Gene Regulatory Functions Research Articles

A comprehensive review of cancer: CRISPR and its application in lung cancer with a potential in overcoming EGFR mutations

As a comprehensive review of cancer, oncogenes, and CRISPR, this review explores the transformative potential of CRISPR technology in combating cancer, particularly focusing on lung cancer and oncogenes. CRISPR’s precision gene-editing capabilities offer a promising avenue for developing more targeted cancer therapies, overcoming the limitations of current treatments such as chemotherapy and radiation, which often fail due to cancer cell heterogeneity and resistance. Additionally, this review proposes the potential use of CRISPR to target specific oncogenes like EGFR and its significant promise for improving treatment outcomes in lung cancer by directly modifying or regulating gene function. This review synthesizes current research, highlighting the integration of CRISPR with other therapeutic modalities to enhance the effectiveness of cancer treatments, delivery systems that improve targeting and minimize off-target effects, and the future directions in CRISPR-mediated lung cancer therapy.

METTL3 drives heart failure by regulating Spp1 and Fos m6A modification in myocardial infarction

Abstract Background While m6A modification has been reported in myocardial infarction (MI), the detailed mechanism by which METTL3 regulates the progression of the disease has not yet been elucidated, and it remains unclear why m6A modification increases after MI. Purpose To investigate the role and mechanism of METTL3 in regulating its targets through m6A methylation in MI. The research results will not only add new content to the basic mechanism of myocardial protection but also provide new ideas and new targets for the prevention and treatment of MI. Methods Through MeRIP-seq and extensive bioinformatics analysis, the target genes SPP1 and FOS with the most significant m6A modification and differential expression in MI were screened. We successfully constructed heart specific Mettl3 knockout mice (Mettl3CKO) to verify that METTL3 promotes the deterioration of cardiac function after MI. We performed complementary molecular methods to assess protein quantity and interactions to identify mechanisms regulating this response. We manipulated select molecular pathways using both genetic and pharmacological methods to validate these mechanisms. Results Here, we showed that METTL3 exerted methyltransferase activity-dependent functions in gene regulation in MI, and a significant transcription factor HuR assisted the function of METTL3. and demonstrated that METTL3 was critical for the promotion of heart failure after MI. More specifically, METTL3 directly interacted with HuR through its nuclear localization domain in the cell nucleus under normoxia condition. When hypoxia developed, METTL3 separated from HuR and deposited m6A into 5’UTR of Spp1 and Fos mRNA to maintain their stability. In contrast, HuR bound to the ARE domain of 3’UTR of Spp1 and Fos mRNA to take them to the cytosol, maintaining their stability. Moreover, HIF-1α directly interacted with the HRE domain of Mettl3 to promote its transcription, and HuR bound to the ARE domain of 3’UTR of Mettl3 mRNA to maintain its stability to promote following translation. Conclusions Collectively, our studies revealed previously unappreciated functions of METTL3 with the help of HuR, and a direct target of HIF-1α under normoxia condition, which together contribute to its essential function in MI, suggesting therapeutic potential for targeting the METTL3/HuR/Spp1(Fos) mRNA axis.Figure1

Gene polymorphisms of miR-323b and miR-1343 that regulate kininogen L are associated with schizophrenia susceptibility: A preliminary population‑based study.

miR-SNP is a type of functional single nucleotide polymorphism (SNP) that affects the regulatory functions of miRNA genes, miRNA binding sites, or components of miRNA biogenesis. This study aimed to explore the relationship between miRNA gene polymorphisms that regulate the kininogen L protein and schizophrenia (SCZ). Bioinformatics methods predicted miRNA gene polymorphism sites regulating the kininogen L protein. The polymorphisms of rs56103835, rs6513496, rs651349, and rs2986407 were detected using imLDR multiple SNP typing technologies in 513 SCZ patients and 509 controls. The association of miR-SNP variations with SCZ susceptibility and symptoms was evaluated using SNPstat to determine the optimal inheritance model. Generalized multifactor dimensionality reduction (GMDR) analysis and logistic regression were used to calculate miR-SNP interactions. The association between hsa-miR-323b-rs56103835 and SCZ was statistically significant under the dominant model. The result of gene-gene interaction showed that the three-factor model (rs56103835 / rs2986407 / rs2155248) was the best, but it could not be considered significantly related to SCZ. Additionally, SCZ patients with the CC or CT genotype on rs2986407 were more likely to experience auditory hallucinations than those with the TT genotype. Our data revealed that the mutation of hsa-miR-323b-rs56103835 from C to T was associated with susceptibility to SCZ. The mutation of hsa-miR-1343-rs2986407 from T to C increases the risk of auditory hallucinations in SCZ patients.

