Alternative Splicing Transcripts Research Articles

Alternative Splicing and Nonsense-Mediated Decay of a Zebrafish GABA Receptor Subunit Transcript.

The superfamily of Cys-loop ionotropic neurotransmitter receptors includes those that detect GABA, glutamate, glycine, and acetylcholine. There is ample evidence that many Cys-loop receptor subunit genes include alternatively spliced exons. In this study, we report a novel example of alternative splicing (AS): we show that the 68-bp exon 3 in the zebrafish gabrr2b gene-which codes for the ρ2b GABAAR subunit-is an alternative cassette exon. Skipping of gabrr2b exon 3 results in a downstream frame shift and a premature termination codon (PTC). We provide evidence in larval zebrafish that transcripts containing the PTC are subject to degradation through nonsense-mediated decay. We also compile reports of AS of homologous exons in other Cys-loop receptor genes in multiple species. Our data add to a large body of research demonstrating that exon 3 in Cys-loop receptor genes is a conserved site for AS, the effects of which can vary from novel splice-isoform generation to downregulation of gene expression through transcript degradation.

CsTT8 regulates anthocyanin accumulation in blood orange through alternative splicing transcription.

A homologous gene of basic-helix-loop-helix AtTT8 in Arabidopsis thaliana was identified in juice sac cells of pulp tissues from blood orange (Citrus sinensis cv 'Tarocco'), which was designated as CsTT8 in this study. Additionally, the mRNA levels of TT8 with the full-length open reading frame were significantly higher in 'Tarocco' than in mutant fruit lacking pigment in pulp or peel tissues. However, an alternative splicing transcript, Δ15-TT8, with the fourth exon skipped, was also identified from transcripts different in length from that in 'Tarocco'. The mRNA levels of Δ15-TT8 were higher in mutant fruit lacking pigment in pulp or peel tissues than in the wild type. Therefore, the TT8/Δ15-TT8 mRNA level ratio was found to be crucial for sufficient pigment in either pulp or peel tissues. TT8 from blood orange fruit demonstrated the capacity for nucleus localization and binding to other proteins. In contrast, Δ15-TT8, lacking the fourth exon, lost its ability to interact with RUBY1 and to localize at the nucleus. Using a dual luciferase reporter assay and transient overexpression in tobacco, we proved that two regulatory complexes formed by a functional TT8 with different MYB(v-myb avian myeloblastosis viral oncogene homolog)-type partners significantly promoted expression of an anthocyanin biosynthetic gene and a proton pumping gene, leading to anthocyanin and citrate production. Our findings suggest that TT8, rather than dysfunctional Δ15-TT8, is possibly involved in modulating anthocyanin biosynthesis and its transport into vacuoles by proton gradients. However, increased mRNA levels of the dysfunctional alternative splicing transcript may act as a negative feedback to downregulate TT8 expression and limit anthocyanin accumulation in blood oranges.

A heterozygous splicing variant IVS9-7A > T in intron 9 of the MAPT gene in a patient with right-temporal variant frontotemporal dementia with atypical 4 repeat tauopathy

Right temporal variant frontotemporal dementia, also called right-predominant semantic dementia, often has an unclear position within the framework of the updated diagnostic criteria for behavioral variant frontotemporal dementia or primary progressive aphasia. Recent studies have suggested that this population may be clinically, neuropathologically, and genetically distinct from those with behavioral variant frontotemporal dementia or left-predominant typical semantic variant primary progressive aphasia. Here we describe a Japanese case of right temporal variant frontotemporal dementia with novel heterozygous MAPT mutation Adenine to Thymidine in intervening sequence (IVS) 9 at position -7 from 3ʹ splicing site of intron 9/exon 10 boundary (MAPT IVS9-7A > T). Postmortem neuropathological analysis revealed a predominant accumulation of 4 repeat tau, especially in the temporal lobe, amygdala, and substantia nigra, but lacked astrocytic plaques or tufted astrocytes. Immunoelectron microscopy of the tau filaments extracted from the brain revealed a ribbon-like structure. Moreover, a cellular MAPT splicing assay confirmed that this novel variant promoted the inclusion of exon 10, resulting in the predominant production of 4 repeat tau. These data strongly suggest that the MAPT IVS9-7 A > T variant found in our case is a novel mutation that stimulates the inclusion of exon 10 through alternative splicing of MAPT transcript and causes predominant 4 repeat tauopathy which clinically presents as right temporal variant frontotemporal dementia.

