Short Sequence Motifs Research Articles

Shared cis-regulatory architecture identified across defense response genes is associated with broad-spectrum quantitative resistance in rice

Plant disease resistance that is durable and effective against diverse pathogens (broad-spectrum) is essential to stabilize crop production. Such resistance is frequently controlled by Quantitative Trait Loci (QTL), and often involves differential regulation of Defense Response (DR) genes. In this study, we sought to understand how expression of DR genes is orchestrated, with the long-term goal of enabling genome-wide breeding for more effective and durable resistance. We identified short sequence motifs in rice promoters that are shared across Broad-Spectrum DR (BS-DR) genes co-expressed after challenge with three major rice pathogens (Magnaporthe oryzae, Rhizoctonia solani, and Xanthomonas oryzae pv. oryzae) and several chemical elicitors. Specific groupings of these BS-DR-associated motifs, called cis-Regulatory Modules (CRMs), are enriched in DR gene promoters, and the CRMs include cis-elements known to be involved in disease resistance. Polymorphisms in CRMs occur in promoters of genes in resistant relative to susceptible BS-DR haplotypes providing evidence that these CRMs have a predictive role in the contribution of other BS-DR genes to resistance. Therefore, we predict that a CRM signature within BS-DR gene promoters can be used as a marker for future breeding practices to enrich for the most responsive and effective BS-DR genes across the genome.

Transcriptomic analyses reveal groups of co-expressed, syntenic lncRNAs in four species of the genus Caenorhabditis

ABSTRACTLong non-coding RNAs (lncRNAs) are a heterogeneous class of genes that do not code for proteins. Since lncRNAs (or a fraction thereof) are expected to be functional, many efforts have been dedicated to catalog lncRNAs in numerous organisms, but our knowledge of lncRNAs in non vertebrate species remains very limited. Here, we annotated lncRNAs using transcriptomic data from the same larval stage of four Caenorhabditis species. The number of annotated lncRNAs in self-fertile nematodes was lower than in out-crossing species. We used a combination of approaches to identify putatively homologous lncRNAs: synteny, sequence conservation, and structural conservation. We classified a total of 1,532 out of 7,635 genes from the four species into families of lncRNAs with conserved synteny and expression at the larval stage, suggesting that a large fraction of the predicted lncRNAs may be species specific. Despite both sequence and local secondary structure seem to be poorly conserved, sequences within families frequently shared BLASTn hits and short sequence motifs, which were more likely to be unpaired in the predicted structures. We provide the first multi-species catalog of lncRNAs in nematodes and identify groups of lncRNAs with conserved synteny and expression, that share exposed motifs.

SgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing

SummaryCas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targeting sequence. Here, we discovered that two short sequence motifs at the 3′ end of the targeting sequence are almost exclusively present in inefficient sgRNAs of published sgRNA-activity datasets. By specific knock-in of sgRNA target sequences with or without these motifs and quantitative measurement of knockout efficiency, we show that the presence of these motifs in sgRNAs per se results in a 10-fold reduction of gene knockout frequencies. Mechanistically, the cause of the low efficiency differs between the two motifs. These sequence motifs are relevant for future sgRNA design approaches and studies of Cas9-DNA interactions.

Transcription factor Sp2 potentiates binding of the TALE homeoproteins Pbx1:Prep1 and the histone-fold domain protein Nf-y to composite genomic sites

Different transcription factors operate together at promoters and enhancers to regulate gene expression. Transcription factors either bind directly to their target DNA or are tethered to it by other proteins. The transcription factor Sp2 serves as a paradigm for indirect genomic binding. It does not require its DNA-binding domain for genomic DNA binding and occupies target promoters independently of whether they contain a cognate DNA-binding motif. Hence, Sp2 is strikingly different from its closely related paralogs Sp1 and Sp3, but how Sp2 recognizes its targets is unknown. Here, we sought to gain more detailed insights into the genomic targeting mechanism of Sp2. ChIP-exo sequencing in mouse embryonic fibroblasts revealed genomic binding of Sp2 to a composite motif where a recognition sequence for TALE homeoproteins and a recognition sequence for the trimeric histone-fold domain protein nuclear transcription factor Y (Nf-y) are separated by 11 bp. We identified a complex consisting of the TALE homeobox protein Prep1, its partner PBX homeobox 1 (Pbx1), and Nf-y as the major partners in Sp2-promoter interactions. We found that the Pbx1:Prep1 complex together with Nf-y recruits Sp2 to co-occupied regulatory elements. In turn, Sp2 potentiates binding of Pbx1:Prep1 and Nf-y. We also found that the Sp-box, a short sequence motif close to the Sp2 N terminus, is crucial for Sp2's cofactor function. Our findings reveal a mechanism by which the DNA binding-independent activity of Sp2 potentiates genomic loading of Pbx1:Prep1 and Nf-y to composite motifs present in many promoters of highly expressed genes.

Functional Analysis of Short Linear Motifs in the Plant Cyclin-Dependent Kinase Inhibitor SIAMESE.

Endoreplication, a modified cell cycle in which DNA is replicated without subsequent cell division, plays an important but poorly understood role in plant growth and in plant responses to biotic and abiotic stress. The Arabidopsis (Arabidopsis thaliana) SIAMESE (SIM) gene encodes the first identified member of the SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors. SIM controls endoreplication during trichome development, and sim mutant trichomes divide several times instead of endoreplicating their DNA. The SMR family is defined by several short linear amino acid sequence motifs of largely unknown function, and family members have little sequence similarity to any known protein functional domains. Here, we investigated the roles of the conserved motifs in SIM site-directed Arabidopsis mutants using several functional assays. We identified a potential cyclin-dependent kinase (CDK)-binding site, which bears no resemblance to other known CDK interaction motifs. We also identified a potential site of phosphorylation and two redundant nuclear localization sequences. Surprisingly, the only motif with similarity to the other family of plant CDK inhibitors, the INHIBITOR/INTERACTOR OF CDC2 KINASE/KIP-RELATED PROTEIN proteins, is not required for SIM function in vivo. Because even highly divergent members of the SMR family are able to replace SIM function in Arabidopsis trichomes, it is likely that the results obtained here for SIM will apply to other members of this plant-specific family of CDK inhibitors.

Identification of conserved, primary sequence motifs that direct retrovirus RNA fate.

Precise stoichiometry of genome-length transcripts and alternatively spliced mRNAs is a hallmark of retroviruses. We discovered short, guanosine and adenosine sequence motifs in the 5′untranslated region of several retroviruses and ascertained the reasons for their conservation using a representative lentivirus and genetically simpler retrovirus. We conducted site-directed mutagenesis of the GA-motifs in HIV molecular clones and observed steep replication delays in T-cells. Quantitative RNA analyses demonstrate the GA-motifs are necessary to retain unspliced viral transcripts from alternative splicing. Mutagenesis of the GA-motifs in a C-type retrovirus validate the similar downregulation of unspliced transcripts and virion structural protein. The evidence from cell-based co-precipitation studies shows the GA-motifs in the 5′untranslated region confer binding by SFPQ/PSF, a protein co-regulated with T-cell activation. Diminished SFPQ/PSF or mutation of either GA-motif attenuates the replication of HIV. The interaction of SFPQ/PSF with both GA-motifs is crucial for maintaining the stoichiometry of the viral transcripts and does not affect packaging of HIV RNA. Our results demonstrate the conserved GA-motifs direct the fate of retrovirus RNA. These findings have exposed an RNA-based molecular target to attenuate retrovirus replication.

Intra-molecular pathways of allosteric control in Hsp70s.

The 70 kDa heat-shock protein (Hsp70) is undoubtedly the most versatile of all molecular chaperones. Hsp70 is involved in numerous cellular protein folding processes, accompanying proteins throughout their lifespan from de novo folding at the ribosome to degradation at the proteasome, surveilling protein stability and functionality. Several properties of this ATP-dependent chaperone constitute the molecular basis for this versatility. With its substrate binding domain (SBD), Hsp70 transiently interacts with a short degenerative linear sequence motif found practically in all proteins and, in addition, with more folded protein conformers. Binding to polypeptides is tightly regulated by ATP binding and hydrolysis in the nucleotide binding domain, which is coupled to the SBD by an intricate allosteric mechanism. Hsp70 is regulated by a host of J-cochaperones, which act as targeting factors by regulating the ATPase activity of Hsp70 in synergism with the substrates themselves, and by several families of nucleotide exchange factors. In this review, I focus on the allosteric mechanism, which allows Hsp70s to interact with substrates with ultrahigh affinity through a non-equilibrium mode of action and summarize what mutagenesis and structural studies have taught us about the pathways and mechanics of interdomain communication.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

At Long Last, a C-Terminal Bookend for the Ubiquitin Code

The ubiquitin-proteasome system controls the stability of myriad protein substrates via short sequence motifs called degrons. Studies by Koren etal. (2018) and Lin etal. (2018) have uncovered a broad new class of degrons located at the extreme C terminus of substrates.

Preventing Autoimmunity in Transcription‐Dependent CRISPR‐Cas Systems

The CRISPR‐Cas systems are a collection of RNA‐based adaptive immune systems that protect many prokaryotes from invasion by viruses and plasmids. CRISPR‐Cas systems are very diverse and can be organized into six types and more than 15 subtypes. Type III systems are unique as their targeting mechanism requires transcription of the target DNA. Type III effector complexes use small CRISPR RNAs (crRNAs) to identify complementary transcripts. Transcript binding simultaneously activates two nuclease domains, leading to the degradation of both the bound transcript and adjacent DNA. Like all other immune systems, CRISPR‐Cas systems must differentiate between self and non‐self in order to prevent autoimmunity, which could arise from targeting of the host CRISPR‐array. Type III systems use two mechanisms to identify self‐transcripts. Studies of the Type III‐A system from Staphylococcus epidermidis revealed that base pairing between the crRNA and the sequence flanking the 3′ end of RNA target site prevents autoimmunity. However, a study of the Type III‐B system from Pyrococcus furiosus revealed that a short sequence motif flanking the RNA target sequence is required for immunity and the absence of this motif in CRISPR arrays prevents autoimmunity. Neither of these mechanisms is well understood biochemically and it is not clear if these mechanisms are conserved within the subtypes. To address these points, we monitored RNA‐activated DNA cleavage by the Type III‐B effector complex from Thermotoga maritima with purified components. A series of RNA transcripts containing various target 3′‐flanking sequences were tested for their ability to activate DNA cleavage. We find that two factors are important in preventing autoimmunity in the T. maritima Type III‐B system: (1) base pairing between the crRNA and the target 3′‐flanking sequence of the transcript and (2) the presence of a G, which does not base pair with the crRNA, immediately adjacent to the 3′‐end of the RNA target sequence. These data suggest that the mechanisms used to avoid autoimmunity by Type III systems are not subtype‐specific and that identification of self transcripts is complex, involving base pairing and specific sequence recognition.Support or Funding InformationThis work was supported by National Institutes of Health grant GM097330 to S.B.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeted substrate degradation by Kelch controls the actin cytoskeleton during ring canal expansion.

During Drosophila oogenesis, specialized actin-based structures called ring canals form and expand to accommodate growth of the oocyte. Previous work demonstrated that Kelch and Cullin 3 function together in a Cullin 3-RING ubiquitin ligase complex (CRL3Kelch) to organize the ring canal cytoskeleton, presumably by targeting a substrate for proteolysis. Here, we use tandem affinity purification followed by mass spectrometry to identify HtsRC as the CRL3Kelch ring canal substrate. CRISPR-mediated mutagenesis of HtsRC revealed its requirement in the recruitment of the ring canal F-actin cytoskeleton. We present genetic evidence consistent with HtsRC being the CRL3Kelch substrate, as well as biochemical evidence indicating that HtsRC is ubiquitylated and degraded by the proteasome. Finally, we identify a short sequence motif in HtsRC that is necessary for Kelch binding. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system.

The interaction between androgen receptor and semenogelin I: a synthetic LxxLL peptide antagonist inhibits the growth of prostate cancer cells

Background:We previously demonstrated that a seminal plasma protein, semenogelin I (SgI), functioned as an androgen receptor (AR) coactivator. Meanwhile, several short sequence motifs in AR coregulators, such as LxxLL (L=leucine), have been shown to mediate specific interactions with AR.Methods:We investigated the role of the LxxLL motif within SgI in the interactions with AR and cell growth in prostate cancer lines in vitro.Results:A full-length SgI with mutations in the motif (i.e., LxxAA; A=alanine) failed to significantly increase cell proliferation/migration as well as androgen-mediated AR transcription. Co-immunoprecipitation showed no physical interactions between AR and the mutant SgI. In addition, transfection of an 18-amino acid peptide of SgI containing LxxLL, but not LxxAA, resulted in considerable reduction in cell growth and prostate-specific antigen expression in LNCaP and C4-2 lines.Conclusions:The LxxLL motif of SgI could be a novel therapeutic target for both androgen-sensitive and castration-resistant prostate cancers.

NLSdb-major update for database of nuclear localization signals and nuclear export signals.

NLSdb is a database collecting nuclear export signals (NES) and nuclear localization signals (NLS) along with experimentally annotated nuclear and non-nuclear proteins. NES and NLS are short sequence motifs related to protein transport out of and into the nucleus. The updated NLSdb now contains 2253 NLS and introduces 398 NES. The potential sets of novel NES and NLS have been generated by a simple ‘in silico mutagenesis’ protocol. We started with motifs annotated by experiments. In step 1, we increased specificity such that no known non-nuclear protein matched the refined motif. In step 2, we increased the sensitivity trying to match several different families with a motif. We then iterated over steps 1 and 2. The final set of 2253 NLS motifs matched 35% of 8421 experimentally verified nuclear proteins (up from 21% for the previous version) and none of 18 278 non-nuclear proteins. We updated the web interface providing multiple options to search protein sequences for NES and NLS motifs, and to evaluate your own signal sequences. NLSdb can be accessed via Rostlab services at: https://rostlab.org/services/nlsdb/

Sequence requirement of the ade6-4095 meiotic recombination hotspot in Schizosaccharomyces pombe.

Homologous recombination occurs at a greatly elevated frequency in meiosis compared to mitosis and is initiated by programmed double-strand DNA breaks (DSBs). DSBs do not occur at uniform frequency throughout the genome in most organisms, but occur preferentially at a limited number of sites referred to as hotspots. The location of hotspots have been determined at nucleotide-level resolution in both the budding and fission yeasts, and while several patterns have emerged regarding preferred locations for DSB hotspots, it remains unclear why particular sites experience DSBs at much higher frequency than other sites with seemingly similar properties. Short sequence motifs, which are often sites for binding of transcription factors, are known to be responsible for a number of hotspots. In this study we identified the minimum sequence required for activity of one of such motif identified in a screen of random sequences capable of producing recombination hotspots. The experimentally determined sequence, GGTCTRGACC, closely matches the previously inferred sequence. Full hotspot activity requires an effective sequence length of 9.5bp, whereas moderate activity requires an effective sequence length of approximately 8.2bp and shows significant association with DSB hotspots. In combination with our previous work, this result is consistent with a large number of different sequence motifs capable of producing recombination hotspots, and supports a model in which hotspots can be rapidly regenerated by mutation as they are lost through recombination.

High-resolution profiling of linear B-cell epitopes from mucin-associated surface proteins (MASPs) of Trypanosoma cruzi during human infections.

BackgroundThe Trypanosoma cruzi genome bears a huge family of genes and pseudogenes coding for Mucin-Associated Surface Proteins (MASPs). MASP molecules display a ‘mosaic’ structure, with highly conserved flanking regions and a strikingly variable central and mature domain made up of different combinations of a large repertoire of short sequence motifs. MASP molecules are highly expressed in mammal-dwelling stages of T. cruzi and may be involved in parasite-host interactions and/or in diverting the immune response.Methods/Principle findingsHigh-density microarrays composed of fully overlapped 15mer peptides spanning the entire sequences of 232 non-redundant MASPs (~25% of the total MASP content) were screened with chronic Chagasic sera. This strategy led to the identification of 86 antigenic motifs, each one likely representing a single linear B-cell epitope, which were mapped to 69 different MASPs. These motifs could be further grouped into 31 clusters of structurally- and likely antigenically-related sequences, and fully characterized. In contrast to previous reports, we show that MASP antigenic motifs are restricted to the central and mature region of MASP polypeptides, consistent with their intracellular processing. The antigenicity of these motifs displayed significant positive correlation with their genome dosage and their relative position within the MASP polypeptide. In addition, we verified the biased genetic co-occurrence of certain antigenic motifs within MASP polypeptides, compatible with proposed intra-family recombination events underlying the evolution of their coding genes. Sequences spanning 7 MASP antigenic motifs were further evaluated using distinct synthesis/display approaches and a large panel of serum samples. Overall, the serological recognition of MASP antigenic motifs exhibited a remarkable non normal distribution among the T. cruzi seropositive population, thus reducing their applicability in conventional serodiagnosis. As previously observed in in vitro and animal infection models, immune signatures supported the concurrent expression of several MASPs during human infection.Conclusions/SignificanceIn spite of their conspicuous expression and potential roles in parasite biology, this study constitutes the first unbiased, high-resolution profiling of linear B-cell epitopes from T. cruzi MASPs during human infection.

Determinants and Regulation of Protein Turnover in Yeast.

Protein turnover maintains the recycling needs of theproteome, and its malfunction has been linked to aging and age-related diseases. However, not allproteins turnover equally, and the factors that contribute to accelerate or slow down turnover are mostly unknown. We measured turnover rates for 3,160 proteins in exponentially growing yeast and analyzed their dependence on physical, functional, and genetic properties. We found that functional characteristics, including protein localization, complex membership, and connectivity, have greater effect on turnover than sequence elements. We also found that protein turnover and mRNA turnover are correlated. Analysis under nutrient perturbation and osmotic stress revealed that protein turnover highly depends on cellular state and is faster when proteinsare being actively used. Finally, stress-induced changes in protein and transcript abundance correlated with changes in protein turnover. This study provides a resource of protein turnover rates and principles to understand the recycling needs of the proteome under basal conditions and perturbation.

Ionic Current-Based Mapping of Short Sequence Motifs in Single DNA Molecules Using Solid-State Nanopores.

Nanopore sensors show great potential for rapid, single-molecule determination of DNA sequence information. Here, we develop an ionic current-based method for determining the positions of short sequence motifs in double-stranded DNA molecules with solid-state nanopores. Using the DNA-methyltransferase M.TaqI and a biotinylated S-adenosyl-l-methionine cofactor analogue we create covalently attached biotin labels at 5′-TCGA-3′ sequence motifs. Monovalent streptavidin is then added to bind to the biotinylated sites giving rise to additional current blockade signals when the DNA passes through a conical quartz nanopore. We determine the relationship between translocation time and position along the DNA contour and find a minimum resolvable distance between two labeled sites of ∼200 bp. We then characterize a variety of DNA molecules by determining the positions of bound streptavidin and show that two short genomes can be simultaneously detected in a mixture. Our method provides a simple, generic single-molecule detection platform enabling DNA characterization in an electrical format suited for portable devices for potential diagnostic applications.

Short DNA sequence patterns accurately identify broadly active human enhancers

BackgroundEnhancers are DNA regulatory elements that influence gene expression. There is substantial diversity in enhancers’ activity patterns: some enhancers drive expression in a single cellular context, while others are active across many. Sequence characteristics, such as transcription factor (TF) binding motifs, influence the activity patterns of regulatory sequences; however, the regulatory logic through which specific sequences drive enhancer activity patterns is poorly understood. Recent analysis of Drosophila enhancers suggested that short dinucleotide repeat motifs (DRMs) are general enhancer sequence features that drive broad regulatory activity. However, it is not known whether the regulatory role of DRMs is conserved across species.ResultsWe performed a comprehensive analysis of the relationship between short DNA sequence patterns, including DRMs, and human enhancer activity in 38,538 enhancers across 411 different contexts. In a machine-learning framework, the occurrence patterns of short sequence motifs accurately predicted broadly active human enhancers. However, DRMs alone were weakly predictive of broad enhancer activity in humans and showed different enrichment patterns than in Drosophila. In general, GC-rich sequence motifs were significantly associated with broad enhancer activity, and consistent with this enrichment, broadly active human TFs recognize GC-rich motifs.ConclusionsOur results reveal the importance of specific sequence motifs in broadly active human enhancers, demonstrate the lack of evolutionary conservation of the role of DRMs, and provide a computational framework for investigating the logic of enhancer sequences.

Eukaryotic transcription factors: paradigms of protein intrinsic disorder.

Gene-specific transcription factors (TFs) are key regulatory components of signaling pathways, controlling, for example, cell growth, development, and stress responses. Their biological functions are determined by their molecular structures, as exemplified by their structured DNA-binding domains targeting specific cis-acting elements in genes, and by the significant lack of fixed tertiary structure in their extensive intrinsically disordered regions. Recent research in protein intrinsic disorder (ID) has changed our understanding of transcriptional activation domains from 'negative noodles' to ID regions with function-related, short sequence motifs and molecular recognition features with structural propensities. This review focuses on molecular aspects of TFs, which represent paradigms of ID-related features. Through specific examples, we review how the ID-associated flexibility of TFs enables them to participate in large interactomes, how they use only a few hydrophobic residues, short sequence motifs, prestructured motifs, and coupled folding and binding for their interactions with co-activators, and how their accessibility to post-translational modification affects their interactions. It is furthermore emphasized how classic biochemical concepts like allostery, conformational selection, induced fit, and feedback regulation are undergoing a revival with the appreciation of ID. The review also describes the most recent advances based on computational simulations of ID-based interaction mechanisms and structural analysis of ID in the context of full-length TFs and suggests future directions for research in TF ID.

V-ELMpiRNAPred: Identification of human piRNAs by the voting-based extreme learning machine (V-ELM) with a new hybrid feature.

Piwi-interacting RNAs (piRNAs) were recently discovered as endogenous small noncoding RNAs. Some recent research suggests that piRNAs may play an important role in cancer. So the precise identification of human piRNAs is a significant work. In this paper, we introduce a series of new features with 80 dimension called short sequence motifs (SSM). A hybrid feature vector with 1444 dimension can be formed by combining 1364 features of [Formula: see text]-mer strings and 80 features of SSM features. We optimize the 1444 dimension features using the feature score criterion (FSC) and list them in descending order according to the scores. The first 462 are selected as the input feature vector in the classifier. Moreover, eight of 80 SSM features appear in the top 20. This indicates that these eight SSM features play an important part in the identification of piRNAs. Since five of the above eight SSM features are associated with nucleotide A and G ('A*G', 'A**G', 'A***G', 'A****G', 'A*****G'). So, we guess there may exist some biological significance. We also use a neural network algorithm called voting-based extreme learning machine (V-ELM) to identify real piRNAs. The Specificity (Sp) and Sensitivity (Sn) of our method are 95.48% and 94.61%, respectively in human species. This result shows that our method is more effective compared with those of the piRPred, piRNApredictor, Asym-Pibomd, Piano and McRUMs. The web service of V-ELMpiRNAPred is available for free at http://mm20132014.wicp.net:38601/velmprepiRNA/Main.jsp .

Identification of Entry Factors Involved in Hepatitis C Virus Infection Based on Host-Mimicking Short Linear Motifs

Host factors that facilitate viral entry into cells can, in principle, be identified from a virus-host protein interaction network, but for most viruses information for such a network is limited. To help fill this void, we developed a bioinformatics approach and applied it to hepatitis C virus (HCV) infection, which is a current concern for global health. Using this approach, we identified short linear sequence motifs, conserved in the envelope proteins of HCV (E1/E2), that potentially can bind human proteins present on the surface of hepatocytes so as to construct an HCV (envelope)-host protein interaction network. Gene Ontology functional and KEGG pathway analyses showed that the identified host proteins are enriched in cell entry and carcinogenesis functionalities. The validity of our results is supported by much published experimental data. Our general approach should be useful when developing antiviral agents, particularly those that target virus-host interactions.