Nucleic Acids Research
Structure-guided sequence specificity engineering of the modification-dependent restriction endonuclease LpnPI
The eukaryotic Set and Ring Associated (SRA) domains and structurally similar DNA recognition domains of prokaryotic cytosine modification-dependent restriction endonucleases recognize methylated, hydroxymethylated or glucosylated cytosine in various sequence contexts. Here, we report the apo-structure of the N-terminal SRA-like domain of the cytosine modification-dependent restriction enzyme LpnPI that recognizes modified cytosine in the 5'-C(mC)DG-3' target sequence (where mC is 5-methylcytosine or 5-hydroxymethylcytosine and D = A/T/G). Structure-guided mutational analysis revealed LpnPI residues involved in base-specific interactions and demonstrated binding site plasticity that allowed limited target sequence degeneracy. Furthermore, modular exchange of the LpnPI specificity loops by structural equivalents of related enzymes AspBHI and SgrTI altered sequence specificity of LpnPI. Taken together, our results pave the way for specificity engineering of the cytosine modification-dependent restriction enzymes.
Ion-mediated interaction is critical to the structure and stability of nucleic acids. Recent experiments suggest that the multivalent ion-induced aggregation of double-stranded (ds) RNAs and DNAs may strongly depend on the topological nature of helices, while there is still lack of an understanding on the relevant ion-mediated interactions at atomistic level. In this work, we have directly calculated the potentials of mean force (PMF) between two dsRNAs and between two dsDNAs in Co(NH3)63+ (Co-Hex) solutions by the atomistic molecular dynamics simulations. Our calculations show that at low [Co-Hex], the PMFs between B-DNAs and between A-RNAs are both (strongly) repulsive. However, at high [Co-Hex], the PMF between B-DNAs is strongly attractive, while those between A-RNAs and between A-DNAs are still (weakly) repulsive. The microscopic analyses show that for A-form helices, Co-Hex would become ‘internal binding’ into the deep major groove and consequently cannot form the evident ion-bridge between adjacent helices, while for B-form helices without deep grooves, Co-Hex would exhibit ‘external binding’ to strongly bridge adjacent helices. In addition, our further calculations show that, the PMF between A-RNAs could become strongly attractive either at very high [Co-Hex] or when the bottom of deep major groove is fixed with a layer of water.
RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally-occurring RNA thermosensors are heat-inducible, have long sequences, and function by sequestering the ribosome binding site in a hairpin structure at lower temperatures. Here, we demonstrate the de novo design of short, heat-repressible RNA thermosensors. These thermosensors contain a cleavage site for RNase E, an enzyme native to Escherichia coli and many other organisms, in the 5' untranslated region of the target gene. At low temperatures, the cleavage site is sequestered in a stem–loop, and gene expression is unobstructed. At high temperatures, the stem–loop unfolds, allowing for mRNA degradation and turning off expression. We demonstrated that these thermosensors respond specifically to temperature and provided experimental support for the central role of RNase E in the mechanism. We also demonstrated the modularity of these RNA thermosensors by constructing a three-input composite circuit that utilizes transcriptional, post-transcriptional, and post-translational regulation. A thorough analysis of the 24 thermosensors allowed for the development of design guidelines for systematic construction of similar thermosensors in future applications. These short, modular RNA thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.
Synthesis and triplex-forming properties of oligonucleotides capable of recognizing corresponding DNA duplexes containing four base pairs
A triplex-forming oligonucleotide (TFO) could be a useful molecular tool for gene therapy and specific gene modification. However, unmodified TFOs have two serious drawbacks: low binding affinities and high sequence-dependencies. In this paper, we propose a new strategy that uses a new set of modified nucleobases for four-base recognition of TFOs, and thereby overcome these two drawbacks. TFOs containing a 2’-deoxy-4N-(2-guanidoethyl)-5-methylcytidine (dgC) residue for a C-G base pair have higher binding and base recognition abilities than those containing 2’-OMe-4N-(2-guanidoethyl)-5-methylcytidine (2’-OMegC), 2’-OMe-4N-(2-guanidoethyl)-5-methyl-2-thiocytidine (2’-OMegCs), dgC and 4S-(2-guanidoethyl)-4-thiothymidine (gsT). Further, we observed that N-acetyl-2,7-diamino-1,8-naphtyridine (DANac) has a higher binding and base recognition abilities for a T-A base pair compared with that of dG and the other DNA derivatives. On the basis of this knowledge, we successfully synthesized a fully modified TFO containing DANac, dgC, 2’-OMe-2-thiothymidine (2’-OMesT) and 2’-OMe-8-thioxoadenosine (2’-OMesA) with high binding and base recognition abilities. To the best of our knowledge, this is the first report in which a fully modified TFO accurately recognizes a complementary DNA duplex having a mixed sequence under neutral conditions.