Transcriptional Regulation Associated with Subcutaneous Adipogenesis in Porcine ACSL1 Gene.

Long-chain acyl-CoA synthetase 1 (ACSL1) plays an important role in fatty acid metabolism and fat deposition. The transcription of the ACSL1 gene is regulated specifically among cells and physiological processes, and transcriptional regulation of ACSL1 in adipogenesis remains elusive. Here, we characterize transcription factors (TFs) associated with adipogenesis in the porcine ACSL1 gene. CCAAT-enhancer binding protein (C/EBP)α, a well-known adipogenic marker, was found to enhance the expression of the ACSL1 gene via binding two tandem motifs in the promoter. Further, we demonstrate that ACSL1 mediates C/EBPα effects on adipogenesis in preadipocytes cultured from subcutaneous fat tissue of pigs via gain- and loss-of-function analyses. The cAMP-response element binding protein, another TF involved in adipogenesis, was also identified in the regulation of ACSL1 gene expression. Additionally, single nucleotide polymorphisms (SNPs) were screened in the promoter of ACSL1 among four breeds including the Chinese indigenous Min, and Duroc, Berkshire, and Yorkshire pigs through sequencing of PCR products. Two tightly linked SNPs, -517G>T and -311T>G, were found exclusively in Min pigs. The haplotype mutation decreases promoter activity in PK-15 and ST cells, and in vivo the expression of ACSL1, illustrating a possible role in adipogenesis regulated by C/EBPα/ACSL1 axis. Additionally, a total of 24 alternative splicing transcripts were identified, indicating the complexity of alternative splicing in the ACSL1 gene. The results will contribute to further revealing the regulatory mechanisms of ACSL1 during adipogenesis and to the characterization of molecular markers for selection of fat deposition in pigs.

HnRNPL expression dynamics in the embryo and placenta

Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is a conserved RNA binding protein (RBP) that plays an important role in the alternative splicing of gene transcripts, and thus in the generation of specific protein isoforms. Global deficiency in hnRNPL in mice results in preimplantation embryonic lethality at embryonic day (E) 3.5. To begin to understand the contribution of hnRNPL-regulated pathways in the normal development of the embryo and placenta, we determined hnRNPL expression profile and subcellular localization throughout development. Proteome and Western blot analyses were employed to determine hnRNPL abundance between E3.5 and E17.5. Histological analyses supported that the embryo and implantation site display distinct hnRNPL localization patterns. In the fully developed mouse placenta, nuclear hnRNPL was observed broadly in trophoblasts, whereas within the implantation site a discrete subset of cells showed hnRNPL outside the nucleus. In the first-trimester human placenta, hnRNPL was detected in the undifferentiated cytotrophoblasts, suggesting a role for this factor in trophoblast progenitors. Parallel in vitro studies utilizing Htr8 and Jeg3 cell lines confirmed expression of hnRNPL in cellular models of human trophoblasts. These studies [support] coordinated regulation of hnRNPL during the normal developmental program in the mammalian embryo and placenta.

Genome-wide analysis of fatty acid desaturase genes in chia (Salvia hispanica) reveals their crucial roles in cold response and seed oil formation

