neoplastic diseases. In the present study, we aimed to determine the role of LPHNs in the progression of prostate cancer. We assessed the actions of LPHNs, including LPHN1, LPHN2, and LPHN3, in human prostate cancer lines via their ligand (e.g., α-LTX, FLRT3) treatment or shRNA infection, as well as in surgical specimens. In androgen receptor (AR)-positive LNCaP/C4-2/22Rv1 cells, dihydrotestosterone considerably increased the expression levels of LPHNs, while chromatin immunoprecipitation assay revealed the binding of endogenous ARs, including AR-V7, to the promoter region of each LPHN. Treatment with α-LTX or FLRT3 resulted in induction in the cell viability and migration of both AR-positive and AR-negative lines. α-LTX and FLRT3 also enhanced the expression of Bcl-2 and phosphorylated forms of JAK2 and STAT3. Meanwhile, the knockdown of each LPHN showed opposite effects on all of those mediated by ligand treatment. Immunohistochemistry in radical prostatectomy specimens further showed the significantly elevated expression of each LPHN in prostate cancer, compared with adjacent normal-appearing prostate, which was associated with a significantly higher risk of postoperative biochemical recurrence in both univariate and multivariable settings. These findings indicate that LPHNs function as downstream effectors of ARs and promote the growth of androgen-sensitive, castration-resistant, or even AR-negative prostate cancer.

Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer's adaptation to produce another small RNA class-microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in Dicer ΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. Dicer ΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo., (© 2024. The Author(s).)

Creating long-lasting memories requires learning-induced changes in gene expression, which are impacted by epigenetic modifications of DNA and associated histone proteins. Post-translational modifications (PTMs) of histones are key regulators of transcription, with different PTMs producing unique effects on gene activity and behavior. Although recent studies implicate histone variants as novel regulators of memory, effects of PTMs on the function of histone variants are rarely considered. We previously showed that the histone variant H2A.Z suppresses memory, but it is unclear if this role is impacted by H2A.Z acetylation, a PTM that is typically associated with positive effects on transcription and memory. To answer this question, we used a mutation approach to manipulate acetylation on H2A.Z without impacting acetylation of other histone types. Specifically, we used adeno-associated virus (AAV) constructs to overexpress mutated H2A.Z.1 isoforms that either mimic acetylation (acetyl-mimic) by replacing lysines 4, 7 and 11 with glutamine (KQ), or H2A.Z.1 with impaired acetylation (acetyl-defective) by replacing the same lysines with alanine (KA). Expressing the H2A.Z.1 acetyl-mimic (H2A.Z.1 KQ ) improved memory under weak learning conditions, whereas expressing the acetyl-defective H2A.Z.1 KA generally impaired memory, indicating that the effect of H2A.Z.1 on memory depends on its acetylation status. RNA sequencing showed that H2A.Z.1 KQ and H2A.Z.1 KA uniquely impact the expression of different classes of genes in both females and males. Specifically, H2A.Z.1 KA preferentially impacts genes involved in synaptic function, suggesting that acetyl-defective H2A.Z.1 impairs memory by altering synaptic regulation. Finally, we describe, for the first time, that H2A.Z is also involved in alternative splicing of neuronal genes, whereby H2A.Z depletion, as well as expression of H2A.Z.1 lysine mutants influence transcription and splicing of different gene targets, suggesting that H2A.Z.1 can impact behavior through effects on both splicing and gene expression. This is the first study to demonstrate that direct manipulation of H2A.Z post-translational modifications regulates memory, whereby acetylation adds another regulatory layer by which histone variants can fine tune higher brain functions through effects on gene expression and splicing., (© 2024. The Author(s).)