Key components of the translational apparatus, i.e. ribosomes, elongation factor EF-Tu and most aminoacyl-tRNA synthetases, are stereoselective and prevent incorporation of d-amino acids (d-aa) into polypeptides. The rare appearance of d-aa in natural polypeptides arises from post-translational modifications or non-ribosomal synthesis. We introduce an in vitro translation system that enables single incorporation of 17 out of 18 tested d-aa into a polypeptide; incorporation of two or three successive d-aa was also observed in several cases. The system consists of wild-type components and d-aa are introduced via artificially charged, unmodified tRNAGly that was selected according to the rules of ‘thermodynamic compensation’. The results reveal an unexpected plasticity of the ribosomal peptidyltransferase center and thus shed new light on the mechanism of chiral discrimination during translation. Furthermore, ribosomal incorporation of d-aa into polypeptides may greatly expand the armamentarium of in vitro translation towards the identification of peptides and proteins with new properties and functions.
High-Throughput (HT) SELEX combines SELEX (Systematic Evolution of Ligands by EXponential Enrichment), a method for aptamer discovery, with massively parallel sequencing technologies. This emerging technology provides data for a global analysis of the selection process and for simultaneous discovery of a large number of candidates but currently lacks dedicated computational approaches for their analysis. To close this gap, we developed novel in-silico methods to analyze HT-SELEX data and utilized them to study the emergence of polymerase errors during HT-SELEX. Rather than considering these errors as a nuisance, we demonstrated their utility for guiding aptamer discovery. Our approach builds on two main advancements in aptamer analysis: AptaMut—a novel technique allowing for the identification of polymerase errors conferring an improved binding affinity relative to the ‘parent’ sequence and AptaCluster—an aptamer clustering algorithm which is to our best knowledge, the only currently available tool capable of efficiently clustering entire aptamer pools. We applied these methods to an HT-SELEX experiment developing aptamers against Interleukin 10 receptor alpha chain (IL-10RA) and experimentally confirmed our predictions thus validating our computational methods.
An implementation of the Gillespie algorithm for RNA kinetics with logarithmic time update
In this paper I outline a fast method called KFOLD for implementing the Gillepie algorithm to stochastically sample the folding kinetics of an RNA molecule at single base-pair resolution. In the same fashion as the KINFOLD algorithm, which also uses the Gillespie algorithm to predict folding kinetics, KFOLD stochastically chooses a new RNA secondary structure state that is accessible from the current state by a single base-pair addition/deletion following the Gillespie procedure. However, unlike KINFOLD, the KFOLD algorithm utilizes the fact that many of the base-pair addition/deletion reactions and their corresponding rates do not change between each step in the algorithm. This allows KFOLD to achieve a substantial speed-up in the time required to compute a prediction of the folding pathway and, for a fixed number of base-pair moves, performs logarithmically with sequence size. This increase in speed opens up the possibility of studying the kinetics of much longer RNA sequences at single base-pair resolution while also allowing for the RNA folding statistics of smaller RNA sequences to be computed much more quickly.
Global transcription network incorporating distal regulator binding reveals selective cooperation of cancer drivers and risk genes
Global network modeling of distal regulatory interactions is essential in understanding the overall architecture of gene expression programs. Here, we developed a Bayesian probabilistic model and computational method for global causal network construction with breast cancer as a model. Whereas physical regulator binding was well supported by gene expression causality in general, distal elements in intragenic regions or loci distant from the target gene exhibited particularly strong functional effects. Modeling the action of long-range enhancers was critical in recovering true biological interactions with increased coverage and specificity overall and unraveling regulatory complexity underlying tumor subclasses and drug responses in particular. Transcriptional cancer drivers and risk genes were discovered based on the network analysis of somatic and genetic cancer-related DNA variants. Notably, we observed that the risk genes were functionally downstream of the cancer drivers and were selectively susceptible to network perturbation by tumorigenic changes in their upstream drivers. Furthermore, cancer risk alleles tended to increase the susceptibility of the transcription of their associated genes. These findings suggest that transcriptional cancer drivers selectively induce a combinatorial misregulation of downstream risk genes, and that genetic risk factors, mostly residing in distal regulatory regions, increase transcriptional susceptibility to upstream cancer-driving somatic changes.