Chia (Salvia hispanica) is a functional food crop with high α-linolenic acid (ALA), the omega-3 essential fatty acid, but its worldwide plantation is limited by cold-intolerance and strict short-photoperiod flowering feature. Fatty acid desaturases (FADs) are responsible for seed oil accumulation, and play important roles in cold stress tolerance of plants. To date, there is no report on systemically genome-wide analysis of FAD genes in chia (ShiFADs). In this study, 31 ShiFAD genes were identified, 3 of which contained 2 alternative splicing transcripts, and they were located in 6 chromosomes of chia. Phylogenetic analysis classified the ShiFAD proteins into 7 groups, with conserved gene structure and MEME motifs within each group. Tandem and segmental duplications coursed the expansion of ShiFAD genes. Numerous cis-regulatory elements, including hormone response elements, growth and development elements, biotic/abiotic stress response elements, and transcription factor binding sites, were predicted in ShiFAD promoters. 24 miRNAs targeting ShiFAD genes were identified at whole-genome level. In total, 15 SSR loci were predicted in ShiFAD genes/promoters. RNA-seq data showed that ShiFAD genes were expressed in various organs with different levels. qRT-PCR detection revealed the inducibility of ShiSAD2 and ShiSAD7 in response to cold stress, and validated the seed-specific expression of ShiSAD11a. Yeast expression of ShiSAD11a confirmed the catalytic activity of its encoded protein, and its heterologous expression in Arabidopsis thaliana significantly increased seed oleic acid content. This work lays a foundation for molecular dissection of chia high-ALA trait and functional study of ShiFAD genes in cold tolerance.

A cell-type-specific alternative splicing regulator shapes synapse properties in a trans-synaptic manner

The specification of synaptic properties is fundamental for the function of neuronal circuits. "Terminal selector" transcription factors coordinate terminal gene batteries that specify cell-type-specific properties. Moreover, pan-neuronal splicing regulators have been implicated in directing neuronal differentiation. However, the cellular logic of how splicing regulators instruct specific synaptic properties remains poorly understood. Here, we combine genome-wide mapping of mRNA targets and cell-type-specific loss-of-function studies to uncover the contribution of the RNA-binding protein SLM2 to hippocampal synapse specification. Focusing on pyramidal cells and somatostatin (SST)-positive GABAergic interneurons, we find that SLM2 preferentially binds and regulates alternative splicing of transcripts encoding synaptic proteins. In the absence of SLM2, neuronal populations exhibit normal intrinsic properties, but there are non-cell-autonomous synaptic phenotypes and associated defects in a hippocampus-dependent memory task. Thus, alternative splicing provides a critical layer of gene regulation that instructs specification of neuronal connectivity in a trans-synaptic manner.

Frequency and molecular basis of CD36 deficiency among platelet donors in Kunming, China

CD36 is a multifunctional receptor expressed on the surface of many cell types. Among healthy individuals, CD36 may be absent on platelets and monocytes (type I deficiency) or platelets alone (type II deficiency). However, the exact molecular mechanisms underlying CD36 deficiency remain unclear. In this study, we aimed to identify individuals with CD36 deficiency and investigate the molecular basis underlying it. Blood samples were collected from platelet donors at Kunming Blood Center. Platelets and monocytes were isolated and CD36-expression levels were analyzed using flow cytometry. DNA from whole blood and mRNA isolated from monocytes and platelets of individuals with CD36 deficiency were analyzed using polymerase chain reaction (PCR) testing. The PCR products were cloned and sequenced. Among the 418 blood donors,7 (1.68%) were CD36 deficient: 1 (0.24%) with type I deficiency and 6(1.44%) with type II deficiency. Six heterozygous mutations occurred, including c.268C>T (in type I individuals), c.120 + 1 G>T, c.268C>T, c.329_330del/AC, c.1156 C>T, c.1163A>C, and c.1228_1239del/ATTGTGCCTATT (in type II individuals). Mutations were not detected in one type II individual . At the cDNA level, only mutant, but not wild-type, transcripts were detected in the platelets and monocytes of type I individual. In type II individuals, only mutant transcripts were found in platelets, whereas monocytes possessed wild-type and mutant transcripts. Interestingly, only alternative splicing transcripts were observed in the individual without mutation. We report the incidence rates of type I and II CD36 deficiencies among platelet donors in Kunming. Molecular genetic analyses of DNA and cDNA demonstrated that homozygous mutations on the cDNA level in platelets and monocytes or platelets alone identified type I and II deficiencies, respectively. Furthermore, alternatively spliced products also potentially contribute to the mechanism of CD36 deficiency.

Epidermal phyB requires RRC1 to promote light responses by activating the circadian rhythm.

Phytochrome B (phyB) expressed in the epidermis is sufficient to promote red light responses, including the inhibition of hypocotyl elongation and hypocotyl negative gravitropism. Nonetheless, the downstream mechanism of epidermal phyB in promoting light responses had been elusive. Here, we mutagenized the epidermis-specific phyB-expressing line (MLB) using ethyl methanesulfonate (EMS) and characterized a novel mutant allele of RRC1 (rrc1-689), which causes reduced epidermal phyB-mediated red light responses. The rrc1-689 mutation increases the alternative splicing of major clock gene transcripts, including PRR7 and TOC1, disrupting the rhythmic expression of the entire clock and clock-controlled genes. Combined with the result that MLB/prr7 exhibits the same red-hyposensitive phenotypes as MLB/rrc1-689, our data support that the circadian clock is required for the ability of epidermal phyB to promote light responses. We also found that, unlike phyB, RRC1 preferentially acts in the endodermis to maintain the circadian rhythm by suppressing the alternative splicing of core clock genes. Together, our results suggest that epidermal phyB requires RRC1 to promote light responses by activating the circadian rhythm in Arabidopsis thaliana.

Post-Transcriptional Modification by Alternative Splicing and Pathogenic Splicing Variants in Cardiovascular Development and Congenital Heart Defects.

Advancements in genomics, bioinformatics, and genome editing have uncovered new dimensions in gene regulation. Post-transcriptional modifications by the alternative splicing of mRNA transcripts are critical regulatory mechanisms of mammalian gene expression. In the heart, there is an expanding interest in elucidating the role of alternative splicing in transcriptome regulation. Substantial efforts were directed toward investigating this process in heart development and failure. However, few studies shed light on alternative splicing products and their dysregulation in congenital heart defects (CHDs). While elegant reports showed the crucial roles of RNA binding proteins (RBPs) in orchestrating splicing transitions during heart development and failure, the impact of RBPs dysregulation or genetic variation on CHDs has not been fully addressed. Herein, we review the current understanding of alternative splicing and RBPs' roles in heart development and CHDs. Wediscuss the impact of perinatal splicing transition and its dysregulation in CHDs. We further summarize the discoveries made of causal splicing variants in key transcription factors that are implicated in CHDs. An improved understanding of the roles of alternative splicing in heart development and CHDs may potentially inform novel preventive and therapeutic advancements for newborn infants with CHDs.

New insights into genome annotation in Podospora anserina through re-exploiting multiple RNA-seq data

BackgroundPublicly available RNA-seq datasets are often underused although being helpful to improve functional annotation of eukaryotic genomes. This is especially true for filamentous fungi genomes which structure differs from most well annotated yeast genomes. Podospora anserina is a filamentous fungal model, which genome has been sequenced and annotated in 2008. Still, the current annotation lacks information about cis-regulatory elements, including promoters, transcription starting sites and terminators, which are instrumental to integrate epigenomic features into global gene regulation strategies.ResultsHere we took advantage of 37 RNA-seq experiments that were obtained in contrasted developmental and physiological conditions, to complete the functional annotation of P. anserina genome. Out of the 10,800 previously annotated genes, 5’UTR and 3’UTR were defined for 7554, among which, 3328 showed differential transcriptional signal starts and/or transcriptional end sites. In addition, alternative splicing events were detected for 2350 genes, mostly due alternative 3’splice sites and 1732 novel transcriptionally active regions (nTARs) in unannotated regions were identified.ConclusionsOur study provides a comprehensive genome-wide functional annotation of P. anserina genome, including chromatin features, cis-acting elements such as UTRs, alternative splicing events and transcription of non-coding regions. These new findings will likely improve our understanding of gene regulation strategies in compact genomes, such as those of filamentous fungi. Characterization of alternative transcripts and nTARs paves the way to the discovery of putative new genes, alternative peptides or regulatory non-coding RNAs.

Expression dynamics of periodic transcripts during cancer cell cycle progression and their correlation with anticancer drug sensitivity

BackgroundThe cell cycle is at the center of cellular activities and is orchestrated by complex regulatory mechanisms, among which transcriptional regulation is one of the most important components. Alternative splicing dramatically expands the regulatory network by producing transcript isoforms of genes to exquisitely control the cell cycle. However, the patterns of transcript isoform expression in the cell cycle are unclear. Therapies targeting cell cycle checkpoints are commonly used as anticancer therapies, but none of them have been designed or evaluated at the alternative splicing transcript level. The utility of these transcripts as markers of cell cycle-related drug sensitivity is still unknown, and studies on the expression patterns of cell cycle-targeting drug-related transcripts are also rare.MethodsTo explore alternative splicing patterns during cell cycle progression, we performed sequential transcriptomic assays following cell cycle synchronization in colon cancer HCT116 and breast cancer MDA-MB-231 cell lines, using flow cytometry and reference cell cycle transcripts to confirm the cell cycle phases of samples, and we developed a new algorithm to describe the periodic patterns of transcripts fluctuating during the cell cycle. Genomics of Drug Sensitivity in Cancer (GDSC) drug sensitivity datasets and Cancer Cell Line Encyclopedia (CCLE) transcript datasets were used to assess the correlation of genes and their transcript isoforms with drug sensitivity. We identified transcripts associated with typical drugs targeting cell cycle by determining correlation coefficients. Cytotoxicity assays were used to confirm the effect of ENST00000257904 against cyclin dependent kinase 4/6 (CDK4/6) inhibitors. Finally, alternative splicing transcripts associated with mitotic (M) phase arrest were analyzed using an RNA synthesis inhibition assay and transcriptome analysis.ResultsWe established high-resolution transcriptome datasets of synchronized cell cycle samples from colon cancer HCT116 and breast cancer MDA-MB-231 cells. The results of the cell cycle assessment showed that 43,326, 41,578 and 29,244 transcripts were found to be periodically expressed in HeLa, HCT116 and MDA-MB-231 cells, respectively, among which 1280 transcripts showed this expression pattern in all three cancer cell lines. Drug sensitivity assessments showed that a large number of these transcripts displayed a higher correlation with drug sensitivity than their corresponding genes. Cell cycle-related drug screening showed that the level of the CDK4 transcript ENST00000547281 was more significantly associated with the resistance of cells to CDK4/6 inhibitors than the level of the CDK4 reference transcript ENST00000257904. The transcriptional inhibition assay following M phase arrest further confirmed the M-phase-specific expression of the splicing transcripts. Combined with the cell cycle-related drug screening, the results also showed that a set of periodic transcripts, for example, ENST00000314392 (a dolichyl-phosphate mannosyltransferase polypeptide 2 isoform transcript), was more associated with drug sensitivity than the levels of their corresponding gene transcripts.ConclusionsIn summary, we identified a panel of cell cycle-related periodic transcripts and found that the levels of transcripts of drug target genes showed different values for predicting drug sensitivity, providing novel insights into alternative splicing-related drug development and evaluation.

Genetic variation in P-element dysgenic sterility is associated with double-strand break repair and alternative splicing of TE transcripts.

The germline mobilization of transposable elements (TEs) by small RNA mediated silencing pathways is conserved across eukaryotes and critical for ensuring the integrity of gamete genomes. However, genomes are recurrently invaded by novel TEs through horizontal transfer. These invading TEs are not targeted by host small RNAs, and their unregulated activity can cause DNA damage in germline cells and ultimately lead to sterility. Here we use hybrid dysgenesis-a sterility syndrome of Drosophila caused by transposition of invading P-element DNA transposons-to uncover host genetic variants that modulate dysgenic sterility. Using a panel of highly recombinant inbred lines of Drosophila melanogaster, we identified two linked quantitative trait loci (QTL) that determine the severity of dysgenic sterility in young and old females, respectively. We show that ovaries of fertile genotypes exhibit increased expression of splicing factors that suppress the production of transposase encoding transcripts, which likely reduces the transposition rate and associated DNA damage. We also show that fertile alleles are associated with decreased sensitivity to double-stranded breaks and enhanced DNA repair, explaining their ability to withstand high germline transposition rates. Together, our work reveals a diversity of mechanisms whereby host genotype modulates the cost of an invading TE, and points to genetic variants that were likely beneficial during the P-element invasion.

Tau Isoforms: Gaining Insight into MAPT Alternative Splicing

Tau microtubule-associated proteins, encoded by the MAPT gene, are mainly expressed in neurons participating in axonal transport and synaptic plasticity. Six major isoforms differentially expressed during cell development and differentiation are translated by alternative splicing of MAPT transcripts. Alterations in the expression of human Tau isoforms and their aggregation have been linked to several neurodegenerative diseases called tauopathies, including Alzheimer's disease, progressive supranuclear palsy, Pick's disease, and frontotemporal dementia with parkinsonism linked to chromosome 17. Great efforts have been dedicated in recent years to shed light on the complex regulatory mechanism of Tau splicing, with a perspective to developing new RNA-based therapies. This review summarizes the most recent contributions to the knowledge of Tau isoform expression and experimental models, highlighting the role of cis-elements and ribonucleoproteins that regulate the alternative splicing of Tau exons.

De Novo Germline DHX38 Variant Associated with Alternative Splicing of Multiple Transcripts in Iron-Related Pathways in a Patient with Atypical Congenital Dyserythropoietic Anemia with Ring Sideroblasts

Congenital Dyserythropoietic Anemias (CDAs) are a heterogeneous group of rare hereditary anemias caused by ineffective and dyspoietic erythropoiesis, with the pathognomonic feature of binuclear and/or multinuclear erythroid precursors in the bone marrow. The clinical diagnosis of CDA is based on identifying the characteristic morphology of erythroblasts in the bone marrow of patients presenting with chronic anemia, frequently with evidence of hemolysis but suboptimal reticulocytosis, and iron overload. CDAR (ClinicalTrials.gov identifier: NCT02964494), a registry for patients with CDA in North America, has been established with the goal of providing a longitudinal database and associated biorepository to facilitate natural history studies and research on the pathogenetic mechanisms in this heterogenous disease. A 2-year-old female, enrolled in CDAR, had transfusion-dependent anemia since birth. She demonstrated accelerated iron overload and had failure to thrive despite regular transfusions maintaining trough hemoglobin higher than 9.5 g/dL. Bone marrow evaluation revealed dyserythropoiesis along with ring sideroblasts (RS), resembling acquired myelodysplastic syndrome (MDS) with the characteristics of Refractory Anemia with RS (RARS). Whole-exome sequencing for the proband and her parents in trio analysis identified a de novo, heterozygous germline DHX38 missense variant c.3310A>C, p.Thr1104Pro in the proband. In silico prediction programs suggest this rare variant is deleterious. DEAH box RNA helicase DHX38 is an RNA helicase, homolog to the yeast Prp16 that has been shown to play a dual role in the spliceosome complex: an ATP-independent role in the first catalytic step mediating proofreading of the branchpoint sequence, and an ATP-dependent role in the second step, mediating removal of the lariat and joining the exons to complete the splicing (Tseng et al, RNA 2011). DHX38 is highly expressed during terminal erythropoiesis similarly to SF3B1, the splicing factor gene most frequently mutated in MDS-RARS. RNA-seq analysis of CD71+ reticulocytes isolated from the proband at three different time points compared to three different normal control samples, using AltAnalyze software, revealed approximately 3,000 differentially expressed genes. More than 1,500 differentially spliced events were identified, affecting over 200 genes, including SLC25A37, coding for an iron importer localized in the mitochondrial inner membrane and PPOX, coding for protoporphyrinogen oxidase (Figure 1A), both essential for the synthesis of mitochondrial heme. Mis-splicing of the same genes is a characteristic finding in MDS-RARS associated with SF3B1 pathogenic variants, most commonly p.Lys700Glu, and may contribute to RS formation. Moreover, DHX38 is known to interact with centromeric noncoding RNA, regulating chromosome segregation during mitosis, and its depletion in vitro causes abnormal grape-shaped nuclear morphology (Nishimura et al, Genes Cells 2019). Review of the patient's bone marrow studies revealed several erythroblasts with grape-shaped nuclei (Figure 1B). Erythropoiesis cultures from patient-derived iPSCs were utilized to phenocopy the disease in vitro. Cytospins of the generated erythroblasts revealed dyserythropoiesis that was verified quantitatively by increased frequency of nuclear abnormalities in erythroblasts produced in vitro from patient-derived iPSCs analyzed by imaging flow cytometry. Extra-hematopoietic abnormalities of the nucleus and mitotic defects were explored in fibroblasts generated from the patient's skin biopsy, which also tested positive for DHX38 p.Thr1104Pro. Grape-shaped nuclei were detected in cultures of patient's fibroblasts indicating improper mitotic exit due to aberrant chromosome segregation. Our findings indicate that DHX38 is likely the causative gene for this CDA with a RARS phenotype, causing mis-splicing of a multitude of genes critical for iron metabolism and erythropoiesis as well as defects in chromosome segregation and cell division. Out of approximately 250,000 alleles in the gnomAD database, the DHX38 p.Thr1104Pro variant has been detected in 4 alleles; however, SF3B1 p.Lys700Glu is also detected in 22. Therefore, the rare presence of this DHX38 variant in "healthy" population may indicate that acquired DHX38 pathogenic variants exist and could be responsible for SF3B1-negative MDS-RARS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Integrated top-down and bottom-up proteomics mass spectrometry for the characterization of endogenous ribosomal protein heterogeneity