Vitamin D (VD) is produced by the skin upon exposure to sunlight or is obtained from dietary sources. Several risk factors are associated with VD deficiency including mutations and post-translational modifications in its transport protein known as vitamin D binding protein (VDBP) or GC-globulin. The two common single nucleotide polymorphisms rs7041 and rs4588 create three major isoforms of VDBP, including GC-1F also called wild type, GC1S, and GC-2. The 3D models for both GC-1F and GC-2 were constructed in their glycosylated states to decipher the effect of these mutations on the overall conformational changes and VD-binding affinity. The binding affinities were estimated using the Molecular Mechanics Poison-Boltzmann surface area (MM-PBSA) method and conformational changes were investigated after free energy landscapes estimations. Total free energies suggest that GC-1F exhibits stronger affinity (ΔE = -116.09 kJ/mol) than GC-2 (ΔE = -95 kJ/mol) variant with VD. The GC-1F isoforms had more streamlined motion compared to GC-2 isoforms, predicting a trade-off between cross-talk residues that significantly impacts protein structural stability. The data suggest that glycation at Thr418 plays a vital role in the overall VDBP-VD affinity by stabilizing the N-T loop that holds the domain I (VD-pocket) and domain III intact. The loss of glycation in GC-2 has a pivotal role in the inter-domain conformational stability of VDBP, which may ultimately affect VD transportation and maturation. These findings describe a novel mechanism in how mutations distant from the VD-active site change the overall conformational of the VDBP and abrogate the VDBP-VD interaction.Communicated by Ramaswamy H. Sarma.

Static gene expression programs have been extensively characterized in stem cells and mature human cells. However, the dynamics of RNA isoform changes upon cell-state-transitions during cell differentiation, the determinants and functional consequences have largely remained unclear. Here, we established an improved model for human neurogenesis in vitro that is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct differentiation stages. RNA isoforms mainly arise from exon skipping and the alternative usage of transcription start and polyadenylation sites during human neurogenesis. The transcript isoform changes can remodel the identity and functions of protein isoforms. Finally, our study identifies a set of RNA binding proteins as a potential determinant of differentiation stage-specific global isoform changes. This work supports the view of regulated isoform changes that underlie state-transitions during neurogenesis., (© 2024. The Author(s).)

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family and has the ability to catalyze the cross-linking of extracellular matrix collagen and elastin. High expression of LOXL2 is related to tumor cell proliferation, invasion, and metastasis. LOXL2 contains 14 exons. Previous studies have found that LOXL2 has abnormal alternative splicing and exon skipping in a variety of tissues and cells, resulting in a new alternatively spliced isoform denoted LOXL2Δ13. LOXL2Δ13 lacks LOXL2WT exon 13, but its encoded protein has greater ability to induce tumor cell proliferation, invasion, and metastasis. However, the molecular events that produce LOXL2Δ13 are still unclear. In this study, we found that overexpression of the splicing factor hnRNPA1 in cells can regulate the alternative splicing of LOXL2 and increase the expression of LOXL2Δ13. The exonic splicing silencer exists at the 3' splice site and 5' splice site of LOXL2 exon 13. HnRNPA1 can bind to the exonic splicing silencer and inhibit the inclusion of exon 13. The RRM domain of hnRNPA1 and phosphorylation of hnRNPA1 at S91 and S95 are important for the regulation of LOXL2 alternative splicing. These results show that hnRNPA1 is a splicing factor that enhances the production of LOXL2Δ13., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Autophagy is a pivotal regulatory and catabolic process, induced under various stressful conditions, including hypoxia. However, little is known about alternative splicing of autophagy genes in the hypoxic landscape in breast cancer. Our research unravels the hitherto unreported alternative splicing of BNIP3L, a crucial hypoxia-induced autophagic gene. We showed that BNIP3L, under hypoxic condition, forms two isoforms, a full-length isoform (BNIP3L-F) and a shorter isoform lacking exon 1 (BNIP3L-Δ1). The hypoxia-induced BNIP3L-F promotes autophagy, while under normoxia, the BNIP3L-Δ1 inhibits autophagy. We discovered a novel dimension of hypoxia-mediated epigenetic modification that regulates the alternative splicing of BNIP3L. Here, we showed differential DNA methylation of BNIP3L intron 1, causing reciprocal binding of epigenetic factor CCCTC-binding factor (CTCF) and its paralog BORIS. Additionally, we highlighted the role of CTCF and BORIS impacting autophagy in breast cancer. The differential binding of CTCF and BORIS results in alternative splicing of BNIP3L forming BNIP3L-F and BNIP3L-Δ1, respectively. The binding of CTCF on unmethylated BNIP3L intron 1 under hypoxia results in RNA Pol-II pause and inclusion of exon 1, promoting BNIP3L-F and autophagy. Interestingly, the binding of BORIS on methylated BNIP3L intron 1 under normoxia also results in RNA Pol-II pause but leads to the exclusion of exon 1 from BNIP3L mRNA. Finally, we reported the critical role of BORIS-mediated RNA Pol-II pause, which subsequently recruits SRSF6, redirecting the proximal splice-site selection, promoting BNIP3L-Δ1, and inhibiting autophagy. Our study provides novel insights into the potential avenues for breast cancer therapy by targeting autophagy regulation, specifically under hypoxic condition., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In recent years, in the development of emerging immunotherapy, B7-H3 is also termed as CD276 and has become a novel chimeric antigen receptor (CAR)-T target against glioma and other tumours, and aroused extensive attention. However, B7-H3 has three isoforms (2, 3 and 4Ig) with the controversial expression and elusive function in tumour especially glioma. The current study mainly focuses on the regulatory factors and related mechanisms of generation of different B7-H3 isoforms. First, we have determined that 2Ig is dominant in glioma with high malignancy, and 4Ig is widely expressed, whereas 3Ig shows negative expression in all glioma. Next, we have further found that RNA binding protein annexin A2 (ANXA2) is essential for B7-H3 isoform maintenance, but fail to determine the choice of 4Ig or 2Ig. RNA methyltransferase NOP2/Sun RNA methyltransferase 2 (NSUN2) and 5-methylcytosine reader Y-box binding protein 1 (YBX1) facilitate the production of 2Ig. Our findings have uncovered a series of factors (ANXA2/NSUN2/YBX1) that can determine the alternative generation of different isoforms of B7-H3 in glioma. Our result aims to help peers gain a clearer understanding of the expression and regulatory mechanisms of B7H3 in tumour patients, and to provide better strategies for designing B7H3 as a target in immunotherapy., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Alternative splicing is one of the major cellular processes that determine the tissue-specific expression of protein variants. However, it remains challenging to identify physiologically relevant and tissue-selective proteins that are generated by alternative splicing. Hence, we investigated the target spectrum of the splicing factor Rbfox1 in the cardiac muscle context in more detail. By using a combination of in silico target prediction and in-cell validation, we identified several focal adhesion proteins as alternative splicing targets of Rbfox1. We focused on the alternative splicing patterns of vinculin (metavinculin isoform) and paxillin (extended paxillin isoform) and identified both as potential Rbfox1 targets. Minigene analyses suggested that both isoforms are promoted by Rbfox1 due to binding in the introns. Focal adhesions play an important role in the cardiac muscle context, since they mainly influence cell shape, cytoskeletal organization, and cell-matrix association. Our data confirmed that depletion of Rbfox1 changed cardiomyoblast morphology, cytoskeletal organization, and multinuclearity after differentiation, which might be due to changes in alternative splicing of focal adhesion proteins. Hence, our results indicate that Rbfox1 promotes alternative splicing of focal adhesion genes in cardiac muscle cells, which might contribute to heart disease progression, where downregulation of Rbfox1 is frequently observed., (© The Author(s) (2024). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