Significant expansion of the REST/NRSF cistrome in human versus mouse embryonic stem cells: potential implications for neural development
Recent studies have employed cross-species comparisons of transcription factor binding, reporting significant regulatory network ‘rewiring’ between species. Here, we address how a transcriptional repressor targets and regulates neural genes differentially between human and mouse embryonic stem cells (ESCs). We find that the transcription factor, Repressor Element 1 Silencing Transcription factor (REST; also called neuron restrictive silencer factor) binds to a core group of ~1200 syntenic genomic regions in both species, with these conserved sites highly enriched with co-factors, selective histone modifications and DNA hypomethylation. Genes with conserved REST binding are enriched with neural functions and more likely to be upregulated upon REST depletion. Interestingly, we identified twice as many REST peaks in human ESCs compared to mouse ESCs. Human REST cistrome expansion involves additional peaks in genes targeted by REST in both species and human-specific gene targets. Genes with expanded REST occupancy in humans are enriched for learning or memory functions. Analysis of neurological disorder associated genes reveals that Amyotrophic Lateral Sclerosis and oxidative stress genes are particularly enriched with human-specific REST binding. Overall, our results demonstrate that there is substantial rewiring of human and mouse REST cistromes, and that REST may have human-specific roles in brain development and functions.
N protein from lambdoid phages transforms NusA into an antiterminator by modulating NusA-RNA polymerase flap domain interactions
Interaction of the lambdoid phage N protein with the bacterial transcription elongation factor NusA is the key component in the process of transcription antitermination. A convex surface of E. coli NusA-NTD, located opposite to its RNA polymerase-binding domain (the β-flap domain), directly interacts with N in the antitermination complex. We hypothesized that this N-NusA interaction induces allosteric effects on the NusA-RNAP interaction leading to transformation of NusA into a facilitator of the antitermination process. Here we showed that mutations in β-flap domain specifically defective for N antitermination exhibited altered NusA-nascent RNA interaction and have widened RNA exit channel indicating an intricate role of flap domain in the antitermination. The presence of N reoriented the RNAP binding surface of NusA-NTD, which changed its interaction pattern with the flap domain. These changes caused significant spatial rearrangement of the β-flap as well as the β' dock domains to form a more constricted RNA exit channel in the N-modified elongation complex (EC), which might play key role in converting NusA into a facilitator of the N antitermination. We propose that in addition to affecting the RNA exit channel and the active center of the EC, β-flap domain rearrangement is also a mechanistic component in the N antitermination process.
Yeast high mobility group protein HMO1 stabilizes chromatin and is evicted during repair of DNA double strand breaks
DNA is packaged into condensed chromatin fibers by association with histones and architectural proteins such as high mobility group (HMGB) proteins. However, this DNA packaging reduces accessibility of enzymes that act on DNA, such as proteins that process DNA after double strand breaks (DSBs). Chromatin remodeling overcomes this barrier. We show here that the Saccharomyces cerevisiae HMGB protein HMO1 stabilizes chromatin as evidenced by faster chromatin remodeling in its absence. HMO1 was evicted along with core histones during repair of DSBs, and chromatin remodeling events such as histone H2A phosphorylation and H3 eviction were faster in absence of HMO1. The facilitated chromatin remodeling in turn correlated with more efficient DNA resection and recruitment of repair proteins; for example, inward translocation of the DNA-end-binding protein Ku was faster in absence of HMO1. This chromatin stabilization requires the lysine-rich C-terminal extension of HMO1 as truncation of the HMO1 C-terminal tail phenocopies hmo1 deletion. Since this is reminiscent of the need for the basic C-terminal domain of mammalian histone H1 in chromatin compaction, we speculate that HMO1 promotes chromatin stability by DNA bending and compaction imposed by its lysine-rich domain and that it must be evicted along with core histones for efficient DSB repair.
Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits
Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the α, β and isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1β emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits.