Native Circular RNA Pulldown Method to Simultaneously Profile RNA and Protein Interactions.

Circular RNAs (circRNAs) are a widespread, cell-, tissue-, and disease-specific class of largely non-coding RNA transcripts. These single-stranded, covalently-closed transcripts arise through non-canonical splicing of pre-mRNA, a process called back-splicing. Back-splicing results in circRNAs which are distinguishable from their cognate mRNA as they possess a unique sequence of nucleic acids called the backsplice junction (BSJ). CircRNAs have been shown to play key functional roles in various cellular contexts and achieve this through their interaction with other macromolecules, particularly other RNA molecules and proteins. To elucidate the molecular mechanisms underlying circRNA function, it is necessary to identify these interacting partners. Herein, we present an optimized strategy for the simultaneous purification of the circRNA interactome within eukaryotic cells, allowing the identification of both circRNA-RNA and circRNA-protein interactions.

Versatile toolkit for highly-efficient and scarless overexpression of circular RNAs.

Circular RNAs (circRNAs) are a class of single-stranded, covalently closed RNA that contain a unique back-splice junction (bsj) sequence created by the ligation of their 5' and 3' ends via spliceosome-catalyzed back-splicing. A key step in illuminating the cellular roles of specific circRNAs is via increasing their expression. This is frequently done by transfecting cells with plasmid DNA containing cloned exons from which the circRNA is transcribed, flanked by sequences that promote back-splicing. We observed that commonly used plasmids lead to the production of circRNAs with molecular scars at the circRNA bsj. Stepwise redesign of the cloning vector corrected this problem, ensuring bona fide circRNAs are produced with their natural bsj at high efficiency. The fidelity of circRNAs produced from this new construct was validated by RNA sequencing and also functionally validated. To increase the utility of this modified resource for expressing circRNA, we developed an expanded set of vectors incorporating this design that (i) enables selection with a variety of antibiotics and fluorescent proteins, (ii) employs a range of promoters varying in promoter strength and (iii) generated a complementary set of lentiviral plasmids for difficult-to-transfect cells. These resources provide a novel and versatile toolkit for high-efficiency and scarless overexpression of circular RNAs that fulfill a critical need for the investigation of circRNA function.

Circular RNAs drive oncogenic chromosomal translocations within the MLL recombinome in leukemia.

The first step of oncogenesis is the acquisition of a repertoire of genetic mutations to initiate and sustain the malignancy. An important example of this initiation phase in acute leukemias is the formation of a potent oncogene by chromosomal translocations between the mixed lineage leukemia (MLL) gene and one of 100 translocation partners, known as the MLL recombinome. Here, we show that circular RNAs (circRNAs)-a family of covalently closed, alternatively spliced RNA molecules-are enriched within the MLL recombinome and can bind DNA, forming circRNA:DNA hybrids (circR loops) at their cognate loci. These circR loops promote transcriptional pausing, proteasome inhibition, chromatin re-organization, and DNA breakage. Importantly, overexpressing circRNAs in mouse leukemia xenograft models results in co-localization of genomic loci, de novo generation of clinically relevant chromosomal translocations mimicking the MLL recombinome, and hastening of disease onset. Our findings provide fundamental insight into the acquisition of chromosomal translocations by endogenous RNA carcinogens in leukemia.

The RNA interactome in the Hallmarks of Cancer.

Ribonucleic acid (RNA) molecules are indispensable for cellular homeostasis in healthy and malignant cells. However, the functions of RNA extend well beyond that of a protein-coding template. Rather, both coding and non-coding RNA molecules function through critical interactions with a plethora of cellular molecules, including other RNAs, DNA, and proteins. Deconvoluting this RNA interactome, including the interacting partners, the nature of the interaction, and dynamic changes of these interactions in malignancies has yielded fundamental advances in knowledge and are emerging as a novel therapeutic strategy in cancer. Here, we present an RNA-centric review of recent advances in the field of RNA-RNA, RNA-protein, and RNA-DNA interactomic network analysis and their impact across the Hallmarks of Cancer. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Global mapping of RNA homodimers in living cells.