Alternative splicing significantly enhances the diversity of the G protein-coupled receptor (GPCR) family, including the histamine H 3 receptor (H 3 R). This post-transcriptional modification generates multiple H 3 R isoforms with potentially distinct pharmacological and physiological profiles. H 3 R is primarily involved in the presynaptic inhibition of neurotransmitter release in the central nervous system. Despite the approval of pitolisant for narcolepsy (Wakix ® ) and daytime sleepiness in adults with obstructive sleep apnea (Ozawade ® ) and ongoing clinical trials for other H 3 R antagonists/inverse agonists, the functional significance of the numerous H 3 R isoforms remains largely enigmatic. Recent publicly available RNA sequencing data have confirmed the expression of multiple H 3 R isoforms in the brain, with some isoforms exhibiting unique tissue-specific distribution patterns hinting at isoform-specific functions and interactions within neural circuits. In this review, we discuss the complexity of H 3 R isoforms with a focus on their potential roles in central nervous system (CNS) function. Comparative analysis across species highlights evolutionary conservation and divergence in H 3 R splicing, suggesting species-specific regulatory mechanisms. Understanding the functionality of H 3 R isoforms is crucial for the development of targeted therapeutics. This knowledge will inform the design of more precise pharmacological interventions, potentially enhancing therapeutic efficacy and reducing adverse effects in the treatment of neurological and psychiatric disorders.

