Alternative Transcription Initiation Research Articles

ASAPA: a bioinformatic pipeline based on Iso-Seq that identifies the links among alternative splicing, alternative transcription initiation and alternative polyadenylation.

Although the events associated with alternative splicing (AS), alternative polyadenylation (APA) and alternative transcription initiation (ATI) can be identified by many approaches based on isoform sequencing (Iso-Seq), these analyses are generally independent of each other and the links between these events are still rarely mentioned. However, an interdependency analysis can be achieved because the transcriptional start site, splice sites and polyA site could be simultaneously included in a long, full-length read from Iso-Seq. We create ASAPA pipeline that enables streamlined analysis for a robust detection of potential links among AS, ATI and APA using Iso-Seq data. We tested this pipeline using Arabidopsis data and found some interesting results: some adjacent introns tend to be simultaneously spliced or retained; coupling between AS and ATI or APA is limited to the initial or terminal intron; and ATI and APA are potentially linked in some special cases. Our pipeline enables streamlined analysis for a robust detection of potential links among AS, ATI and APA using Iso-Seq data, which is conducive to a better understanding of transcription landscape generation.

Ubiquitination-mediated Golgi-to-endosome sorting determines the toxin-antidote duality of fission yeast wtf meiotic drivers

Killer meiotic drivers (KMDs) skew allele transmission in their favor by killing meiotic progeny not inheriting the driver allele. Despite their widespread presence in eukaryotes, the molecular mechanisms behind their selfish behavior are poorly understood. In several fission yeast species, single-gene KMDs belonging to the wtf gene family exert selfish killing by expressing a toxin and an antidote through alternative transcription initiation. Here we investigate how the toxin and antidote products of a wtf-family KMD gene can act antagonistically. Both the toxin and the antidote are multi-transmembrane proteins, differing only in their N-terminal cytosolic tails. We find that the antidote employs PY motifs (Leu/Pro-Pro-X-Tyr) in its N-terminal cytosolic tail to bind Rsp5/NEDD4 family ubiquitin ligases, which ubiquitinate the antidote. Mutating PY motifs or attaching a deubiquitinating enzyme transforms the antidote into a toxic protein. Ubiquitination promotes the transport of the antidote from the trans-Golgi network to the endosome, thereby preventing it from causing toxicity. A physical interaction between the antidote and the toxin enables the ubiquitinated antidote to translocate the toxin to the endosome and neutralize its toxicity. We propose that post-translational modification-mediated protein localization and/or activity changes may be a common mechanism governing the antagonistic duality of single-gene KMDs.

Core promoterome of barley embryo

Precise localization and dissection of gene promoters are key to understanding transcriptional gene regulation and to successful bioengineering applications. The core RNA polymerase II initiation machinery is highly conserved among eukaryotes, leading to a general expectation of equivalent underlying mechanisms. Still, less is known about promoters in the plant kingdom. In this study, we employed cap analysis of gene expression (CAGE) at three embryonic developmental stages in barley to accurately map, annotate, and quantify transcription initiation events. Unsupervised discovery of de novo sequence clusters grouped promoters based on characteristic initiator and position-specific core-promoter motifs. This grouping was complemented by the annotation of transcription factor binding site (TFBS) motifs. Integration with genome-wide epigenomic data sets and gene ontology (GO) enrichment analysis further delineated the chromatin environments and functional roles of genes associated with distinct promoter categories. The TATA-box presence governs all features explored, supporting the general model of two separate genomic regulatory environments. We describe the extent and implications of alternative transcription initiation events, including those that are specific to developmental stages, which can affect the protein sequence or the presence of regions that regulate translation. The generated promoterome dataset provides a valuable genomic resource for enhancing the functional annotation of the barley genome. It also offers insights into the transcriptional regulation of individual genes and presents opportunities for the informed manipulation of promoter architecture, with the aim of enhancing traits of agronomic importance.