RNA homodimerization is important for various physiological processes, including the assembly of membraneless organelles, RNA subcellular localization, and packaging of viral genomes. However, understanding RNA dimerization has been hampered by the lack of systematic in vivo detection methods. Here, we show that CLASH, PARIS, and other RNA proximity ligation methods detect RNA homodimers transcriptome-wide as "overlapping" chimeric reads that contain more than one copy of the same sequence. Analyzing published proximity ligation data sets, we show that RNA:RNA homodimers mediated by direct base-pairing are rare across the human transcriptome, but highly enriched in specific transcripts, including U8 snoRNA, U2 snRNA, and a subset of tRNAs. Mutations in the homodimerization domain of U8 snoRNA impede dimerization in vitro and disrupt zebrafish development in vivo, suggesting an evolutionarily conserved role of this domain. Analysis of virus-infected cells reveals homodimerization of SARS-CoV-2 and Zika genomes, mediated by specific palindromic sequences located within protein-coding regions of N gene in SARS-CoV-2 and NS2A gene in Zika. We speculate that regions of viral genomes involved in homodimerization may constitute effective targets for antiviral therapies.

Science: priceless, but costly.

Working as a researcher is very satisfying. However, it comes with a price. This is a story about growing up as a scientist in the field of molecular biology. Starting as a young, rather naive researcher, I learned, step by step, not only the facts about my favorite RNA molecules but also the demands and downsides of academia. Going through my recent "scientific awakening," I fully acknowledged the rules of the game: to write, to publish, to patent, to apply for grants and awards, and finally, to engage in all forms of coscientific endeavors. After going through a divorce, single parenting, immigration, and being scooped, I became a scientist who finally takes her career in her own hands and navigates through, but does not succumb to, the difficulties in science. This is my monument to resilience.

SRRM4 Expands the Repertoire of Circular RNAs by Regulating Microexon Inclusion.

High-throughput RNA sequencing (RNA-seq) and dedicated bioinformatics pipelines have synergized to identify an expansive repertoire of unique circular RNAs (circRNAs), exceeding 100,000 variants. While the vast majority of these circRNAs comprise canonical exonic and intronic sequences, microexons (MEs)-which occur in 30% of functional mRNA transcripts-have been entirely overlooked. CircRNAs which contain these known MEs (ME-circRNAs) could be identified with commonly utilized circRNA prediction pipelines, CIRCexplorer2 and CIRI2, but were not previously recognized as ME-circRNAs. In addition, when employing a bespoke bioinformatics pipeline for identifying RNA chimeras, called Hyb, we could also identify over 2000 ME-circRNAs which contain novel MEs at their backsplice junctions, that are uncalled by either CIRCexplorer2 or CIRI2. Analysis of circRNA-seq datasets from gliomas of varying clinical grades compared with matched control tissue has shown circRNAs have potential as prognostic markers for stratifying tumor from healthy tissue. Furthermore, the abundance of microexon-containing circRNAs (ME-circRNAs) between tumor and normal tissues is correlated with the expression of a splicing associated factor, Serine/arginine repetitive matrix 4 (SRRM4). Overexpressing SRRM4, known for regulating ME inclusion in mRNAs critical for neural differentiation, in human HEK293 cells resulted in the biogenesis of over 2000 novel ME-circRNAs, including ME-circEIF4G3, and changes in the abundance of many canonical circRNAs, including circSETDB2 and circLBRA. This shows SRRM4, in which its expression is correlated with poor prognosis in gliomas, acts as a bona fide circRNA biogenesis factor. Given the known roles of MEs and circRNAs in oncogenesis, the identification of these previously unrecognized ME-circRNAs further increases the complexity and functional purview of this non-coding RNA family.

Mitochondrial Transcriptome Control and Intercompartment Cross-Talk During Plant Development.