The androgen receptor (AR) is a ligand-dependent nuclear transcription factor belonging to the steroid hormone nuclear receptor family. Due to its roles in regulating cell proliferation and differentiation, AR is tightly regulated to maintain proper levels of itself and the many genes it controls. AR dysregulation is a driver of many human diseases including prostate cancer. Though this dysregulation often occurs at the RNA level, there are many unknowns surrounding post-transcriptional regulation of AR mRNA, particularly the role that RNA secondary structure plays. Thus, a comprehensive analysis of AR transcript secondary structure is needed. We address this through the computational and experimental analyses of two key isoforms, full length (AR-FL) and truncated (AR-V7). Here, a combination of in-cell RNA secondary structure probing experiments (targeted DMS-MaPseq) and computational predictions were used to characterize the static structural landscape and conformational dynamics of both isoforms. Additionally, in-cell assays were used to identify functionally relevant structures in the 5' and 3' UTRs of AR-FL. A notable example is a conserved stem loop structure in the 5'UTR of AR-FL that can bind to Poly(RC) Binding Protein 2 (PCBP2). Taken together, our results reveal novel features that regulate AR expression., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In Drosophila coordinated proliferation of two neural stem cells, neuroblasts (NB) and neuroepithelial (NE) cells, is pivotal for proper larval brain growth that ultimately determines the final size and performance of an adult brain. The larval brain growth displays two phases based on behaviors of NB and NEs: the first one in early larval stages, influenced by nutritional status and the second one in the last larval stage, promoted by ecdysone signaling after critical weight checkpoint. Mutations of the baboon (babo) gene that produces three isoforms (BaboA-C), all acting as type-I receptors of Activin-type transforming growth factor β (TGF-β) signaling, cause a small brain phenotype due to severely reduced proliferation of the neural stem cells. In this study we show that loss of babo function severely affects proliferation of NBs and NEs as well as conversion of NEs from both phases. By analyzing babo-null and newly generated isoform-specific mutants by CRISPR mutagenesis as well as isoform-specific RNAi knockdowns in a cell- and stage-specific manner, our data support differential contributions of the isoforms for these cellular events with BaboA playing the major role. Stage-specific expression of EcR-B1 in the brain is also regulated primarily by BaboA along with function of the other isoforms. Blocking EcR function in both neural stem cells results in a small brain phenotype that is more severe than baboA-knockdown alone. In summary, our study proposes that the Babo-mediated signaling promotes proper behaviors of the neural stem cells in both phases and achieves this by acting upstream of EcR-B1 expression in the second phase., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The actin cytoskeleton is one of the most important players in cell motility, adhesion, division, and functioning. The regulation of specific microfilament formation largely determines cellular functions. The main actin-binding protein in animal cells is tropomyosin (Tpm). The unique structural and functional diversity of microfilaments is achieved through the diversity of Tpm isoforms. In our work, we studied the properties of the cytoplasmic isoforms Tpm1.8 and Tpm1.9. The results showed that these isoforms are highly thermostable and differ in the stability of their central and C -terminal fragments. The properties of these isoforms were largely determined by the 6th exons. Thus, the strength of the end-to-end interactions, as well as the affinity of the Tpm molecule for F-actin, differed between the Tpm1.8 and Tpm1.9 isoforms. They were determined by whether an alternative internal exon, 6a or 6b, was included in the Tpm isoform structure. The strong interactions of the Tpm1.8 and Tpm1.9 isoforms with F-actin led to the formation of rigid actin filaments, the stiffness of which was measured using an optical trap. It is quite possible that the structural and functional features of the Tpm isoforms largely determine the appearance of these isoforms in the rigid actin structures of the cell cortex.

Cytokinesis is the final step of the cell division cycle that leads to the formation of two new cells. Successful cytokinesis requires significant remodelling of the plasma membrane by spatially distinct β- and γ-actin networks. These networks are generated by the formin family of actin nucleators, DIAPH3 and DIAPH1 respectively. Here we show that β- and γ-actin perform specialized and non-redundant roles in cytokinesis and cannot substitute for one another. Expression of hybrid DIAPH1 and DIAPH3 proteins with altered actin isoform specificity relocalized cytokinetic actin isoform networks within the cell, causing cytokinetic failure. Consistent with this we show that β-actin networks, but not γ-actin networks, are required for the maintenance of non-muscle myosin II and RhoA at the cytokinetic furrow. These data suggest that independent and spatially distinct actin isoform networks form scaffolds of unique interactors that facilitate localized biochemical activities to ensure successful cell division., (© 2024. The Author(s).)