Ribosomes are abundant, large RNA-protein complexes that are the sites of all protein synthesis in cells. Defects in ribosomal proteins (RPs), including proteoforms arising from genetic variations, alternative splicing of RNA transcripts, post-translational modifications and alterations of protein expression level, have been linked to a diverse range of diseases, including cancer and aging. Comprehensive characterization of ribosomal proteoforms is challenging but important for the discovery of potential disease biomarkers or protein targets. In the present work, using E.coli 70S RPs as an example, we first developed a top-down proteomics approach on a Waters Synapt G2 Si mass spectrometry (MS) system, and then applied it to the HeLa 80S ribosome. The results were complemented by a bottom-up approach. In total, 50 out of 55 RPs were identified using the top-down approach. Among these, more than 30 RPs were found to have their N-terminal methionine removed. Additional modifications such as methylation, acetylation, and hydroxylation were also observed, and the modification sites were identified by bottom-up MS. In a HeLa 80S ribosomal sample, we identified 98 ribosomal proteoforms, among which multiple truncated 80S ribosomal proteoforms were observed, the type of information which is often overlooked by bottom-up experiments. Although their relevance to diseases is not yet known, the integration of top-down and bottom-up proteomics approaches paves the way for the discovery of proteoform-specific disease biomarkers or targets.

Sequence and expression regulation of the BCL2L2 gene in pigs

B cell lymphoma-2-like 2 (BCL2L2), an important regulator of apoptosis, plays vital roles in several physiological processes, as revealed by studies in humans and mice. However, reports on pig BCL2L2 are few, and the encoding gene has not been identified experimentally. This study was designed to clone the porcine BCL2L2 gene and its alternative splicing (AS) transcripts using molecular biological techniques and to analyze the regulatory mechanisms underlying transcription and translation. The BCL2L2 cDNA (V1) was 807 bp in length and encoded a polypeptide of 193 aa containing four BCL-2 homology domains. A total of nine AS transcripts were obtained, among which V2 and V3 differed from V1 in the 5′ untranslated region (UTR). The core promoter was mapped to a range of −1102 to −759 bp (the first nucleotide of the start codon was designated as +1). There were several functional cis-elements, including one SP1 and two C/EBPα binding sites at around −759 bp. AS in the 5′ UTR is involved in the regulation of gene expression, as revealed by dual-luciferase reporter and western blot analysis, and the secondary structure of the 5′ UTRs may be the reason for the differential expression of V1–3. At the same time, an upstream open reading frame (ORF) existed in each of the three 5′ UTRs, was found to repress the expression of the main ORF. Additionally, the roles of porcine BCL2L2 in cell proliferation and apoptosis were preliminarily analyzed. The results will contribute to further characterizing the role of BCL2L2.

CaaX-less lamins: Lophotrochozoa provide a glance at the playground of evolution

Nuclear lamins are the main components of the nuclear lamina in many eukaryotes. They are members of the intermediate filament (IF) protein family. Lamins differ from cytoplasmic IF proteins by the presence of a nuclear localisation sequence (NLS) and a C-terminal tetrapeptide, the CaaX motif. The CaaX motif is target of post-translational modifications including isoprenylation, proteolytic processing, and carboxyl-methylation. These modifications, in conjunction with the NLS, direct lamins to the inner nuclear membrane where they assemble into filaments. Lamins lacking a CaaX motif are unable to associate independently with nuclear membranes and remain in the nucleoplasm. So far, three species have been reported to exclusively express CaaX-less lamins. All three belong to the lophotrochozoan lineage. To find out whether they represent rare exceptions, we analysed lamins of representatives of 17 lophotrochozoan phyla. Here we report that all four clades of Rotifera as well as individual taxa of Mollusca and Annelida lack CaaX-lamins, but express lamins with alternative C-termini. Of note, the respective mollusc and annelid groups occupy very different phylogenetic ranks. Most of these alternative C-termini are rich in aromatic residues. A possible function of these residues in membrane association is discussed. Alternative splicing of terebellid lamin transcripts gives rise to two lamin variants, one with a CaaX motif and one with an alternative C-terminus. A similar situation is found in Arenicolidae, Opheliidae, Capitellidae, and Echiura. This points a way, how the switch from lamins carrying a CaaX motif to lamins with alternative C-termini may have occurred.

Genomic organization, intragenic tandem duplication, and expression analysis of chicken TGFBR2 gene

Transforming growth factor beta receptor Ⅱ (TGFBR2), a core member of the transforming growth factor-β (TGF-β) signaling pathway. To date, chicken TGFBR2 (cTGFBR2) genomic structure has not been fully explored. Here, the complete sequences of cTGFBR2 transcript isoforms were determined by 5′ and 3′ rapid amplification of cDNA ends (5′ & 3′ RACE) and reverse transcription polymerase chain reaction (RT-PCR); the tissue expression profiling of cTGFBR2 transcript isoforms was performed using quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that cTGFBR2 gene produced 3 transcript isoforms though alternative transcription initiation, splicing, and polyadenylation, which were designated as cTGFBR2-1, cTGFBR2-2, and cTGFBR2-3, respectively. These 3 cTGFBR2 transcript isoforms encoded 3 protein isoforms: cTGFBR2-1, cTGFBR2-2, and cTGFBR2-3. Duplication analysis revealed that, unlike other animal species, cTGFBR2 gene harbored a 5.5-kb intragenic tandem duplication. Tissue expression profiling in the 4-wk-old Arbor Acres (AA) broiler chickens showed that cTGFBR2-1 was ubiquitously expressed, with high expression in abdominal fat, subcutaneous fat, lung, gizzard, and muscle; cTGFBR2-2 was highly expressed in heart, kidney, gizzard, and muscle; cTGFBR2-3 was weakly expressed in all the tested chicken tissues. Tissue expression profiling in the 7-wk-old broiler chickens of the fat and lean lines of Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) showed that cTGFBR2-1 was significantly differentially expressed in all the tested tissues except heart, cTGFBR2-2 was significantly differentially expressed in all the tested tissues except subcutaneous fat and liver, and cTGFBR2-3 was significantly differentially expressed in all the tested tissues between the lean and fat lines. Intriguingly, in the fat line, the 3 cTGFBR2 transcript isoforms were expressed to varying degrees in all the 3 tested fat tissues, while in the lean line, only cTGFBR2-1 was expressed in all the 3 tested fat tissues. This is the first report of intragenic tandem duplication within TGFBR2 gene. Our findings pave the way for further studies on the functions and regulation of cTGFBR2 gene.

An auto-inhibited state of protein kinase G and implications for selective activation.

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iβ in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.