De novo genome assemblies of two cryptodiran turtles with ZZ/ZW and XX/XY sex chromosomes provide insights into patterns of genome reshuffling and uncover novel 3D genome folding in amniotes.

Understanding the evolution of chromatin conformation among species is fundamental to elucidate the architecture and plasticity of genomes. Nonrandom interactions of linearly distant loci regulate gene function in species-specific patterns, affecting genome function, evolution, and, ultimately, speciation. Yet, data from nonmodel organisms are scarce. To capture the macroevolutionary diversity of vertebrate chromatin conformation, here we generate de novo genome assemblies for two cryptodiran (hidden-neck) turtles via Illumina sequencing, chromosome conformation capture, and RNA-seq: Apalone spinifera (ZZ/ZW, 2n = 66) and Staurotypus triporcatus (XX/XY, 2n = 54). We detected differences in the three-dimensional (3D) chromatin structure in turtles compared to other amniotes beyond the fusion/fission events detected in the linear genomes. Namely, whole-genome comparisons revealed distinct trends of chromosome rearrangements in turtles: (1) a low rate of genome reshuffling in Apalone (Trionychidae) whose karyotype is highly conserved when compared to chicken (likely ancestral for turtles), and (2) a moderate rate of fusions/fissions in Staurotypus (Kinosternidae) and Trachemys scripta (Emydidae). Furthermore, we identified a chromosome folding pattern that enables "centromere-telomere interactions" previously undetected in turtles. The combined turtle pattern of "centromere-telomere interactions" (discovered here) plus "centromere clustering" (previously reported in sauropsids) is novel for amniotes and it counters previous hypotheses about amniote 3D chromatin structure. We hypothesize that the divergent pattern found in turtles originated from an amniote ancestral state defined by a nuclear configuration with extensive associations among microchromosomes that were preserved upon the reshuffling of the linear genome.

Atomistic Insights into Sequence-Mediated Spontaneous Association of Short RNA Chains.

RNA-RNA association and phase separation appear to be essential for the assembly of stress granules and underlie RNA foci formation in repeat expansion disorders. RNA molecules are found to play a significant role in gene-regulatory functions via condensate formation among themselves or with RNA-binding proteins. The interplay between driven versus spontaneous processes is likely to be an important factor for controlling the formation of RNA-mediated biomolecular condensate. However, the sequence-specific interactions and molecular mechanisms that drive the spontaneous RNA-RNA association and help to form RNA-mediated phase-separated condensate remain unclear. With microseconds-long atomistic molecular simulations here, we report how essential aspects of RNA chains, namely, base composition, metal ion binding, and hydration properties, contribute to the association of the series of simplest biologically relevant homopolymeric and heteropolymeric short RNA chains. We show that spontaneous processes make the key contributions governed by the sequence-intrinsic properties of RNA chains, where the definite roles of base-specific hydrogen bonding and stacking interactions are prominent in the association of the RNA chains. Purine versus pyrimidine contents of RNA chains can directly influence the association properties of RNA chains by modulating hydrogen bonding and base stacking interactions. This study determines the impact of ionic environment in sequence-specific spontaneous association of short RNA chains, hydration features, and base-specific interactions of Na+, K+, and Mg2+ ions with RNA chains.