Abstract 2076: Novel framework for systematically detecting alternative transcript initiation by integrating ATAC-seq and RNA-seq

Abstract Background: Alternative transcription initiation (ATI) has been frequently observed in cancer suggesting that it contributes to the malignant transformation of the cells. However, ATI remains largely unexplored mainly due to the lack of tools for detecting ATI. We propose a computational method for integrating bulk ATAC-seq and bulk RNA-seq to identify ATI and understand their differential usage between tissues. We hypothesize that differential ATAC-seq intensity can be used as a guide for looking for differential promoter usage, which might enable the identification of novel ATI events in a transcript-agnostic way. Methods: We recently published methods for RNA-seq base-level analysis for identifying structural variations in transcripts. Building on this, we developed a supervised sparse non-negative matrix factorization approach that integrates base-level RNA-seq and ATAC-seq, which aims to identify ATIs as well as characterize the latent structure of the underlying isoforms. This method equips many unique features that can be useful for the identification of novel ATIs. Because the method scans an entire collection of DNA accessible regions provided by ATAC-seq, it enables a comprehensive screening of novel ATI candidates that are not limited to the known promoters. Additionally, the uncompressed view of base-level RNA-seq allows us to infer the structure of individual isoforms independently of known gene annotation. The predicted isoforms can be further used to deconvolute base-level RNA-seq of individual cases into the isoforms and thus infer expression levels of each isoform. Results: We applied this method to a sub-cohort (N=350) of TCGA pan-cancer samples in which both ATAC-seq and RNA-seq are available. Empiric comparison to existing methods confirmed known true positive ATIs including important cancer genes such as CDKN2A and ALK. In particular, the method successfully identified the ATIs including some challenging cases such as ATIs located at internal introns or at constitutive exons in other transcripts. The deconvolution analysis applied to the extended cohort to all available ~10,000 TCGA pan-caner samples across 32 tissue types revealed that ATIs were predominantly differentiated across tissue types. By applying the method to a set of transcription regulator genes, we identified ~1% of the genes had ATIs including the novel cancer-specific genes with known ATIs that had not been previously reported as relevant to cancer. Additionally, we provide examples in which the isoform’s function relative to cancer appears mechanistically interesting. Conclusion: We propose a multi-omics integration method that is independent of known gene annotation, enabling a robust identification of ATIs. Our results strongly demonstrate our ability to pick up known as well as novel ATIs that are otherwise difficult to identify by existing methods. Citation Format: Hyo Young Choi, Won-Young Choi, David N. Hayes. Novel framework for systematically detecting alternative transcript initiation by integrating ATAC-seq and RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2076.

Proliferation-associated 2G4 P48 is stabilized by malignant T-cell amplified sequence 1 and promotes the proliferation of head and neck squamous cell carcinoma

Background/purposeProliferation-associated protein 2G4 (PA2G4) has alternative transcriptional and translational initiation. One dominant transcript ENST00000303305 could be translated into two protein isoforms (PA2G4-P42 and PA2G4-P48). In this study, we aimed to explore the effects of PA2G4-P42 and PA2G4-P48 on the proliferation of head and neck squamous cell carcinoma (HNSCC) and the mechanisms regulating PA2G4-P48 stability. Materials and methodsHNSCC cell lines HSC2 and SCC25 with relatively low PA2G4 expression were used for in-vitro cell studies. PA2G4-P42 and PA2G4-P48 overexpression lentiviruses were generated. In vitro cell proliferation was assessed by CCK-8 and colony formation. In vivo tumor cell proliferation was assessed by HSC2 cell-derived xenograft tumors. Liquid chromatography-mass spectrometry (LC-MS)/MS and co-immunoprecipitation (co-IP) assays were applied to check PA2G4-P48 interacting partners. Cycloheximide (CHX) chase and ubiquitin-based co-IP assays were also performed. ResultsPA2G4-P48 was the dominant isoform, with substantially higher expression than PA2G4-P42 in HNSCC. PA2G4-P48 overexpression enhanced HNSCC cell proliferation, but PA2G4-P42 overexpression slowed the proliferation. MCTS1 interacted with PA2G4-P48, but not PA2G4-P42. PA2G4 protein but not its mRNA expression was decreased in cells with MCTS1 knockdown. MG132 treatment abrogated this alteration. MCTS1 overexpression significantly elevated the half-life of PA2G4-P48, while its knockdown drastically reduced the half-life compared with the control cells. In addition, MCTS1 overexpression significantly decreased the polyubiquitination of exogenous flag-tagged PA2G4-P48. MCTS1 overexpression-induced cell proliferation was hampered by knocking down of PA2G4-P48. ConclusionPA2G4-P42 and PA2G4-P48 exert growth-suppressive and growth-promoting effects in HNSCC, respectively. MCTS1 can interact with PA2G4-P48 and prolong its half-life by reducing its poly-ubiquitination.

