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Methods in Molecular Biology (Clifton,... 2024Structural changes in RNAs are an important contributor to controlling gene expression not only at the posttranscriptional stage but also during transcription. A...
Structural changes in RNAs are an important contributor to controlling gene expression not only at the posttranscriptional stage but also during transcription. A subclass of riboswitches and RNA thermometers located in the 5' region of the primary transcript regulates the downstream functional unit - usually an ORF - through premature termination of transcription. Not only such elements occur naturally, but they are also attractive devices in synthetic biology. The possibility to design such riboswitches or RNA thermometers is thus of considerable practical interest. Since these functional RNA elements act already during transcription, it is important to model and understand the dynamics of folding and, in particular, the formation of intermediate structures concurrently with transcription. Cotranscriptional folding simulations are therefore an important step to verify the functionality of design constructs before conducting expensive and labor-intensive wet lab experiments. For RNAs, full-fledged molecular dynamics simulations are far beyond practical reach because of both the size of the molecules and the timescales of interest. Even at the simplified level of secondary structures, further approximations are necessary. The BarMap approach is based on representing the secondary structure landscape for each individual transcription step by a coarse-grained representation that only retains a small set of low-energy local minima and the energy barriers between them. The folding dynamics between two transcriptional elongation steps is modeled as a Markov process on this representation. Maps between pairs of consecutive coarse-grained landscapes make it possible to follow the folding process as it changes in response to transcription elongation. In its original implementation, the BarMap software provides a general framework to investigate RNA folding dynamics on temporally changing landscapes. It is, however, difficult to use in particular for specific scenarios such as cotranscriptional folding. To overcome this limitation, we developed the user-friendly BarMap-QA pipeline described in detail in this contribution. It is illustrated here by an elaborate example that emphasizes the careful monitoring of several quality measures. Using an iterative workflow, a reliable and complete kinetics simulation of a synthetic, transcription-regulating riboswitch is obtained using minimal computational resources. All programs and scripts used in this contribution are free software and available for download as a source distribution for Linux or as a platform-independent Docker image including support for Apple macOS and Microsoft Windows.
Topics: RNA Folding; Transcription, Genetic; Molecular Dynamics Simulation; Nucleic Acid Conformation; Riboswitch; RNA; Software
PubMed: 38780738
DOI: 10.1007/978-1-0716-3519-3_14 -
Methods in Molecular Biology (Clifton,... 2024Although RNA molecules are synthesized via transcription, little is known about the general impact of cotranscriptional folding in vivo. We present different...
Although RNA molecules are synthesized via transcription, little is known about the general impact of cotranscriptional folding in vivo. We present different computational approaches for the simulation of changing structure ensembles during transcription, including interpretations with respect to experimental data from literature. Specifically, we analyze different mutations of the E. coli SRP RNA, which has been studied comparatively well in previous literature, yet the details of which specific metastable structures form as well as when they form are still under debate. Here, we combine thermodynamic and kinetic, deterministic, and stochastic models with automated and visual inspection of those systems to derive the most likely scenario of which substructures form at which point during transcription. The simulations do not only provide explanations for present experimental observations but also suggest previously unnoticed conformations that may be verified through future experimental studies.
Topics: RNA Folding; Nucleic Acid Conformation; RNA, Bacterial; Thermodynamics; Escherichia coli; Transcription, Genetic; Signal Recognition Particle; Kinetics; Computational Biology; Mutation; Models, Molecular
PubMed: 38780737
DOI: 10.1007/978-1-0716-3519-3_13 -
Methods in Molecular Biology (Clifton,... 2024Generating accurate alignments of non-coding RNA sequences is indispensable in the quest for understanding RNA function. Nevertheless, aligning RNAs remains a...
Generating accurate alignments of non-coding RNA sequences is indispensable in the quest for understanding RNA function. Nevertheless, aligning RNAs remains a challenging computational task. In the twilight-zone of RNA sequences with low sequence similarity, sequence homologies and compatible, favorable (a priori unknown) structures can be inferred only in dependency of each other. Thus, simultaneous alignment and folding (SA&F) remains the gold-standard of comparative RNA analysis, even if this method is computationally highly demanding. This text introduces to the recent release 2.0 of the software package LocARNA, focusing on its practical application. The package enables versatile, fast and accurate analysis of multiple RNAs. For this purpose, it implements SA&F algorithms in a specific, lightweight flavor that makes them routinely applicable in large scale. Its high performance is achieved by combining ensemble-based sparsification of the structure space and banding strategies. Probabilistic banding strongly improves the performance of LocARNA 2.0 even over previous releases, while simplifying its effective use. Enabling flexible application to various use cases, LocARNA provides tools to globally and locally compare, cluster, and multiply aligned RNAs based on optimization and probabilistic variants of SA&F, which optionally integrate prior knowledge, expressible by anchor and structure constraints.
Topics: Software; Algorithms; RNA Folding; RNA; Computational Biology; Nucleic Acid Conformation; Sequence Alignment; Sequence Analysis, RNA
PubMed: 38780734
DOI: 10.1007/978-1-0716-3519-3_10 -
Methods in Molecular Biology (Clifton,... 2024Nucleotide modifications are occurrent in all types of RNA and play an important role in RNA structure formation and stability. Modified bases not only possess the...
Nucleotide modifications are occurrent in all types of RNA and play an important role in RNA structure formation and stability. Modified bases not only possess the ability to shift the RNA structure ensemble towards desired functional confirmations. By changes in the base pairing partner preference, they may even enlarge or reduce the conformational space, i.e., the number and types of structures the RNA molecule can adopt. However, most methods to predict RNA secondary structure do not provide the means to include the effect of modifications on the result. With the help of a heavily modified transfer RNA (tRNA) molecule, this chapter demonstrates how to include the effect of different base modifications into secondary structure prediction using the ViennaRNA Package. The constructive approach demonstrated here allows for the calculation of minimum free energy structure and suboptimal structures at different levels of modified base support. In particular we, show how to incorporate the isomerization of uridine to pseudouridine ( ) and the reduction of uridine to dihydrouridine (D).
Topics: Nucleic Acid Conformation; RNA; RNA, Transfer; Nucleotides; Base Pairing; Computational Biology; Thermodynamics; Software; Uridine; Models, Molecular; Pseudouridine
PubMed: 38780732
DOI: 10.1007/978-1-0716-3519-3_8 -
Methods in Molecular Biology (Clifton,... 2024Analysis of the folding space of RNA generally suffers from its exponential size. With classified Dynamic Programming algorithms, it is possible to alleviate this burden...
Analysis of the folding space of RNA generally suffers from its exponential size. With classified Dynamic Programming algorithms, it is possible to alleviate this burden and to analyse the folding space of RNA in great depth. Key to classified DP is that the search space is partitioned into classes based on an on-the-fly computed feature. A class-wise evaluation is then used to compute class-wide properties, such as the lowest free energy structure for each class, or aggregate properties, such as the class' probability. In this paper we describe the well-known shape and hishape abstraction of RNA structures, their power to help better understand RNA function and related methods that are based on these abstractions.
Topics: RNA; Algorithms; Nucleic Acid Conformation; RNA Folding; Computational Biology; Software; Thermodynamics
PubMed: 38780730
DOI: 10.1007/978-1-0716-3519-3_6 -
Methods in Molecular Biology (Clifton,... 2024A number of analyses require estimates of the folding free energy changes of specific RNA secondary structures. These predictions are often based on a set of nearest...
A number of analyses require estimates of the folding free energy changes of specific RNA secondary structures. These predictions are often based on a set of nearest neighbor parameters that models the folding stability of a RNA secondary structure as the sum of folding stabilities of the structural elements that comprise the secondary structure. In the software suite RNAstructure, the free energy change calculation is implemented in the program efn2. The efn2 program estimates the folding free energy change and the experimental uncertainty in the folding free energy change. It can be run through the graphical user interface for RNAstructure, from the command line, or a web server. This chapter provides detailed protocols for using efn2.
Topics: RNA; RNA Folding; Software; Nucleic Acid Conformation; Thermodynamics; Computational Biology; Models, Molecular
PubMed: 38780725
DOI: 10.1007/978-1-0716-3519-3_1 -
BMC Genomics May 2024Long non-coding RNAs (lncRNAs) are crucial modulators of post-transcriptional gene expression regulation, cell fate determination, and disease development. However,...
Long non-coding RNAs (lncRNAs) are crucial modulators of post-transcriptional gene expression regulation, cell fate determination, and disease development. However, lncRNA functions during short-term heat stress in adult worker bees are poorly understood. Here, we performed deep sequencing and bioinformatic analyses of honeybee lncRNAs. RNA interference was performed by using siRNA targeting the most highly expressed lncRNA. The silencing effect on lncRNA and the relative expression levels of seven heat shock protein (HSP) genes, were subsequently examined. Overall, 7,842 lncRNAs and 115 differentially expressed lncRNAs (DELs) were identified in adult worker bees following heat stress exposure. Structural analysis revealed that the overall expression abundance, length of transcripts, exon number, and open reading frames of lncRNAs were lower than those of mRNAs. GO analysis revealed that the target genes were mainly involved in "metabolism," "protein folding," "response to stress," and "signal transduction" pathways. KEGG analysis indicated that the "protein processing in endoplasmic reticulum" and "longevity regulating pathway-multiple species" pathways were most enriched. Quantitative real-time polymerase chain reaction (qRT-PCR) detection of the selected DELs confirmed the reliability of the sequencing data. Moreover, the siRNA experiment indicated that feeding siRNA yielded a silencing efficiency of 77.51% for lncRNA MSTRG.9645.5. Upon silencing this lncRNA, the expression levels of three HSP genes were significantly downregulated (p < 0.05), whereas those of three other HSP genes were significantly upregulated (p < 0.05). Our results provide a new perspective for understanding the regulatory mechanisms of lncRNAs in adult worker bees under short-term heat stress.
Topics: Animals; Bees; RNA, Long Noncoding; Heat-Shock Response; Heat-Shock Proteins; Gene Expression Profiling; Gene Expression Regulation; RNA Interference; High-Throughput Nucleotide Sequencing; Computational Biology
PubMed: 38778290
DOI: 10.1186/s12864-024-10399-8 -
RNA (New York, N.Y.) May 2024Residing in the 5' untranslated region of the mRNA, the 2'-deoxyguanosine (2'-dG) riboswitch mRNA element adopts an alternative structure with the binding of the 2'-dG...
Residing in the 5' untranslated region of the mRNA, the 2'-deoxyguanosine (2'-dG) riboswitch mRNA element adopts an alternative structure with the binding of the 2'-dG molecule, which terminates transcription. In general, RNA conformations are strongly affected by positively charged metal ions (especially Mg2+). We have quantitatively explored the combined effect of ligand (2'-dG) and Mg2+ binding on the energy landscape of the aptamer domain of the 2'-dG riboswitch with both explicit solvent all atom molecular dynamics simulations and SHAPE biochemical probing experiments. We show that both ligand and Mg2+ are required for the stabilization of the aptamer domain; however, the two factors function in different ways. While the addition of Mg2+ remodels the energy landscape and reduces its frustration by the formation of additional contacts, the binding of 2'-dG eliminates the metastable states by building a compact core for the aptamer domain. In particular, Mg2+ ions and ligand binding are required to stabilize the most unstable helix P1 (which needs to unfold to activate the transcription platform), and the riboswitch core formed by the backbone of the P2 and P3 helices. They also facilitate a more compact structure in the three-way junction region.
PubMed: 38777381
DOI: 10.1261/rna.079767.123 -
Journal of Virology Jun 2024Enteroviruses are the causative agents associated with several human and animal diseases, posing a significant threat to human and animal health. As one of the host...
Enteroviruses are the causative agents associated with several human and animal diseases, posing a significant threat to human and animal health. As one of the host immune defense strategies, innate immunity plays a crucial role in defending against invading pathogens, where the host utilizes a variety of mechanisms to inhibit or eliminate the pathogen. Here, we report a new strategy for the host to repress enterovirus replication by the 78 kDa glucose-regulated protein (GRP78), also known as heat shock protein family A member 5 (HSPA5). The GRP78 recognizes the EV-encoded RNA-dependent RNA polymerases (RdRPs) 3D protein and interacts with the nuclear factor kappa B kinase complex (CHUK) and subunit beta gene (IKBKB) to facilitate the phosphorylation and nuclear translocation of NF-κB, which induces the production of inflammatory factors and leads to a broad inhibition of enterovirus replication. These findings demonstrate a new role of GRP78 in regulating host innate immunity in response to viral infection and provide new insights into the mechanism underlying enterovirus replication and NF-κB activation.IMPORTANCEGRP78 is known as a molecular chaperone for protein folding and plays a critical role in maintaining protein folding and participating in cell proliferation, cell survival, apoptosis, and metabolism. However, the functions of GRP78 to participate in enterovirus genome replication and innate immune responses are rarely documented. In this study, we explored the functions of the EV-3D-interacting protein GRP78 and found that GRP78 inhibits enterovirus replication by activating NF-κB through binding to EV-F 3D and interacting with the NF-κB signaling molecules CHUK/IKBKB. This is the first report that GRP78 interacts with CHUK/IKBKB to activate the NF-κB signaling pathway, which leads to the expression of the proinflammatory cytokines and inhibition of enterovirus replication. These results demonstrate a unique mechanism of virus replication regulation by GRP78 and provide insights into the prevention and treatment of viral infections.
Topics: Endoplasmic Reticulum Chaperone BiP; Humans; Virus Replication; NF-kappa B; Heat-Shock Proteins; Immunity, Innate; Enterovirus; Host-Pathogen Interactions; HEK293 Cells; Enterovirus Infections; Animals; Phosphorylation; RNA-Dependent RNA Polymerase; Signal Transduction
PubMed: 38775480
DOI: 10.1128/jvi.00268-24 -
Proteins May 2024A propeptide is removed from a precursor protein to generate its active or mature form. Propeptides play essential roles in protein folding, transportation, and...
A propeptide is removed from a precursor protein to generate its active or mature form. Propeptides play essential roles in protein folding, transportation, and activation and are present in about 2.3% of reviewed proteins in the UniProt database. They are often found in secreted or membrane-bound proteins including proteolytic enzymes, hormones, and toxins. We identified a variety of globular and nonglobular Pfam domains in protein sequences designated as propeptides, some of which form intramolecular interactions with other domains in the mature proteins. Propeptide-containing enzymes mostly function as proteases, as they are depleted in other enzyme classes such as hydrolases acting on DNA and RNA, isomerases, and lyases. We applied AlphaFold to generate structural models for over 7000 proteins with propeptides having no less than 20 residues. Analysis of residue contacts in these models revealed conformational changes for over 300 proteins before and after the cleavage of the propeptide. Examples of conformation change occur in several classes of proteolytic enzymes in the families of subtilisins, trypsins, aspartyl proteases, and thermolysin-like metalloproteases. In most of the observed cases, cleavage of the propeptide releases the constraints imposed by the covalent bond between the propeptide and the mature protein, and cleavage enables stronger interactions between the propeptide and the mature protein. These findings suggest that post-cleavage propeptides could play critical roles in regulating the activity of mature proteins.
PubMed: 38775337
DOI: 10.1002/prot.26702