7615 Genomic Consequences of GRHL2 Overexpression in ER+ Breast Cancer Cells

Abstract Disclosure: K.O. Allen: None. C. Zheng: None. N. Solodin: None. M.S. Ozers: Owner/Co-Owner; Self; Proteovista, LLC. C.L. Warren: None. P.E. Messa: None. D.T. Vogt: None. E.T. Alarid: None. Breast cancer progression is characterized by the presence and prevalence of metastatic disease. At a genomic level, metastasis can be facilitated via rearrangement of the chromatin landscape to expose oncogenic genes for transcription. Grainyhead like protein 2 (GRHL2) is a nuclear transcription factor implicated in epithelial cell differentiation. The GRHL2 motif has been found near activated estrogen receptor (ER) binding sites, and elevated levels of GRHL2 have been linked to worse prognosis in hormone receptor positive breast cancer patients. In its capacity to contribute to a more severe disease state when overexpressed, GRHL2 may function as either a transcription or pioneer factor to aid in the expression of metastasis-associated genes. Our lab previously showed that GRHL2 genomic binding precedes ER binding at co-regulated binding sites. Also, overexpression of GRHL2 in MCF7 cells for 24 hours did not appreciably alter gene expression by RNA-Seq. These data taken together indicate that GRHL2 may be exhibiting pioneer factor activity. However, the specific role of GRHL2 overexpression in hormone positive breast cancer progression remains unknown. In order to examine the mechanism of GRHL2 action, GRHL2 genomic binding was investigated using a tetracycline-inducible MCF7 model that allows controlled overexpression of GRHL2. ChIP studies were performed to determine GRHL2 binding intensity at specific and genome wide GRHL2 binding sites. Specific focus was given to established GRHL2 binding sites near oncogenic genes responsible for epithelial to mesenchymal transition (EMT), dormancy, and proto-oncogenic behavior. A similar analysis examined H3K27 acetylation to validate active gene transcription. An orthogonal technology, Specificity and Affinity for Protein (SNAP) microarrays, which characterizes direct GRHL2 binding to tiled genomic regions identified in ChIP-Seq analyses from multiple labs, was utilized to discriminate direct and indirect GRHL2 binding sites. The requirement for GRHL2 transactivation function in regulation of EMT related genes was further interrogated using transactivation GRHL2 dominant negative mutants. This work aims to distinguish the transcriptional and non-transcriptional activities of GRHL2 in breast cancer cells on a genome wide level and thereby, provide a deeper understanding of how overexpressed GRHL2 contributes to hormone positive breast cancer. Presentation: 6/3/2024

9366 The Rnf20 Histone Modifier Is A Crucial Mediator Of Pancreatic Beta Cell Identity And Function

Abstract Disclosure: T.H. Pierre: None. Y. Liu: None. M.M. Bethea: None. K. Coutinho: None. C. Hunter: None. Diabetes is characterized by the loss of pancreatic β-cell mass. As diabetes incidence continues to elevate, it is critical that we develop a better understanding of the factors that define functional β-cells in order to improve current therapeutics. One approach is through the study of transcription factors (TFs), which are essential for β-cell development and function. Prior work from our lab and others has examined Islet-1 (Isl1) a LIM-homeodomain TF that is required for the maturation of the endocrine pancreas and adult β-cell function. We have also extensively characterized several Isl1-interacting co-regulators (e.g., Ldb1 and SSBP3) that facilitate its roles in maintaining glucose homeostasis. Most recently, we have identified that Isl1 interacts with the ubiquitin ligases Ring Finger 20 (Rnf20) and Rnf40, whose histone H2B monoubiquitation (H2Bub1) activity support the gene regulatory functions of Isl1. With in vitro experiments, we demonstrated that Rnf20 and Rnf40 are important for glucose stimulated insulin secretion (GSIS) and mediate the expression of β-cell identity genes. Based on these findings, we hypothesize that Isl1-Rnf complexes modify histones to regulate β-cell gene expression and function. To address this hypothesis in vivo, we developed an adult inducible β-cell knockout of Rnf20 (Rnf20Δβ-cell). Rnf20Δβ-cell mice display fasting hyperglycemia and severe glucose intolerance that is driven by impaired GSIS, reduced insulin content, and the dysregulation of critical β-cell genes including Ins1, Ucn3, and Slc2a2. Additionally, loss of Rnf20 perturbs insulin processing and overall β-cell maturation as observed by measurement of proinsulin:insulin ratios, C-peptide levels, and whole islet RNA-seq. Phenotypes exhibited by Rnf20Δβ-cell mice are similar to those observed in Isl1 β-cell knockout mice, and comparative transcriptomics revealed an overlap of 570 genes involved in metabolism, zinc homeostasis, and islet proliferation including Slc30a8 and Matn2, both of which we found are occupied by Isl1 and Rnf20. Collectively, our findings demonstrate the importance of the Rnf20 histone modifier in regulating β-cell function and expand our understanding of the Isl1 regulatory network. Presentation: 6/2/2024

Plasmodium berghei HMGB1 controls the host immune responses and splenic clearance by regulating the expression of pir genes

High mobility group box (HMGB) proteins belong to high mobility group (HMG) superfamily of non-histone nuclear proteins that are involved in chromatin remodeling, regulation of gene expression and DNA repair. When extracellular, HMGBs serve as alarmins inducing inflammation and this is attributed to the proinflammatory activity of box B. Here, we show that Plasmodium HMGB1 has key amino acid changes in box B resulting in the loss of TNF-α stimulatory activity. Site-directed mutagenesis of the critical amino acids in box B with respect to mouse HMGB1 renders recombinant Plasmodium berghei (Pb) HMGB1 capable of inducing TNF-α release. Targeted deletion of PbHMGB1 and a detailed in vivo phenotyping show that PbHMGB1 knockout (KO) parasites can undergo asexual stage development. Interestingly, Balb/c mice-infected with PbHMGB1KO parasites display a protective phenotype with subsequent clearance of blood parasitemia, and develop long-lasting protective immunity against the challenges performed with Pb wildtype parasites. The characterization of splenic responses show prominent germinal centres leading to effective humoral responses and enhanced T follicular helper cells. There is also a complete protection from experimental cerebral malaria in CBA/CaJ mice susceptible for cerebral pathogenesis with subsequent parasite clearance. Transcriptomic studies suggest the involvement of PbHMGB1 in pir expression. Our findings highlight the gene regulatory function of parasite HMGB1 and its in vivo significance in modulating the host immune responses. Further, clearance of asexual stages in PbHMGB1KO-infected mice underscores the important role of parasite HMGB1 in host immune evasion. These findings have implications in developing attenuated blood-stage vaccine for malaria.

Comparative Analysis of Circular RNAs Expression and Function between Aortic and Intracranial Aneurysms.

An aneurysm is an abnormal enlargement or bulging of the wall of a blood vessel. Most often, aneurysms occur in large blood vessels - the aorta (Thoracic Aortic Aneurysm (TAA) and Abdominal Aortic Aneurysm (AAA) and brain vessels (Intracranial Aneurysm (IA)). Despite the presence of significant differences in the pathogenesis of the development and progression of IA and TAA/AAA, there are also similarities. For instance, both have been shown to be strongly influenced by shear stress, inflammatory processes, and enzymatic destruction of the elastic lamellae and extracellular matrix (ECM) proteins of the vascular wall. Moreover, although IA and TAA are predominantly considered arteriopathies with different pathological mechanisms, they share risk factors with AAA, such as hypertension and smoking. However, there is a need for a more in- -depth study of the key elements that may influence the formation and progression of a particular aneurysm to find ways of therapeutic intervention or search for a diagnostic tool. Today, it is known that the disruption of gene expression is one of the main mechanisms that contribute to the development of aneurysms. At the same time, growing evidence suggests that aberrant epigenetic regulation of gene function is strongly related to the genesis of aneurysms. Although much has been studied of the known protein-coding genes, circular RNAs (circRNAs), a relatively new and rapidly evolving large family of transcripts, have recently received much scientific attention. CircRNAs regulate gene expression through the sponging of microRNAs (miRNAs) and can also be used as therapeutic targets and biomarkers. Increasing evidence has implicated circRNAs in the pathogenesis of multiple cardiovascular diseases, including the development of aneurysms. However, the mechanism of dysregulation of certain circRNAs in a particular aneurysm remains to be studied. The discovery of circRNAs has recently advanced our understanding of the latest mode of miRNAs/target genes regulation in the development and progression of IA and TAA/AAA. The aim of this study is to compare the expression profiles of circRNAs to search for similar or different effects of certain circRNAs on the formation and progression of IA and TAA/AAA.