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

New start sites for plant immune gene transcription.

Appropriate spatiotemporal regulation of immune gene expression triggered by pathogen-associated molecular patterns (PAMPs) is critical for defense against pathogens. Thieffry et al. recently found that PAMP-triggered widespread alternative transcription initiation and an immediate transcription factor wave are elements of immune gene reprogramming.

Superenhancer drives a tumor-specific splicing variant of MARCO to promote triple-negative breast cancer progression

The transcription variation, leading to various forms of transcripts and protein diversity, remains largely unexplored in triple-negative breast cancers (TNBCs). Here, we presented a comprehensive analysis of RNA splicing in breast cancer to illustrate the biological function and clinical implications of tumor-specific transcripts (TSTs) arising from these splicing junctions. Aberrant RNA splicing or TSTs were frequently harbored in TNBC and were correlated with a poor outcome. We discovered a tumor-specific splicing variant of macrophage receptor with collagenous structure-TST (MARCO-TST), which was distinguished from myeloid cell-specific wild-type MARCO. MARCO-TST expression was associated with poor outcomes in TNBC patients and could promote tumor progression invitro and invivo. Mechanically, MARCO-TST interacted with PLOD2 and enhanced the stability of HIF-1α, which resulted in the metabolic dysregulation of TNBC to form a hypoxic tumor microenvironment. MARCO-TST was initiated from a de novo alternative transcription initiation site that was activated by a superenhancer. Tumors with MARCO-TST expression conferred greater sensitivity to bromodomain and extraterminal protein inhibitors. This treatment strategy was further validated in patient-derived organoids. In conclusion, our results revealed the transcription variation landscape of TNBC, highlighting MARCO-TST as a crucial oncogenic transcript and therapeutic target.

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.

PAMP-triggered genetic reprogramming involves widespread alternative transcription initiation and an immediate transcription factor wave.

Immune responses triggered by pathogen-associated molecular patterns (PAMPs) are key to pathogen defense, but drivers and stabilizers of the growth-to-defense genetic reprogramming remain incompletely understood in plants. Here, we report a time-course study of the establishment of PAMP-triggered immunity (PTI) using cap analysis of gene expression. We show that around 15% of all transcription start sites (TSSs) rapidly induced during PTI define alternative transcription initiation events. From these, we identify clear examples of regulatory TSS change via alternative inclusion of target peptides or domains in encoded proteins, or of upstream open reading frames in mRNA leader sequences. We also find that 60% of PAMP response genes respond earlier than previously thought. In particular, a cluster of rapidly and transiently PAMP-induced genes is enriched in transcription factors (TFs) whose functions, previously associated with biological processes as diverse as abiotic stress adaptation and stem cell activity, appear to converge on growth restriction. Furthermore, examples of known potentiators of PTI, in one case under direct mitogen-activated protein kinase control, support the notion that the rapidly induced TFs could constitute direct links to PTI signaling pathways and drive gene expression changes underlying establishment of the immune state.

Alternative transcription and feedback regulation suggest that SlIDI1 is involved in tomato carotenoid synthesis in a complex way.

Carotenoid pigments confer photoprotection and visual attraction and serve as precursors for many important signaling molecules. Herein, the orange-fruited phenotype of a tomato elite inbred line resulting from sharply reduced carotenoid levels and an increased β-carotene-to-lycopene ratio in fruit was shown to be controlled by a single recessive gene, oft3. BSA-Seq combined with fine mapping delimited the oft3 gene to a 71.23kb interval on chromosome 4, including eight genes. Finally, the oft3 candidate gene SlIDI1, harboring a 116bp deletion mutation, was identified by genome sequence analysis. Further functional complementation and CRISPR-Cas9 knockout experiments confirmed that SlIDI1 was the gene underlying the oft3 locus. qRT-PCR analysis revealed that the expression of SlIDI1 was highest in flowers and fruit and increased with fruit ripening or flower maturation. SlIDI1 simultaneously produced long and short transcripts by alternative transcription initiation and alternative splicing. Green fluorescent protein fusion expression revealed that the long isoform was mainly localized in plastids and that an N-terminal 59-amino acid extension sequence was responsible for plastid targeting. Short transcripts were identified in leaves and fruit by 5' RACE and in fruit by 3' RACE, which produced corresponding proteins lacking transit peptides and/or putative peroxisome targeting sequences, respectively. In SlIDI1 mutant fruit, SlBCH1 transcription involved in β-carotenoid catabolism was obviously suppressed, which may be responsible for the higher β-carotene-to-lycopene ratio and suggested potential feedback regulatory mechanisms involved in carotenoid pathway flux.

FGGY carbohydrate kinase domain containing is expressed and alternatively spliced in skeletal muscle and attenuates MAP kinase and Akt signaling.

Skeletal muscle atrophy can result from a range of physiological conditions, including denervation, immobilization, hindlimb unweighting, and aging. To better characterize the molecular genetic events of atrophy, a microarray analysis revealed that FGGY carbohydrate kinase domain containing (Fggy) is expressed in skeletal muscle and is induced in response to denervation. Bioinformatic analysis of the Fggy gene locus revealed two validated isoforms with alternative transcription initiation sites that we have designated Fggy-L-552 and Fggy-S-387. Additionally, we cloned two novel alternative splice variants, designated Fggy-L-482 and Fggy-S-344, from cultured muscle cells suggesting that at least four Fggy splice variants are expressed in skeletal muscle. Quantitative RT-PCR was performed using RNA isolated from muscle cells and primers designed to distinguish the four alternative Fggy transcripts and found that the Fggy-L transcripts are more highly expressed during myoblast differentiation, while the Fggy-S transcripts show relatively stable expression in proliferating myoblasts and differentiated myotubes. Confocal fluorescent microscopy revealed that the Fggy-L variants appear to localize evenly throughout the cytoplasm, while the Fggy-S variants produce a more punctuate cytoplasmic localization pattern in proliferating muscle cells. Finally, ectopic expression of Fggy-L-552 and Fggy-S-387 resulted in inhibition of muscle cell differentiation and attenuation of the MAP kinase and Akt signaling pathways. The identification and characterization of novel genes such as Fggy helps to improve our understanding of the molecular and cellular events that lead to atrophy and may eventually result in the identification of new therapeutic targets for the treatment of muscle wasting.

GABAB receptors in GtoPdb v.2021.2

Functional GABAB receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on GABAB receptors [11, 71]) are formed from the heterodimerization of two similar 7TM subunits termed GABAB1 and GABAB2 [11, 70, 28, 71, 87]. GABAB receptors are widespread in the CNS and regulate both pre- and postsynaptic activity. The GABAB1 subunit, when expressed alone, binds both antagonists and agonists, but the affinity of the latter is generally 10-100-fold less than for the native receptor. Co-expression of GABAB1 and GABAB2 subunits allows transport of GABAB1 to the cell surface and generates a functional receptor that can couple to signal transduction pathways such as high-voltage-activated Ca2+ channels (Cav2.1, Cav2.2), or inwardly rectifying potassium channels (Kir3) [12, 11, 5]. The GABAB1 subunit harbours the GABA (orthosteric)-binding site within an extracellular domain (ECD) venus flytrap module (VTM), whereas the GABAB2 subunit mediates G protein-coupled signalling [11, 70, 40, 39]. The cryo-electron microscopy structures of the human full-length GABAB1-GABAB2 heterodimer have been solved in the inactive apo state, two intermediate agonist-bound forms and an active state in which the heterodimer is bound to an agonist and a positive allosteric modulator [81]. The positive allosteric modulator binds to the transmembrane dimerization interface and stabilizes the active state. Recent evidence indicates that higher order assemblies of GABAB receptor comprising dimers of heterodimers occur in recombinant expression systems and in vivo and that such complexes exhibit negative functional cooperativity between heterodimers [69, 22]. Adding further complexity, KCTD (potassium channel tetramerization proteins) 8, 12, 12b and 16 associate as tetramers with the carboxy terminus of the GABAB2 subunit to impart altered signalling kinetics and agonist potency to the receptor complex [86, 3, 79] and are reviewed by [72]. The molecular complexity of GABAB receptors is further increased through association with trafficking and effector proteins [80] and reviewed by [68]. The predominant GABAB1a and GABAB1b isoforms, which are most prevalent in neonatal and adult brain tissue respectively, differ in their ECD sequences as a result of the use of alternative transcription initiation sites. GABAB1a-containing heterodimers localise to distal axons and mediate inhibition of glutamate release in the CA3-CA1 terminals, and GABA release onto the layer 5 pyramidal neurons, whereas GABAB1b-containing receptors occur within dendritic spines and mediate slow postsynaptic inhibition [74, 91]. Amyloid precursor protein (APP) and soluble APP (sAPP) bind to the N- terminal sushi domain of the GABAB1a isoform to regulate axonal trafficking of GABAB receptors and release of neurotransmitters [76].

PSDX: A Comprehensive Multi-Omics Association Database of Populus trichocarpa With a Focus on the Secondary Growth in Response to Stresses.

Populus trichocarpa (P. trichocarpa) is a model tree for the investigation of wood formation. In recent years, researchers have generated a large number of high-throughput sequencing data in P. trichocarpa. However, no comprehensive database that provides multi-omics associations for the investigation of secondary growth in response to diverse stresses has been reported. Therefore, we developed a public repository that presents comprehensive measurements of gene expression and post-transcriptional regulation by integrating 144 RNA-Seq, 33 ChIP-seq, and six single-molecule real-time (SMRT) isoform sequencing (Iso-seq) libraries prepared from tissues subjected to different stresses. All the samples from different studies were analyzed to obtain gene expression, co-expression network, and differentially expressed genes (DEG) using unified parameters, which allowed comparison of results from different studies and treatments. In addition to gene expression, we also identified and deposited pre-processed data about alternative splicing (AS), alternative polyadenylation (APA) and alternative transcription initiation (ATI). The post-transcriptional regulation, differential expression, and co-expression network datasets were integrated into a new P. trichocarpa Stem Differentiating Xylem (PSDX) database (http://forestry.fafu.edu.cn/db/SDX), which further highlights gene families of RNA-binding proteins and stress-related genes. The PSDX also provides tools for data query, visualization, a genome browser, and the BLAST option for sequence-based query. Much of the data is also available for bulk download. The availability of PSDX contributes to the research related to the secondary growth in response to stresses in P. trichocarpa, which will provide new insights that can be useful for the improvement of stress tolerance in woody plants.

