Sequence Motifs Research Articles

Phosphoproteomic analysis reveals CDK5-Mediated phosphorylation of MTDH inhibits protein synthesis in microglia

CDK5 plays a crucial role in maintaining normal central nervous system (CNS) development and synaptic function, while microglia are the primary immune cells present in the CNS and play vital physiological roles in CNS development, immune surveillance, and regulation of synaptic plasticity. Despite this, our understanding of both the substrate proteins and functional mechanisms of CDK5 in microglia remains limited. To address this, we utilized CRISPR-Cas9 knockout of Cdk5 in BV2 cells and conducted quantitative phosphoproteomics analysis to systematically screen potential CDK5 substrates in microglia. Our findings identified 335 phosphorylation sites on 234 proteins as potential CDK5 substrates in microglia based on the reported sequence motif. Through in vitro kinase assay and intracellular inhibition and knockout of CDK5 experiments, we confirmed that ER proteins MTDH (protein LYRIC) and Calnexin are novel substrate proteins of CDK5. Moreover, we demonstrated for the first time a critical mechanism for regulating protein synthesis in microglia, that the phosphorylation of S565 site on MTDH, a key protein mediating cell growth, by CDK5 inhibits protein synthesis. Our data provide valuable insights for the discovery of new substrate proteins of CDK5 and the in-depth investigation of the function and mechanism of CDK5 in microglia.

Therapeutic applications of RNA nanostructures.

RNA-based therapeutics have gained wide public interest in recent years. RNA is a versatile molecule that exists in many forms including mRNA, siRNA, miRNA, ribozymes, and other non-coding RNAs and is primarily applied for gene therapy. RNA is also used as a modular building block to construct RNA nanostructures. The programmable nature of RNA nanostructures enables the generation of simple, modulable, and multi-functional RNA-based therapeutics. Although the therapeutic application of RNA may be limited due to its structural instability, advances in RNA nanotechnology have improved the stability of RNA nanostructures for greater application. Various strategies have been developed to enhance the stability of RNA nanostructures enabling their application in vivo. In this review, we examine the therapeutic applications of RNA nanostructures. Non-immunogenic RNA nanostructures can be rationally designed with functional RNA molecules to modulate gene expression for gene therapy. On the other hand, nucleic acids can be sensed by cellular receptors to elicit an innate immune response, for which certain DNA and RNA motifs can function as adjuvants. Taking advantage of this adjuvant potential, RNA nanostructures can be used for immunotherapy and be designed for cancer vaccines. Thus, we examine the therapeutic application of immunogenic RNA nanostructures for cancer immunotherapy. RNA nanostructures represent promising platforms to design new nanodrugs, gene therapeutics, immunotherapeutic adjuvants, and cancer vaccines. Ongoing research in the field of RNA nanotechnology will continue to empower the development of RNA nanostructure-based therapeutics with high efficacy and limited toxicity.

Unsupervised decomposition of natural monkey behavior into a sequence of motion motifs

Nonhuman primates (NHPs) exhibit complex and diverse behavior that typifies advanced cognitive function and social communication, but quantitative and systematical measure of this natural nonverbal processing has been a technical challenge. Specifically, a method is required to automatically segment time series of behavior into elemental motion motifs, much like finding meaningful words in character strings. Here, we propose a solution called SyntacticMotionParser (SMP), a general-purpose unsupervised behavior parsing algorithm using a nonparametric Bayesian model. Using three-dimensional posture-tracking data from NHPs, SMP automatically outputs an optimized sequence of latent motion motifs classified into the most likely number of states. When applied to behavioral datasets from common marmosets and rhesus monkeys, SMP outperformed conventional posture-clustering models and detected a set of behavioral ethograms from publicly available data. SMP also quantified and visualized the behavioral effects of chemogenetic neural manipulations. SMP thus has the potential to dramatically improve our understanding of natural NHP behavior in a variety of contexts.

Abundant mRNA m1A modification in dinoflagellates: a new layer of gene regulation

Dinoflagellates, a class of unicellular eukaryotic phytoplankton, exhibit minimal transcriptional regulation, representing a unique model for exploring gene expression. The biosynthesis, distribution, regulation, and function of mRNA N1-methyladenosine (m1A) remain controversial due to its limited presence in typical eukaryotic mRNA. This study provides a comprehensive map of m1A in dinoflagellate mRNA and shows that m1A, rather than N6-methyladenosine (m6A), is the most prevalent internal mRNA modification in various dinoflagellate species, with an asymmetric distribution along mature transcripts. In Amphidinium carterae, we identify 6549 m1A sites characterized by a non-tRNA T-loop-like sequence motif within the transcripts of 3196 genes, many of which are involved in regulating carbon and nitrogen metabolism. Enriched within 3′UTRs, dinoflagellate mRNA m1A levels negatively correlate with translation efficiency. Nitrogen depletion further decreases mRNA m1A levels. Our data suggest that distinctive patterns of m1A modification might influence the expression of metabolism-related genes through translational control.

An Efficient Exact Algorithm for Planted Motif Search on Large DNA Sequence Datasets.

DNA motif is the pattern shared by similar fragments in DNA sequences, which plays a key role in regulating gene expression, and DNA motif discovery has become a key research topic. Exact planted ( l, d )-motif search (PMS) is one of the motif discovery approaches, which aims to find from t sequences all the ( l, d )-motifs that are motifs of l length appearing in at least qt sequences with at most d mismatches. The existing exact PMS algorithms are only suitable for small datasets of DNA sequences. The development of high-throughput sequencing technology generates vast amount of DNA sequence data, which brings challenges to solving exact PMS problems efficiently. Therefore, we propose an efficient exact PMS algorithm called PMmotif for large datasets of DNA sequences, after analyzing the time complexity of the existing exact PMS algorithms. PMmotif finds ( l, d )-motifs with strategy by searching the branches on the pattern tree that may contain ( l, d )-motifs. It is verified by experiments that the running time ratio of some existing excellent PMS algorithms to PMmotif is between 14.83 and 58.94. In addition, for the first time, PMmotif can solve the ( 15,5 )and ( 17,6 ) challenge problem instances on large DNA sequence datasets (3000 sequences of length 200) within 24 hours.

Replicative δ Polymerases from Plants and Animals Possess Very Similar Polymerase, Proofreading and Regulatory Domains

In eukaryotes, genome replication starts with the initiation of replication by the primases, followed by the synthesis of the leading- and lagging-strands by two different replicative DNA polymerases (pols), viz. ε and δ, respectively. The polymerase and proofreading (PR) active sites of the δ pols are analyzed from various animal and plant sources by multiple sequence alignment (MSA). The animal and plant δ pols are found to possess almost identical polymerase and PR domains. However, the BLASTp analysis has shown only 56.57% identity between the plant (Arabidopsis thaliana) and animal (human) δ pols. The template-binding pair (–YG-), the catalytic amino acid (K) and the nucleotide selection amino acid (Q) are found to be the same in both plant and animal δ pols. The δ pols from plant and animal sources contain a typical Mg2+-binding motif, (-YGDTD-) in the polymerase domain and 2 possible Zn2+-binding motifs (ZBMs) in their carboxy terminal domain (CTD). One of the ZBMs binds to the 4Fe-4S cluster and is suggested to be involved in the regulation of replication. Interestingly, the invariant –SLYPS- and -YGDTD- motifs which are found in the δ pols are not found in the other replicative pol ε. Furthermore, both animal and plant δ pols use the same PR exonuclease active site amino acids, and thus, belong to the DEDD(Y)-superfamily of exonucleases, as found in other DNA pols. Besides, many specialized, conserved sequence motifs are also identified and discussed.