We address here organellar genetic regulation and intercompartment genome coordination. We developed earlier a strategy relying on a tRNA-like shuttle to mediate import of nuclear transgene-encoded custom RNAs into mitochondria in plants. In the present work, we used this strategy to drive trans-cleaving hammerhead ribozymes into the organelles, to knock down specific mitochondrial RNAs and analyze the regulatory impact. In a similar approach, the tRNA mimic was used to import into mitochondria in Arabidopsis thaliana the orf77, an RNA associated with cytoplasmic male sterility in maize and possessing sequence identities with the atp9 mitochondrial RNA. In both cases, inducible expression of the transgenes allowed to characterise early regulation and signaling responses triggered by these respective manipulations of the organellar transcriptome. The results imply that the mitochondrial transcriptome is tightly controlled by a "buffering" mechanism at the early and intermediate stages of plant development, a control that is released at later stages. On the other hand, high throughput analyses showed that knocking down a specific mitochondrial mRNA triggered a retrograde signaling and an anterograde nuclear transcriptome response involving a series of transcription factor genes and small RNAs. Our results strongly support transcriptome coordination mechanisms within the organelles and between the organelles and the nucleus.

COMRADES determines in vivo RNA structures and interactions.

The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.

RNA dactyloscopy.

Despite the wealth of data on RNA secondary structure, conformational dynamics and tertiary structure in vitro and in vivo, predicting RNA biological activity in cellular environments remains difficult. Here, we present a comparison between in silico RNA fingerprinting and published experimental data that sheds light on efficient design of the hammerhead ribozyme molecules with a high intracellular efficiency. Our method, which we call RNA dactyloscopy, is a reliable tool for assessing the catalytic properties, modeling and design of RNA.

Inhibition of HIV-1 gp41 expression with hammerhead ribozymes.

Despite great progress in the treatment of AIDS, HIV-1 remains one of the major concerns as a human pathogen. One of the therapeutic strategies against viral infections is the application of catalytic ribonucleic acids (ribozymes) that can significantly reduce expression of a target gene by site-specific hydrolysis of its mRNA. In the present paper, we report a study on the activity of several variants of hammerhead ribozymes targeting a conserved region within mRNA encoding HIV-1 envelope glycoprotein gp41. On the basis of the data from in vitro assays and gene silencing in the cultured cells, we propose a new hammerhead ribozyme targeting the gp41-encoding sequence that can be potentially used as a therapeutic agent in AIDS treatment. Moreover, we demonstrate that the hydrolytic activity of the ribozyme in the intracellular environment cannot be inferred solely from the results of in vitro experiments.

Spiegelzymes® mirror-image hammerhead ribozymes and mirror-image DNAzymes, an alternative to siRNAs and microRNAs to cleave mRNAs in vivo?

With the discovery of small non-coding RNA (ncRNA) molecules as regulators for cellular processes, it became intriguing to develop technologies by which these regulators can be applied in molecular biology and molecular medicine. The application of ncRNAs has significantly increased our knowledge about the regulation and functions of a number of proteins in the cell. It is surprising that similar successes in applying these small ncRNAs in biotechnology and molecular medicine have so far been very limited. The reasons for these observations may lie in the high complexity in which these RNA regulators function in the cells and problems with their delivery, stability and specificity. Recently, we have described mirror-image hammerhead ribozymes and DNAzymes (Spiegelzymes®) which can sequence-specifically hydrolyse mirror-image nucleic acids, such as our mirror-image aptamers (Spiegelmers) discovered earlier. In this paper, we show for the first time that Spiegelzymes are capable of recognising complementary enantiomeric substrates (D-nucleic acids), and that they efficiently hydrolyse them at submillimolar magnesium concentrations and at physiologically relevant conditions. The Spiegelzymes are very stable in human sera, and do not require any protein factors for their function. They have the additional advantages of being non-toxic and non-immunogenic. The Spiegelzymes can be used for RNA silencing and also as therapeutic and diagnostic tools in medicine. We performed extensive three-dimensional molecular modelling experiments with mirror-image hammerhead ribozymes and DNAzymes interacting with D-RNA targets. We propose a model in which L/D-double helix structures can be formed by natural Watson-Crick base pairs, but where the nucleosides of one of the two strands will occur in an anticlinal conformation. Interestingly enough, the duplexes (L-RNA/D-RNA and L-DNA/D-RNA) in these models can show either right- or left-handedness. This is a very new observation, suggesting that molecular symmetry of enantiomeric nucleic acids is broken down.

[The world of double helix--"it did not escape our notice"]. / Swiat podwójnej helisy--"nie uszlo naszej uwadze".

One of the key questions of biology is the nature and mechanisms of gene function. It has been 60 years since proposing the right-handed model of DNA double helix in 1953. This discovery was honored with Nobel Prize in 1962 and become a breakthrough in knowing and understanding mechanisms of heredity and genetic code. Since that time a great deal of data have been gathered considering functions, structure and DNA application. It became the basis of modern molecular biology, chemical biology and biotechnology. Today we know, that double helix is characterized by its dynamics and plasticity, which depend on its nucleotide sequence. Chromatin structure and DNA mediated charge transport have a crucial role in understanding mechanisms of its damage and repair. Progress in epigenetics allowed to identify new DNA bases, such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine. Design of new catalytic nucleic acids and the nanotechnology field of DNA origami reveal its application potential.

[Functional architecture of the hammerhead ribozyme]. / Architektura funkcjonalna rybozymu hammerhead.

Hammerhead ribozyme is the smallest naturally occurring catalytic RNA. It is a perfect model for structure-function relation studies. Initially, it was identified as an autocatalytic part of viroid and virusoid genomic RNA. It exists within the genomes of many organisms including human, which makes it the most common autocatalytic motif in the nature. After 25 years of intensive research, there are a lot of data considering its structure, conformational dynamics and an influence of tertiary stabilizing motifs on its stability and properties. Structure of the hammerhead ribozyme is a system of elements that influence each other. The knowledge of ribozyme architecture is outstandingly interesting in the context of rules and logic of design, construction and application of such molecules as spatial molecular constructions. Presence of additional structural motifs distinguishes extended hammerhead ribozyme from the minimal one. Hammerhead ribozyme recognizes complementary RNA and catalyses transesterification after the 5'-NUH-3' sequence. Reaction efficiency depends on an arrangement of atoms of the catalytic core presence of metal ions and other intracellular factors. Innovative and potentially better derivatives of the hammerhead ribozyme are objects of extensive research in the field of molecular medicine.

Prediction of hammerhead ribozyme intracellular activity with the catalytic core fingerprint.

Hammerhead ribozyme is a versatile tool for down-regulation of gene expression in vivo. Owing to its small size and high activity, it is used as a model for RNA structure-function relationship studies. In the present paper we describe a new extended hammerhead ribozyme HH-2 with a tertiary stabilizing motif constructed on the basis of the tetraloop receptor sequence. This ribozyme is very active in living cells, but shows low activity in vitro. To understand it, we analysed tertiary structure models of substrate-ribozyme complexes. We calculated six unique catalytic core geometry parameters as distances and angles between particular atoms that we call the ribozyme fingerprint. A flanking sequence and tertiary motif change the geometry of the general base, general acid, nucleophile and leaving group. We found almost complete correlation between these parameters and the decrease of target gene expression in the cells. The tertiary structure model calculations allow us to predict ribozyme intracellular activity. Our approach could be widely adapted to characterize catalytic properties of other RNAs.

[DNA: from Miescher to Venter and beyond]. / DNA: od Mieschera do Ventera i dalej.

DNA, one of the most famous molecules is 140-years-old. Its history has engaged three centuries of experiments, leading us to a point, where the Homo sapiens genome sequence is known. The "DNA breakthrough" is dated on 1953, when James Watson and Francis Crick proposed the model of molecular structure of DNA. But the origin of that great achievement goes back to 1869 and early efforts of Friedrich Miescher, the Swiss doctor, who isolated DNA (than termed nuclein) for the first time. Since that time wealth information on "nuclein", its functions, structure and usage has been collected and formed a basis for modern molecular biology, chemical biology and biotechnology. This article describes the events and circumstances of the most important DNA discoveries since its first isolation up to completing the human genome project and deep DNA sequencing techniques application.