Global miRNA dosage control of embryonic germ layer specification

MicroRNAs (miRNAs) have essential functions during embryonic development, and their dysregulation causes cancer1,2. Altered global miRNA abundance is found in different tissues and tumours, which implies that precise control of miRNA dosage is important1,3,4, but the underlying mechanism(s) of this control remain unknown. The protein complex Microprocessor, which comprises one DROSHA and two DGCR8 proteins, is essential for miRNA biogenesis5-7. Here we identify a developmentally regulated miRNA dosage control mechanism that involves alternative transcription initiation (ATI) of DGCR8. ATI occurs downstream of a stem-loop in DGCR8 mRNA to bypass an autoregulatory feedback loop during mouse embryonic stem (mES)cell differentiation. Deletion of the stem-loop causes imbalanced DGCR8:DROSHA protein stoichiometry that drives irreversible Microprocessor aggregation, reduced primary miRNA processing, decreased mature miRNA abundance, and widespread de-repression of lipid metabolic mRNA targets. Although global miRNA dosage control is not essential for mES cells to exit from pluripotency, its dysregulation alters lipid metabolic pathways and interferes with embryonic development by disrupting germ layer specification in vitro and in vivo. This miRNA dosage control mechanism is conserved in humans. Our results identify a promoter switch that balances Microprocessor autoregulation and aggregation to precisely control global miRNA dosage and govern stem cell fate decisions during early embryonic development.

English

Chia (Salvia hispanica), originated in Mexico, has outstanding nutritional and health-promoting values, but it is the only ancient American Indian staple crop failed in introduction to the Old Continents. After the rediscovery and revival of chia as a new crop in recent years, the short-day (SD) habit is a crucial limitation for its worldwide cultivation. The circadian oscillator GIGNATEA (GI) is an important transcription factor regulating flowering time through photoperiod-pathway. In this study, we cloned the full-length cDNAs of two GI genes from chia, and analyzed the molecular characteristics of the genes and the encoded proteins. Alternative transcription initiation sites, alternative poly A tailing sites, and 5’-UTR intron retention exist in some of their mRNAs. The origin of GI gene accompanied with the transition from aquatic to terrestrial habits during plant evolution. GI duplication events occurred at order, family and genus levels in angiosperms. ShGI-1 and ShGI-2 were similar to each other in organ specificity with peak expression in small buds. In mature leaf, ShGI-2 is dominant over ShGI-1 in terms of expression level with highest expression in the afternoon, but on the Autumnal Equinox day ShGI-1 is dominant over ShGI-2 with peaks at noon and in the evening. KT, BR, GA3 and IAA upregulated the expression of ShGI-1 and ShGI-2 in long-days (LDs) and inhibited their expression in SDs, with GA3 being the most effective phytohormone. Under most abiotic stresses, ShGI expression fluctuated and returned to near-basal levels. ShGI expression was upregulated by low temperature. SA sharply upregulated ShGI expression after 24 h of treatment. This is the first report of GI genes from the order Lamiales, which will promote the dissection of flowering mechanism of chia and other Lamiales plants, enrich the evolution and expression characteristics of plant GIs, and promote the study on interaction between photoperiod and hormone pathways in flowering time control. © 2021 Friends Science Publishers

Correlation of novel ALKATI with ALK immunohistochemistry and clinical outcomes in metastatic melanoma.

Recently, a novel isoform of anaplastic lymphoma kinase, with alternative transcription initiation (ALKATI ), has been described in melanoma and is susceptible to targeted ALK-inhibitor therapy. Clinical outcomes of patients with ALKATI mutated melanoma as well as correlation with immunohistochemical (IHC) methods have not yet been described. Clinicopathological characteristics were abstracted for 324 patients with metastatic melanoma (MM). IHC, fluorescence in-situ hybridisation and RNA-based digital molecular analysis assays were performed on archival tissue from 173 stage III and 192 stage IV tumours. ALKATI was identified in 12.7 and 4.8% stage III and IV tumours, respectively. Discrete presentations of the ALKATI are seen: isolated ALKATI (n=20) and mixed ALKATI (combined ALKATI and ALKWT ; n=7). Isolated ALKWT expression (n=4) was seen with no ALK fusions. Stage III patients showed improved survival with ALKATI expression compared to those with ALKWT or no expression [5-year survival 80, 95% confidence interval (CI)=57-100% versus 43%, 95% CI=34-55%, P=0.013]. Clinicopathological characteristics were not statistically significant. Strong diffuse cytoplasmic staining of ALK IHC (n=12) has a sensitivity of 52.2%, specificity 100%, PPV of 100% and NPV of 92.5% of detecting isolated ALKATI . Presence of ALKATI is a good prognostic indicator in MM. ALK IHC and digital molecular analysis can be incorporated into MM evaluation to identify patients with ALKATI for targeted therapy.