P2X7 receptor activation by extracellular adenosine triphosphate (eATP) modulates different intracellular pathways, including pro-inflammatory and tumor-promoting cascades. ATP is released by cells and necrotic tissues during stressful conditions and accumulates mainly in the inflammatory and tumoral microenvironments. As a consequence, both the P2X7 blockade and agonism have been proposed as therapeutic strategies in phlogosis and cancer. Nevertheless, most studies have been carried out on the WT fully functional receptor variant. In recent years, the discovery of P2X7 variants derived by alternative splicing mechanisms or single-nucleotide substitutions gave rise to the investigation of these new P2X7 variants' roles in different processes and diseases. Here, we provide an overview of the literature covering the function of human P2X7 splice variants and polymorphisms in diverse pathophysiological contexts, paying particular attention to their role in oncological and neuroinflammatory conditions.

Peroxisome proliferator-activated receptor γ (PPARG) has various splicing variants and plays essential roles in the regulation of adipocyte differentiation and lipogenesis. However, little is known about the expression pattern and effect of the PPARG on milk fat synthesis in the buffalo mammary gland. In this study, we found that only PPARG-X17 and PPARG-X21 of the splicing variant were expressed in the buffalo mammary gland. Amino acid sequence characterization showed that the proteins encoded by PPARG-X17 and PPARG-X21 are endonuclear non-secreted hydrophilic proteins. Protein domain prediction found that only the PPARG-X21 -encoded protein had PPAR ligand-binding domains (NR&#95;LBD&#95;PPAR), which may lead to functional differences between the two splices. RNA interference (RNAi) and the overexpression of PPARG-X17 and PPARG-X21 in buffalo mammary epithelial cells (BMECs) were performed. Results showed that the expression of fatty acid synthesis-related genes ( ACACA , CD36 , ACSL1 , GPAT , AGPAT6 , DGAT1 ) was significantly modified ( p < 0.05) by the RNAi and overexpression of PPARG-X17 and PPARG-X21 . All kinds of FAs detected in this study were significantly decreased ( p < 0.05) after RNAi of PPARG-X17 or PPARG-X21 . Overexpression of PPARG-X17 or PPARG-X21 significantly decreased ( p < 0.05) the SFA content, while significantly increased ( p < 0.05) the UFA, especially the MUFA in the BMECs. In conclusion, there are two PPARG splicing variants expressed in the BMECs that can regulate FA synthesis by altering the expression of diverse fatty acid synthesis-related genes. This study revealed the expression characteristics and functions of the PPARG gene in buffalo mammary glands and provided a reference for further understanding of fat synthesis in buffalo milk.

Alternative splicing contributes to shaping lineage-specific gene expression and phenotypes. In this issue of Cell Genomics, Recinos, Bao, Wang, et al. 1 report that the balance between splicing isoforms of the microtubule-associated protein Tau in the brain is differentially regulated among primates by the RNA-binding protein MBNL2, with consequences for protein aggregation and neurodegeneration in humans., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Full-length p53 (p53α) plays a pivotal role in maintaining genomic integrity and preventing tumor development. Over the years, p53 was found to exist in various isoforms, which are generated through alternative splicing, alternative initiation of translation, and internal ribosome entry site. p53 isoforms, either C-terminally altered or N-terminally truncated, exhibit distinct biological roles compared to p53α, and have significant implications for tumor development and therapy resistance. Due to a lack of part and/or complete C- or N-terminal domains, ectopic expression of some p53 isoforms failed to induce expression of canonical transcriptional targets of p53α like CDKN1A or MDM2, even though they may bind their promoters. Yet, p53 isoforms like Δ40p53α still activate subsets of targets including MDM2 and BAX. Furthermore, certain p53 isoforms transactivate even novel targets compared to p53α. More recently, non-canonical functions of p53α in DNA repair and of different isoforms in DNA replication unrelated to transcriptional activities were discovered, amplifying the potential of p53 as a master regulator of physiological and tumor suppressor functions in human cells. Both regarding canonical and non-canonical functions, alternative p53 isoforms frequently exert dominant negative effects on p53α and its partners, which is modified by the relative isoform levels. Underlying mechanisms include hetero-oligomerization, changes in subcellular localization, and aggregation. These processes ultimately influence the net activities of p53α and give rise to diverse cellular outcomes. Biological roles of p53 isoforms have implications for tumor development and cancer therapy resistance. Dysregulated expression of isoforms has been observed in various cancer types and is associated with different clinical outcomes. In conclusion, p53 isoforms have expanded our understanding of the complex regulatory network involving p53 in tumors. Unraveling the mechanisms underlying the biological roles of p53 isoforms provides new avenues for studies aiming at a better understanding of tumor development and developing therapeutic interventions to overcome resistance., (© 2024. The Author(s).)