In Situ Intracellular Autocatalytic Hairpin Assembly of the Y-Shaped DNA Nanostructure for miR-155 Sensing and Gene Silencing.

miR-155 is a class of cancer markers closely related to cancer metastasis and invasion. Combining in situ detection with gene silencing not only helps to analyze the information on the abundance and spatial location of microRNA expression in the cell but also synergizes the therapy. In this work, we prepared HD@CM vesicles with three hairpin DNAs by using MCF-7 cell membranes. The hairpin DNAs can be triggered by endogenous miR-155, which opens the autocatalytic molecular circuit (ACHA) and obtains Y-shaped DNA nanostructures. This nanostructure not only detects endogenous miR-155 with high sensitivity for in situ imaging but also enables gene regulation of intracellular survivin mRNA. The levels of miR-155 in MDA-MB-231, MCF-7, Hela, and HEK-293T cells are found to be 7703, 3978, 1696, and 1229 copies/cell, respectively, as detected by HD@CMs. The fluorescence produced by HD@CM after coincubation with different cells is found to be proportional to the intracellular miR-155 content by confocal imaging. In addition, the gene regulatory function of the Y-shaped DNA structure resulted in significant inhibition of survivin protein expression and apoptosis rates of up to 83%. We look forward to the future application of our HD@CM platform for the precise diagnosis and programmable treatment of clinical cancers.

Enhancers and super-enhancers as master regulators in cancer

Gene expression regulation is one of the most fundamental cellular processes, enabling the activation of a gene to produce either the translatable protein-coding transcript (mRNA) or a functional non-coding RNA with gene regulatory functions, ultimately determining cell identity and function. Although gene expression regulation can occur at transcriptional, translational, and post-translational levels, transcription initiation is the first and the most important step in gene expression, facilitating the transfer of biological information from DNA to protein. Enhancers and super-enhancers are among the master regulators of tissue- and cell-specific transcription regulation involved in cell differentiation and tumor formation. Despite four decades passing since the first discovery of enhancers in eukaryotes and extensive efforts undertaken to identify enhancers on a genomic scale during the last decade, the discovery of enhancers still faces certain limitations and needs further investigation. The perturbation of enhancer function due to genetic or epigenetic changes is closely linked to a range of human disorders, including the development and progression of cancers. Thus, the detection of early cancer-related enhancer activity and the subsequent normalization of expression abnormalities using enhancer-targeting CRISPR epigenetic editing, as well as enhancer-targeting pharmaceuticals, are regarded as groundbreaking therapeutic tactics in preclinical stages.

DWARF27 and CAROTENOID CLEAVAGE DIOXYGENASE 7 genes regulate release, germination and growth of gemma in Marchantia polymorpha.

Strigolactones (SLs), a class of carotenoid-derived hormones, play a crucial role in flowering plants by regulating underground communication with symbiotic arbuscular mycorrhizal fungi (AM) and controlling shoot and root architecture. While the functions of core SL genes have been characterized in many plants, their roles in non-tracheophyte plants like liverworts require further investigation. In this study, we employed the model liverwort species Marchantia polymorpha, which lacks detectable SL production and orthologs of key SL biosynthetic genes, including CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) and MORE AXILLARY GROWTH 1 (MAX1). However, it retains some SL pathway components, including DWARF27 (D27) and CCD7. To help elucidate the function of these remaining components in M. polymorpha, knockout mutants were generated for MpD27-1, MpD27-2 and MpCCD7. Phenotypic comparisons of these mutants with the wild-type control revealed a novel role for these genes in regulating the release of gemmae from the gemma cup and the germination and growth of gemmae in the dark. Mpd27-1, Mpd27-2, and Mpccd7 mutants showed lower transcript abundance of genes involved in photosynthesis, such as EARLY LIGHT INDUCED (ELI), and stress responses such as LATE EMBRYOGENESIS ABUNDANT (LEA) but exhibited higher transcript levels of ETHYLENE RESPONSE FACTORS (ERFs) and SL and carotenoid related genes, such as TERPENE SYNTHASE (TS), CCD7 and LECITHIN-RETINAL ACYL TRANSFERASE (LRAT). Furthermore, the mutants of M. polymorpha in the SL pathway exhibited increased contents of carotenoid. This unveils a previously unrecognized role for MpD27-1, MpD27-2 and MpCCD7 in controlling release, germination, and growth of gemmae in response to varying light conditions. These discoveries enhance our comprehension of the regulatory functions of SL biosynthesis genes in non-flowering plants.

Viral miRNA delivered by exosomes from Marek's disease virus-transformed lymphoma cell line exerts regulatory function in internalized primary chicken embryo fibroblast cells

In the past decade, research has demonstrated that viral miRNAs encoded by a number of viral genomes, particularly by most of the herpesvirus including Marek's disease virus (MDV), play important regulatory roles in viral infection, replication, and regulation of tumorigenesis. As macrovesicles in cells, exosomes can deliver viral miRNAs and exert gene regulatory functions. Whether the exosomes play a role in the replication, pathogenesis/tumorigenesis of avian herpesviruses such as oncogenic Marek's disease virus (MDV) remains unclear. Herein we extracted and identified the exosomes from MDV-transformed T cell line MSB-1 and demonstrated high abundance of MDV-1 miRNA expression. Using dual luciferase-based reporter assay, we also demonstrated that the exosomes derived from MSB-1 can deliver functional miRNA successfully into primary chicken embryo fibroblasts. These findings provide new insights into the role of exosomes and the mechanisms of how virus-encoded miRNA function in MDV latency/activation switching, viral replication, pathogenesis and/or tumorigenesis.

ETMR-26. MIR-34/449 FAMILY PLAYS A CRUCIAL ROLE IN CHOROID PLEXUS MULTICILIATION AND TUMORIGENESIS

Abstract BACKGROUND Choroid plexus (CP), an epithelial structure within the brain ventricles, produces cerebrospinal fluid and serves barrier functions in the brain. Primary CP neoplasms are rare intracranial tumors that predominantly occurs in childhood. Recent studies revealed multiciliated cells in the CP in humans and mice. In contrast, benign CP papilloma, and malignant CP carcinoma (CPC) in humans are characterized by solitary cilia, and disturbances to multiciliation program directed by transcription factor GMNC. Additionally, most CPC in humans carry mutations in TP53 tumor suppressor. Consistently, Trp53-deficient CPC in mice display singular cilia consequent to defects in GMNC multiciliation program. Thus, the impairment of multiciliation enables tumor cell proliferation. MicroRNAs (miRNAs) are non-coding small RNAs with gene regulatory functions. Specifically, the mir-34/449 family comprises of six miRNAs collaborate to influence multiciliation in diverse tissues but role of mir-34/449 family in the brain is poorly understood. Knowledge of multiciliation program regulation may provide crucial insights into tumor suppression mechanisms in CPC. METHODS To investigate the role of mir-34/449 family in the CP, human CP organoid and mir-34/449 mutant animals were used. RNAscope, In situ hybridization as well as immunofluorescence staining was utilized to identify gene expression in tissues. RESULTS Preliminary studies revealed GMNC program activity and multiciliated cells in CP organoid derived from human induced pluripotent stem cells. Disruption of the mir-34/449 family led to supernumerary cilia and ectopic multiciliation program activity in the CP. Importantly, Trp53-deficient CPC exhibited distinct expression of the mir-34/449 family. Further, sequencing studies revealed an overlap of differentially expressed miRNAs in CP tumors in humans and mice, with an enrichment of the mir-34/449 family. CONCLUSIONS Together, these findings indicate that the mir-34/449 family regulates multiciliation in the CP, whereas the study of its interactions with the multiciliation program holds the key to understanding multiciliation as a tumor suppression pathway in CPC.

Identification of antennal alternative splicing by combining genome and full-length transcriptome analysis in Bactrocera dorsalis.

Alternative splicing is an essential post-transcriptional regulatory mechanism that diversifies gene function by generating multiple protein isoforms from a single gene and act as a crucial role in insect environmental adaptation. Olfaction, a key sense for insect adaptation, relies heavily on the antennae, which are the primary olfactory organs expressing most of the olfactory genes. Despite the extensive annotation of olfactory genes within insect antennal tissues facilitated by high-throughput sequencing technology advancements, systematic analyses of alternative splicing are still relatively less. In this study, we focused on the oriental fruit fly (Bactrocera dorsalis), a significant pest of fruit crops. We performed a detailed analysis of alternative splicing in its antennae by utilizing the full-length transcriptome of its antennal tissue and the insect's genome. The results revealed 8600 non-redundant full-length transcripts identified in the oriental fruit fly antennal full-length transcriptome, spanning 4,145 gene loci. Over 40% of these loci exhibited multiple isoforms. Among these, 161 genes showed sex-biased isoform switching, involving seven different types of alternative splicing. Notably, events involving alternative transcription start sites (ATSS) and alternative transcription termination sites (ATTS) were the most common. Of all the genes undergoing ATSS and ATTS alternative splicing between male and female, 32 genes were alternatively spliced in protein coding regions, potentially affecting protein function. These genes were categorized based on the length of the sex-biased isoforms, with the highest difference in isoform fraction (dIF) associated with the ATSS type, including genes such as BdorABCA13, BdorCAT2, and BdorTSN3. Additionally, transcription factor binding sites for doublesex were identified upstream of both BdorABCA13 and BdorCAT2. Besides being expressed in the antennal tissues, BdorABCA13 and BdorCAT2 are also expressed in the mouthparts, legs, and genitalia of both female and male adults, suggesting their functional diversity. This study reveals alternative splicing events in the antennae of Bactrophora dorsalis from two aspects: odorant receptor genes and other types of genes expressed in the antennae. This study not only provides a research foundation for understanding the regulation of gene function by alternative splicing in the oriental fruit fly but also offers new insights for utilizing olfaction-based behavioral manipulation techniques to manage this pest.

LINE-1 transcription activates long-range gene expression.

Long interspersed nuclear element-1 (LINE-1 or L1) is a retrotransposon group that constitutes 17% of the human genome and shows variable expression across cell types. However, the control of L1 expression and its function in gene regulation are incompletely understood. Here we show that L1 transcription activates long-range gene expression. Genome-wide CRISPR-Cas9 screening using a reporter driven by the L1 5' UTR in human cells identifies functionally diverse genes affecting L1 expression. Unexpectedly, altering L1 expression by knockout of regulatory genes impacts distant gene expression. L1s can physically contact their distal target genes, with these interactions becoming stronger upon L1 activation and weaker when L1 is silenced. Remarkably, L1s contact and activate genes essential for zygotic genome activation (ZGA), and L1 knockdown impairs ZGA, leading to developmental arrest in mouse embryos. These results characterize the regulation and function of L1 in long-range gene activation and reveal its importance in mammalian ZGA.

Exploring functional conservation in silico: a new machine learning approach to RNA-editing.

Around 50years ago, molecular biology opened the path to understand changes in forms, adaptations, complexity, or the basis of human diseases through myriads of reports on gene birth, gene duplication, gene expression regulation, and splicing regulation, among other relevant mechanisms behind gene function. Here, with the advent of big data and artificial intelligence (AI), we focus on an elusive and intriguing mechanism of gene function regulation, RNA editing, in which a single nucleotide from an RNA molecule is changed, with a remarkable impact in the increase of the complexity of the transcriptome and proteome. We present a new generation approach to assess the functional conservation of the RNA-editing targeting mechanism using two AI learning algorithms, random forest (RF) and bidirectional long short-term memory (biLSTM) neural networks with an attention layer. These algorithms, combined with RNA-editing data coming from databases and variant calling from same-individual RNA and DNA-seq experiments from different species, allowed us to predict RNA-editing events using both primary sequence and secondary structure. Then, we devised a method for assessing conservation or divergence in the molecular mechanisms of editing completely in silico: the cross-testing analysis. This novel method not only helps to understand the conservation of the editing mechanism through evolution but could set the basis for achieving a better understanding of the adenosine-targeting mechanism in other fields.

Identification of SMC2 and SMC4 as prognostic markers in breast cancer through bioinformatics analysis.

Breast cancer (BRCA) is one of the most common malignant tumors. The structural maintenance of chromosome (SMC) gene family has been shown to play an important role in human cancers. However, the role of SMC families in BRCA is unclear. This study aimed to explore the role and potential clinical value of whole SMCs in BRCA. TIMER and UALCAN database were used to analysis the expression level. Genetic variations were analyzed by cBioPortal. Promoter methylation and protein level were analyzed by UCLCAN. GO and KEGG were analyzed by Metascape database. Prognostic value of SMCs was analyzed by Kaplan-Meier and multivariate cox regression analyses. Immune infiltration analysis was conducted by CIBERSORT. Immunotherapy outcome prediction was conducted by Cancer Immunome Atlas. Targeted drug therapy outcome prediction was taken by GDSC and R language. The cell viability was tested by CCK8 and migration was tested by wound healing assay. Xenograft model was used to investigate the in vivo role of SMC2. Expression levels of SMC1A, SMC2, SMC4, SMC5 and SMC6 mRNA were increased in BRCA tissues, and negatively correlated with promoter methylation. Overexpression of SMC2 and SMC4 was negatively correlated with survival. Function of SMCs family regulatory genes was mainly related to ATPase activity. Expression of most SMCs was negatively correlated with immunotherapy and drug therapy outcomes. Interfere SMC2 and SMC4 decreased IC50 values of 5-fluorouracil and oxaliplatin and inhibited the migration of MCF7 cells. Tumor growth and weights were significantly decreased in si-SMC2 groups. Combined bioinformatics and clinical specimen analysis verified SMC2 and SMC4 as independent prognostic factors in BRCA, suggesting their significance for the diagnosis and treatment of BRCA.

ProkDBP: Toward more precise identification of prokaryotic DNA binding proteins.

Prokaryotic DNA binding proteins (DBPs) play pivotal roles in governing gene regulation, DNA replication, and various cellular functions. Accurate computational models for predicting prokaryotic DBPs hold immense promise in accelerating the discovery of novel proteins, fostering a deeper understanding of prokaryotic biology, and facilitating the development of therapeutics targeting for potential disease interventions. However, existing generic prediction models often exhibit lower accuracy in predicting prokaryotic DBPs. To address this gap, we introduce ProkDBP, a novel machine learning-driven computational model for prediction of prokaryotic DBPs. For prediction, a total of nine shallow learning algorithms and five deep learning models were utilized, with the shallow learning models demonstrating higher performance metrics compared to their deep learning counterparts. The light gradient boosting machine (LGBM), coupled with evolutionarily significant features selected via random forest variable importance measure (RF-VIM) yielded the highest five-fold cross-validation accuracy. The model achieved the highest auROC (0.9534) and auPRC (0.9575) among the 14 machine learning models evaluated. Additionally, ProkDBP demonstrated substantial performance with an independent dataset, exhibiting higher values of auROC (0.9332) and auPRC (0.9371). Notably, when benchmarked against several cutting-edge existing models, ProkDBP showcased superior predictive accuracy. Furthermore, to promote accessibility and usability, ProkDBP (https://iasri-sg.icar.gov.in/prokdbp/) is available as an online prediction tool, enabling free access to interested users. This tool stands as a significant contribution, enhancing the repertoire of resources for accurate and efficient prediction of prokaryotic DBPs.

Genomic Analysis of Aedes aegypti in the Northern Chihuahuan Desert of Texas and Mexico.

Background: Aedes aegypti, is the primary vector of dengue, Chikungunya, Zika, and yellow fever viruses. Both natural and human-impacted landscapes have selective pressures on Ae. aegypti, resulting in strong genomic structure even within close geographical distances. Materials and Methods: We assess the genetic structure of this medically important mosquito species at the northern leading edge of their distribution in Southwestern USA. Ae. aegypti were collected during 2017 in the urban communities of El Paso and Sparks, Texas (USA) and in the city of Ciudad Juárez, Mexico. Results: Thousands of nuclear loci were sequenced across 260 captured Ae. aegypti. First, we recovered the genetic structure of Ae. aegypti following geography, with all four major collection communities being genetically distinct. Importantly, we found population structure and genetic diversity that suggest rapid expansion through active-short distance dispersals, with Anapra being the likely source for the others. Next, tests of selection recovered eight functional genes across six outliers: calmodulin with olfactory receptor function; the protein superfamily C-type lectin with function in mosquito immune system and development; and TATA box binding protein with function in gene regulation. Conclusion: Despite these populations being documented in the early 2000s, we find that selective pressures on specific genes have already occurred and likely facilitate Ae. aegypti range expansion.