Plastid transit peptides-where do they come from and where do they all belong? Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution.

Subcellular relocalization of proteins determines an organism’s metabolic repertoire and thereby its survival in unique evolutionary niches. In plants, the plastid and its various morphotypes import a large and varied number of nuclear-encoded proteins to orchestrate vital biochemical reactions in a spatiotemporal context. Recent comparative genomics analysis and high-throughput shotgun proteomics data indicate that there are a large number of plastid-targeted proteins that are either semi-conserved or non-conserved across different lineages. This implies that homologs are differentially targeted across different species, which is feasible only if proteins have gained or lost plastid targeting peptides during evolution. In this study, a broad, multi-genome analysis of 15 phylogenetically diverse genera and in-depth analyses of pangenomes from Arabidopsis and Brachypodium were performed to address the question of how proteins acquire or lose plastid targeting peptides. The analysis revealed that random insertions or deletions were the dominant mechanism by which novel transit peptides are gained by proteins. While gene duplication was not a strict requirement for the acquisition of novel subcellular targeting, 40% of novel plastid-targeted genes were found to be most closely related to a sequence within the same genome, and of these, 30.5% resulted from alternative transcription or translation initiation sites. Interestingly, analysis of the distribution of amino acids in the transit peptides of known and predicted chloroplast-targeted proteins revealed monocot and eudicot-specific preferences in residue distribution.

New insights into Arabidopsis transcriptome complexity revealed by direct sequencing of native RNAs.

Arabidopsis thaliana transcriptomes have been extensively studied and characterized under different conditions. However, most of the current ‘RNA-sequencing’ technologies produce a relatively short read length and demand a reverse-transcription step, preventing effective characterization of transcriptome complexity. Here, we performed Direct RNA Sequencing (DRS) using the latest Oxford Nanopore Technology (ONT) with exceptional read length. We demonstrate that the complexity of the A. thaliana transcriptomes has been substantially under-estimated. The ONT direct RNA sequencing identified novel transcript isoforms at both the vegetative (14-day old seedlings, stage 1.04) and reproductive stages (stage 6.00–6.10) of development. Using in-house software called TrackCluster, we determined alternative transcription initiation (ATI), alternative polyadenylation (APA), alternative splicing (AS), and fusion transcripts. More than 38 500 novel transcript isoforms were identified, including six categories of fusion-transcripts that may result from differential RNA processing mechanisms. Aided by the Tombo algorithm, we found an enrichment of m5C modifications in the mobile mRNAs, consistent with a recent finding that m5C modification in mRNAs is crucial for their long-distance movement. In summary, ONT DRS offers an advantage in the identification and functional characterization of novel RNA isoforms and RNA base modifications, significantly improving annotation of the A. thaliana genome.

Targeting of parvulin interactors by diazirine mediated cross-linking discloses a cellular role of human Par14/17 in actin polymerization.

The peptidyl-prolyl cis/trans isomerases (PPIases) Parvulin 14 (Par14) and Parvulin 17 (Par17) result from alternative transcription initiation of the PIN4 gene. Whereas Par14 is present in all metazoan, Par17 is only expressed in Hominidae. Par14 resides mainly within the cellular nucleus, while Par17 is translocated into mitochondria. Using photo-affinity labeling, cross-linking and mass spectrometry (MS) we identified binding partners for both enzymes from HeLa lysates and disentangled their cellular roles. Par14 is involved in biogenesis of ribonucleoprotein (RNP)-complexes, RNA processing and DNA repair. Its elongated isoform Par17 participates in protein transport/translocation and in cytoskeleton organization. Nuclear magnetic resonance (NMR) spectroscopy reveals that Par17 binds to β-actin with its N-terminal region, while both parvulins initiate actin polymerization depending on their PPIase activity as monitored by fluorescence spectroscopy. The knockdown (KD) of Par17 in HCT116 cells results in a defect in cell motility and migration.