Bone formation is a complex process regulated by a variety of pathways that are not yet fully understood. One of the proteins involved in multiple osteogenic pathways is TID (DNAJA3). The aim of this work was to study the association of TID with osteogenesis. Therefore, the expression profiles of the TID splice variants ( TID-L , TID-I ) and their protein products were analyzed during the proliferation and differentiation of bone marrow mesenchymal stromal cells (B-MSCs) into osteoblasts. As the reference, the hFOB1.19 cell line was used. The phenotype of B-MSCs was confirmed by the presence of CD73, CD90, and CD105 surface antigens on ~97% of cells. The osteoblast phenotype was confirmed by increased alkaline phosphatase activity, calcium deposition, and expression of ALPL and SPP1. The effect of silencing the TID gene on the expression of ALPL and SPP1 was also investigated. The TID proteins and the expression of TID splice variants were detected. After differentiation, the expression of TID-L and TID-I increased 5-fold and 3.7-fold, respectively, while their silencing resulted in increased expression of SPP1 . Three days after transfection, the expression of SPP1 increased 7.6-fold and 5.6-fold in B-MSCs and differentiating cells, respectively. Our preliminary study demonstrated that the expression of TID-L and TID-I changes under differentiation of B-MSCs into osteoblasts and may influence the expression of SPP1 . However, for better understanding the functional association of these results with the relevant osteogenic pathways, further studies are needed.

Although alternative splicing (AS) is a major mechanism that adds diversity to gene expression patterns, its precise role in generating variability in ribosomal proteins, known as ribosomal heterogeneity, remains unclear. The ribosomal protein S24 (RPS24) gene, encoding a ribosomal component, undergoes AS; however, in-depth studies have been challenging because of three microexons between exons 4 and 6. We conducted a detailed analysis of RPS24 AS isoforms using a direct approach to investigate the splicing junctions related to these microexons, focusing on four AS isoforms. Each of these isoforms showed tissue specificity and relative differences in expression among cancer types. Significant differences in the proportions of these RPS24 AS isoforms between cancerous and normal tissues across diverse cancer types were also observed. Our study highlighted a significant correlation between the expression levels of a specific RPS24 AS isoform and the epithelial-mesenchymal transition process in lung and breast cancers. Our research contributes to a better understanding of the intricate regulatory mechanisms governing AS of ribosomal protein genes and highlights the biological implications of RPS24 AS isoforms in tissue development and tumorigenesis., (© 2024. The Author(s).)

Purpose: This study evaluated the dysregulation of TCF4 isoforms and differential exon usage (DEU) in corneal endothelial cells (CECs) of Fuchs endothelial corneal dystrophy (FECD) with or without trinucleotide repeat (TNR) expansion in the intron region of the TCF4 gene., Methods: Three RNA-Seq datasets of CECs (our own and two other previously published datasets) derived from non-FECD control and FECD subjects were analyzed to identify TCF4 isoforms and DEU events dysregulated in FECD by comparing control subjects to those with FECD with TNR expansion and FECD without TNR expansion., Results: Our RNA-Seq data demonstrated upregulation of three TCF4 isoforms and downregulation of two isoforms in FECD without TNR expansion compared to the controls. In FECD with TNR expansion, one isoform was upregulated and one isoform was downregulated compared to the control. Additional analysis using two other datasets identified that the TCF4-277 isoform was upregulated in common in all three datasets in FECD with TNR expansion, whereas no isoform was dysregulated in FECD without TNR expansion. DEU analysis showed that one exon (E174) upstream of the TNR, which only encompassed TCF4-277, was upregulated in common in all three datasets, whereas eight exons downstream of the TNR were downregulated in common in all three datasets in FECD with TNR expansion., Conclusions: This study identified TCF4-277 as a dysregulated isoform in FECD with TNR expansion, suggesting a potential contribution of TCF4-277 to FECD pathophysiology.