G-quadruplex forming regions in GCK and TM6SF2 are targets for differential DNA methylation in metabolic disease and hepatocellular carcinoma patients

The alarming increase in global rates of metabolic diseases (MetDs) and their association with cancer risk renders them a considerable burden on our society. The interplay of environmental and genetic factors in causing MetDs may be reflected in DNA methylation patterns, particularly at non-canonical (non-B) DNA structures, such as G-quadruplexes (G4s) or R-loops. To gain insight into the mechanisms of MetD progression, we focused on DNA methylation and functional analyses on intragenic regions of two MetD risk genes, the glucokinase (GCK) exon 7 and the transmembrane 6 superfamily 2 (TM6SF2) intron 2-exon 3 boundary, which harbor non-B DNA motifs for G4s and R-loops.Pyrosequencing of 148 blood samples from a nested cohort study revealed significant differential methylation in GCK and TM6SF2 in MetD patients versus healthy controls. Furthermore, these regions harbor hypervariable and differentially methylated CpGs also in hepatocellular carcinoma versus normal tissue samples from The Cancer Genome Atlas (TCGA). Permanganate/S1 nuclease footprinting with direct adapter ligation (PDAL-Seq), native polyacrylamide DNA gel electrophoresis and circular dichroism (CD) spectroscopy revealed the formation of G4 structures in these regions and demonstrated that their topology and stability is affected by DNA methylation. Detailed analyses including histone marks, chromatin conformation capture data, and luciferase reporter assays, highlighted the cell-type specific regulatory function of the target regions. Based on our analyses, we hypothesize that changes in DNA methylation lead to topological changes, especially in GCK exon 7, and cause the activation of alternative regulatory elements or potentially play a role in alternative splicing.Our analyses provide a new view on the mechanisms underlying the progression of MetDs and their link to hepatocellular carcinomas, unveiling non-B DNA structures as important key players already in early disease stages.

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers.

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Deciphering the Language of Protein-DNA Interactions: A Deep Learning Approach Combining Contextual Embeddings and Multi-Scale Sequence Modeling

Deciphering the mechanisms governing protein-DNA interactions is crucial for understanding key cellular processes and disease pathways. In this work, we present a powerful deep learning approach that significantly advances the computational prediction of DNA-interacting residues from protein sequences.Our method leverages the rich contextual representations learned by pre-trained protein language models, such as ProtTrans, to capture intrinsic biochemical properties and sequence motifs indicative of DNA binding sites. We then integrate these contextual embeddings with a multi-window convolutional neural network architecture, which scans across the sequence at varying window sizes to effectively identify both local and global binding patterns.Comprehensive evaluation on curated benchmark datasets demonstrates the remarkable performance of our approach, achieving an area under the ROC curve (AUC) of 0.89 – a substantial improvement over previous state-of-the-art sequence-based predictors. This showcases the immense potential of pairing advanced representation learning and deep neural network designs for uncovering the complex syntax governing protein-DNA interactions directly from primary sequences.Our work not only provides a robust computational tool for characterizing DNA-binding mechanisms, but also highlights the transformative opportunities at the intersection of language modeling, deep learning, and protein sequence analysis. The publicly available code and data further facilitate broader adoption and continued development of these techniques for accelerating mechanistic insights into vital biological processes and disease pathways.In addition, the code and data for this work are available at https://github.com/B1607/DIRP.

Single cell expression and chromatin accessibility of the Toxoplasma gondii lytic cycle identifies AP2XII-8 as an essential ribosome regulon driver

Sequential lytic cycles driven by cascading transcriptional waves underlie pathogenesis in the apicomplexan parasite Toxoplasma gondii. This parasite’s unique division by internal budding, short cell cycle, and jumbled up classically defined cell cycle stages have restrained in-depth transcriptional program analysis. Here, unbiased transcriptome and chromatin accessibility maps throughout the lytic cell cycle are established at the single-cell level. Correlated pseudo-timeline assemblies of expression and chromatin profiles maps transcriptional versus chromatin level transition points promoting the cell division cycle. Sequential clustering analysis identifies functionally related gene groups promoting cell cycle progression. Promoter DNA motif mapping reveals patterns of combinatorial regulation. Pseudo-time trajectory analysis reveals transcriptional bursts at different cell cycle points. The dominant burst in G1 is driven largely by transcription factor AP2XII-8, which engages a conserved DNA motif, and promotes the expression of 44 ribosomal proteins encoding regulon. Overall, the study provides integrated, multi-level insights into apicomplexan transcriptional regulation.

Genome access is transcription factor-specific and defined by nucleosome position

Mammalian gene expression is controlled by transcription factors (TFs) that engage sequence motifs in a chromatinized genome, where nucleosomes can restrict DNA access. Yet, how nucleosomes affect individual TFs remains unclear. Here, we measure the ability of over one hundred TF motifs to recruit TFs in a defined chromosomal locus in mouse embryonic stem cells. This identifies a set sufficient to enable the binding of TFs with diverse tissue specificities, functions, and DNA-binding domains. These chromatin-competent factors are further classified when challenged to engage motifs within a highly phased nucleosome. The pluripotency factors OCT4-SOX2 preferentially engage non-nucleosomal and entry-exit motifs, but not nucleosome-internal sites, a preference that also guides binding genome wide. By contrast, factors such as BANP, REST, or CTCF engage throughout, causing nucleosomal displacement. This supports that TFs vary widely in their sensitivity to nucleosomes and that genome access is TF specific and influenced by nucleosome position in the cell.

TaCAMTA4 negatively regulates H2O2-dependent wheat leaf rust resistance by activating catalase 1 expression.

Leaf rust, caused by Puccinia triticina Erikss. (Pt), is a serious disease threatening wheat (Triticum aestivum L.) production worldwide. Hydrogen peroxide (H2O2) triggered by Pt infection in resistant wheat cultivars cause oxidative damage directly to biomolecules or is activated by calcium signaling and mediates the hypersensitive response. Calmodulin-binding transcriptional activator 4 (TaCAMTA4) has been reported to negatively regulate wheat resistance to Pt. In this study, we found that TaCAMTA4 was induced by Pt race 165 in its compatible host harboring the Pt resistant locus Lr26, TcLr26, and silencing of TaCAMTA4 increased local H2O2 accumulation and Pt resistance. Subcellular localization and autoactivation tests revealed that TaCAMTA4 is a nucleus-localized transcriptional activator. Furthermore, four DNA motifs recognized by TaCAMTA4 were identified by transcription factor-centered Y1H. Through analyzing the transcriptome database, four gene clusters were identified, each containing a different DNA motif on each promoter. Among them, the expression of catalase 1 (TaCAT1) with motif-1 was highly induced in the compatible interaction and was decreased when TaCAMTA4 was silenced. The results of EMSA, ChIP-qPCR, and RT-qPCR further showed that TaCAMTA4 directly bound motif-1 in the TaCAT1 promoter. Furthermore, silencing of TaCAT1 resulted in enhanced resistance to Pt and increased local H2O2 accumulation in wheat, which is consistent with that of TaCAMTA4. Since CAMTAs are Ca2+ sensors and catalases catalyze the decomposition of H2O2, we hypothesize that Ca2+ regulates the plant immune networks that are controlled by H2O2 and implicate a potential mechanism for Pt to suppress resistance by inducing the expression of the TaCAMTA4-TaCAT1 module, which consequently enhances H2O2 scavenging and attenuates H2O2-dependent resistance.

Novel function of U7 snRNA in the repression of HERV1/LTR12s and lincRNAs in human cells.

U7 snRNA is part of the U7 snRNP complex, required for the 3' end processing of replication-dependent histone pre-mRNAs in S phase of the cell cycle. Here, we show that U7 snRNA plays another function in inhibiting the expression of a subset of long terminal repeats of human endogenous retroviruses (HERV1/LTR12s) and LTR12-containing long intergenic noncoding RNAs (lincRNAs), both bearing sequence motifs that perfectly match the 5' end of U7 snRNA. We demonstrate that U7 snRNA inhibits LTR12 and lincRNA transcription and propose a mechanism in which U7 snRNA hampers the binding/activity of the NF-Y transcription factor to CCAAT motifs within LTR12 elements. Thereby, U7 snRNA plays a protective role in maintaining the silencing of deleterious genetic elements in selected types of cells.

DipR, a GntR/FadR-family transcriptional repressor: regulatory mechanism and widespread distribution of the dip cluster for dipicolinic acid catabolism in bacteria.

Dipicolinic acid is an essential component of bacterial spores for stress resistance, which is released into the environment after spore germination. In a previous study, a dip gene cluster was found to be responsible for the catabolism of dipicolinic acid in Alcaligenes faecalis JQ135. However, the transcriptional regulatory mechanism remains unclear. The present study characterized the new GntR/FadR family transcriptional factor DipR, showing that the dip cluster is transcribed as the six transcriptional units, dipR, dipA, dipBC, dipDEFG, dipHand dipJKLM. The purified DipR protein has six binding sites sharing the 6-bp conserved motif sequence 5'-GWATAC-3'. Site-directed mutations indicated that these motif sequences are essential for DipR binding. Moreover, the four key amino acid residues R63, R67, H196and H218 of DipR, examined by site-directed mutagenesis, played crucial roles in DipR regulation. Bioinformatics analysis showed that dip clusters including dipR genes are widely distributed in bacteria, are taxon-related, and co-evolved with their hosts. This paper provides new insights into the transcriptional regulatory mechanism of dipicolinic acid degradation by DipR in bacteria.

Robust Sequence Design Space for the Isothermal Exponential Amplification of Short Oligonucleotides.

Advances in isothermal amplification techniques have accelerated development in biosensing applications and the design of complex molecular devices. The exponential amplification reaction technique, or EXPAR, is uniquely positioned to process molecular information from short oligonucleotide strands (≈10nucleotides length) typically encountered in molecular computing or microRNA detection. Despite its conceptual simplicity (requiring only a template strand and two enzymes), the issue of nonspecific background amplification has hindered broader adoption. In this work, a new system configuration is established at 37 °C to achieve significantly improved performance. Critical sequence motifs responsible for the excellent signal-to-background profile are identified and generalized as a universal adapter design framework. Orthogonal template sequences generated from the framework are implemented for a triplex reaction and successfully evaluated mixtures of multiple-target inputs in a single-step, one-pot format without the need for exogenous agents.

The ICF syndrome protein CDCA7 harbors a unique DNA binding domain that recognizes a CpG dyad in the context of a non-B DNA.

CDCA7, encoding a protein with a carboxyl-terminal cysteine-rich domain (CRD), is mutated in immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, a disease related to hypomethylation of juxtacentromeric satellite DNA. How CDCA7 directs DNA methylation to juxtacentromeric regions is unknown. Here, we show that the CDCA7 CRD adopts a unique zinc-binding structure that recognizes a CpG dyad in a non-B DNA formed by two sequence motifs. CDCA7, but not ICF mutants, preferentially binds the non-B DNA with strand-specific CpG hemi-methylation. The unmethylated sequence motif is highly enriched at centromeres of human chromosomes, whereas the methylated motif is distributed throughout the genome. At S phase, CDCA7, but not ICF mutants, is concentrated in constitutive heterochromatin foci, and the formation of such foci can be inhibited by exogenous hemi-methylated non-B DNA bound by the CRD. Binding of the non-B DNA formed in juxtacentromeric regions during DNA replication provides a mechanism by which CDCA7 controls the specificity of DNA methylation.

Single-molecule structural and kinetic studies across sequence space.

At the core of molecular biology lies the intricate interplay between sequence, structure, and function. Single-molecule techniques provide in-depth dynamic insights into structure and function, but laborious assays impede functional screening of large sequence libraries. We introduce high-throughput Single-molecule Parallel Analysis for Rapid eXploration of Sequence space (SPARXS), integrating single-molecule fluorescence with next-generation sequencing. We applied SPARXS to study the sequence-dependent kinetics of the Holliday junction, a critical intermediate in homologous recombination. By examining the dynamics of millions of Holliday junctions, covering thousands of distinct sequences, we demonstrated the ability of SPARXS to uncover sequence patterns, evaluate sequence motifs, and construct thermodynamic models. SPARXS emerges as a versatile tool for untangling the mechanisms that underlie sequence-specific processes at the molecular scale.

Structure and rational engineering of the PglX methyltransferase and specificity factor for BREX phage defence

Bacteria have evolved a broad range of systems that provide defence against their viral predators, bacteriophages. Bacteriophage Exclusion (BREX) systems recognise and methylate 6 bp non-palindromic motifs within the host genome, and prevent replication of non-methylated phage DNA that encodes these same motifs. How BREX recognises cognate motifs has not been fully understood. In this study we characterise BREX from pathogenic Salmonella and present X-ray crystallographic structures of the conserved BREX protein, PglX. The PglX N-terminal domain encodes the methyltransferase, whereas the C-terminal domain is for motif recognition. We also present the structure of PglX bound to the phage-derived DNA mimic, Ocr, an inhibitor of BREX activity. Our analyses propose modes for DNA-binding by PglX and indicate that both methyltransferase activity and defence require larger BREX complexes. Through rational engineering of PglX we broaden both the range of phages targeted, and the host motif sequences that are methylated by BREX. Our data demonstrate that PglX is used to recognise specific DNA sequences for BREX activity, contributing to motif recognition for both phage defence and host methylation.

T and Z, partial seed coat patterning genes in common bean, provide insight into the structure and protein interactions of a plant MBW complex.

Flavonoids are secondary metabolites associated with plant seed coat and flower color. These compounds provide health benefits to humans as anti-inflammatory and antioxidant compounds. The expression of the late biosynthetic genes in the flavonoid pathway is controlled by a ternary MBW protein complex consisting of interfacing MYB, beta-helix-loop-helix (bHLH), and WD40 Repeat (WDR) proteins. P, the master regulator gene of the flavonoid expression in common bean (Phaseolus vulgaris L.), was recently determined to encode a bHLH protein. The T and Z genes control the distribution of color in bean seeds and flowers and have historically been considered regulators of the flavonoid gene expression. T and Z candidates were identified using reverse genetics based on genetic mapping, phylogenetic analysis, and mutant analysis. Domain and AlphaFold2 structure analyses determined that T encodes a seven-bladed β-propeller WDR protein, while Z encodes a R2R3 MYB protein. Deletions and SNPs in T and Z mutants, respectively, altered the 3D structure of these proteins. Modeling of the Z MYB/P bHLH/T WDR MBW complex identified interfacing sequence domains and motifs in all three genes that are conserved in dicots. One Z MYB motif is a possible beta-molecular recognition feature (β-MoRF) that only appears in a structured state when Z MYB is modeled as a component of a MBW complex. Complexes containing mutant T and Z proteins changed the interaction of members of the complex in ways that would alter their role in regulating the expression of genes in the flavonoid pathway.

The structural landscape of Microprocessor mediated pri-let-7 miRNA processing.

miRNA biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryo-EM and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has the structural plasticity to accommodate a range of pri-miRNAs. These structures revealed key features of the 5' UG sequence motif, more comprehensively represented as the "fUN" motif. Our analysis explains how cleavage of class-II pri-let-7 members harboring a bulged nucleotide generates a noncanonical precursor with a 1-nt 3' overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 contributes to MP fidelity by interacting with the CNNC-motif and Drosha's PAZ-like domain. Overall, this study sheds light on the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP.