Anna Kurzynska-Kokorniak

Genetic variability: the key problem in the prevention and therapy of RNA-based virus infections.

Despite extraordinary progress that has recently been made in biomedical sciences, viral infectious diseases still remain one of the most serious world health problems. Among the different types of viruses, those using RNA as their genetic material (RNA viruses and retroviruses) are especially dangerous. At present there is no medicine allowing an effective treatment of RNA‐based virus infections. Many RNA viruses and retroviruses need only a few weeks to escape immune response or to produce drug‐resistant mutants. This seems to be the obvious consequence of the unusual genetic variability of RNA‐based viruses. An individual virus does not form a homogenous population but rather a set of similar but not identical variants. In consequence, RNA‐based viruses can easily adapt to environmental changes, also those resulting from immune system response or therapy. The modifications identified within viral genes can be divided into two groups: point mutations and complex genome rearrangements. The former arises mainly during error‐prone replication, whereas RNA recombination and generic reassortment are responsible for the latter. This article shortly describes major strategies used to control virus infections. Then, it presents the various mechanisms generating the genetic diversity of RNA‐based viruses, which are most probably the main cause of clinical problems.

The many faces of Dicer: the complexity of the mechanisms regulating Dicer gene expression and enzyme activities

There is increasing evidence indicating that the production of small regulatory RNAs is not the only process in which ribonuclease Dicer can participate. For example, it has been demonstrated that this enzyme is also involved in chromatin structure remodelling, inflammation and apoptotic DNA degradation. Moreover, it has become increasingly clear that cellular transcript and protein levels of Dicer must be strictly controlled because even small changes in their accumulation can initiate various pathological processes, including carcinogenesis. Accordingly, in recent years, a number of studies have been performed to identify the factors regulating Dicer gene expression and protein activity. As a result, a large amount of complex and often contradictory data has been generated. None of these data have been subjected to an exhaustive review or critical discussion. This review attempts to fill this gap by summarizing the current knowledge of factors that regulate Dicer gene transcription, primary transcript processing, mRNA translation and enzyme activity. Because of the high complexity of this topic, this review mainly concentrates on human Dicer. This review also focuses on an additional regulatory layer of Dicer activity involving the interactions of protein and RNA factors with Dicer substrates.

Human- and Virus-Encoded microRNAs as Potential Targets of Antiviral Therapy

It has recently been demonstrated that short RNA molecules, called microRNAs, are one of the major factors regulating the expression of human genes. There are several lines of evidence that microRNAs also play a key role in host-virus interactions. It is believed that both human- and virus-encoded miRNA will, in the nearest future, become very attractive targets of antiviral therapy.

A New Short Oligonucleotide-Based Strategy for the Precursor-Specific Regulation of microRNA Processing by Dicer

The precise regulation of microRNA (miRNA) biogenesis seems to be critically important for the proper functioning of all eukaryotic organisms. Even small changes in the levels of specific miRNAs can initiate pathological processes, including carcinogenesis. Accordingly, there is a great need to develop effective methods for the regulation of miRNA biogenesis and activity. In this study, we focused on the final step of miRNA biogenesis; i.e., miRNA processing by Dicer. To test our hypothesis that RNA molecules can function not only as Dicer substrates but also as Dicer regulators, we previously identified by SELEX a pool of RNA oligomers that bind to human Dicer. We found that certain of these RNA oligomers could selectively inhibit the formation of specific miRNAs. Here, we show that these specific inhibitors can simultaneously bind both Dicer and pre-miRNAs. These bifunctional riboregulators interfere with miRNA maturation by affecting pre-miRNA structure and sequestering Dicer. Based on these observations, we designed a set of short oligomers (12 nucleotides long) that were capable of influencing pre-miRNA processing in vitro, both in reactions involving recombinant human Dicer and in cytosolic extracts. We propose that the same strategy may be used to develop effective and selective regulators to control the production of any miRNA. Overall, our findings indicate that the interactions between pre-miRNAs and other RNAs may form very complex regulatory networks that modulate miRNA biogenesis and consequently gene expression.

Selection of RNA Oligonucleotides That Can Modulate Human Dicer Activity In Vitro

Human ribonuclease Dicer is an enzyme that excises small regulatory RNAs from perfectly or partially double-stranded RNA precursors. Although Dicer substrates and products have already been quite well characterized, our knowledge about cellular factors regulating the activity of this enzyme is still limited. To learn more about this problem, we attempted to determine whether RNA could function not only as a Dicer substrate but also as its regulator. To this end, we applied an in vitro selection method. We identified 120 RNA oligomers binding human Dicer. Sixteen of them were subjected to more detailed in vitro studies. We found that 6 out of 16 oligomers affected Dicer ability to digest pre-microRNAs (miRNAs), although most of them were cleaved by this enzyme. For the 6 most active oligomers the putative mechanism of Dicer inhibition was determined. Three oligomers were classified as typical competitive inhibitors and one as an allosteric inhibitor. The remaining 2 oligomers acted as selective inhibitors. They affected the production of 1 miRNA, whereas the formation of other miRNAs was hardly influenced. In general, the data obtained suggest that one can modulate the generation of specific miRNAs by using RNA oligomers. Moreover, we found that sequences similar to those of the selected oligomers can be found within the molecules composing human transcriptome.

Revealing a new activity of the human Dicer DUF283 domain in vitro

The ribonuclease Dicer is a multidomain enzyme that plays a fundamental role in the biogenesis of small regulatory RNAs (srRNAs), which control gene expression by targeting complementary transcripts and inducing their cleavage or repressing their translation. Recent studies of Dicer’s domains have permitted to propose their roles in srRNA biogenesis. For all of Dicer’s domains except one, called DUF283 (domain of unknown function), their involvement in RNA substrate recognition, binding or cleavage has been postulated. For DUF283, the interaction with Dicer’s protein partners has been the only function suggested thus far. In this report, we demonstrate that the isolated DUF283 domain from human Dicer is capable of binding single-stranded nucleic acids in vitro. We also show that DUF283 can act as a nucleic acid annealer that accelerates base-pairing between complementary RNA/DNA molecules in vitro. We further demonstrate an annealing activity of full length human Dicer. The overall results suggest that Dicer, presumably through its DUF283 domain, might facilitate hybridization between short RNAs and their targets. The presented findings reveal the complex nature of Dicer, whose functions may extend beyond the biogenesis of srRNAs.

Six Medicago truncatula Dicer-like protein genes are expressed in plant cells and upregulated in nodules

Key messageHere we report the existence of six putative Dicer-likegenes in theMedicago truncatulagenome. They are ubiquitously expressed throughout the plant and significantly induced in root nodules.AbstractOver the past decade, small noncoding RNAs (sncRNA) have emerged as widespread and important regulatory molecules influencing both the structure and expression of plant genomes. One of the key factors involved in sncRNA biogenesis in plants is a group of RNase III-type nucleases known as Dicer-like (DCL) proteins. Based on functional analysis of DCL proteins identified in Arabidopsis thaliana, four types of DCLs were distinguished (DCL1-4). DCL1 mainly produces 21 nt miRNAs. The products generated by DCL2, DCL3, and DCL4 belong to various classes of siRNAs that are 22, 24 and 21 nt in length, respectively. M. truncatula is a model legume plant closely related to many economically important cultivable species. By screening the recent M. truncatula genome assembly, we were able to identify three new DCL genes in addition to the MtDCL1-3 genes that had been earlier characterized. The newly found genes include MtDCL4 and two MtDCL2 homologs. We showed that all six M. truncatula DCL genes are expressed in plant cells. The first of the identified MtDCL2 paralogs encodes a truncated version of the DCL2 protein, while the second undergoes substantial and specific upregulation in the root nodules. Additionally, we identified an alternative splicing variant of MtDCL1 mRNA, similar to the one found in Arabidopsis. Our results indicate that DCL genes are differently activated during Medicago symbiosis with nitrogen fixing bacteria and upon pathogen infection. In addition, we hypothesize that the alternative splicing variant of MtDCL1 mRNA may be involved in tissue-specific regulation of the DCL1 level.

Modeling of the catalytic core of Arabidopsis thaliana Dicer-like 4 protein and its complex with double-stranded RNA

Plant Dicer-like proteins (DCLs) belong to the Ribonuclease III (RNase III) enzyme family. They are involved in the regulation of gene expression and antiviral defense through RNA interference pathways. A model plant, Arabidopsis thaliana encodes four DCL proteins (AtDCL1-4) that produce different classes of small regulatory RNAs. Our studies focus on AtDCL4 that processes double-stranded RNAs (dsRNAs) into 21 nucleotide trans-acting small interfering RNAs. So far, little is known about the structures of plant DCLs and the complexes they form with dsRNA. In this work, we present models of the catalytic core of AtDCL4 and AtDCL4-dsRNA complex constructed by computational methods. We built a homology model of the catalytic core of AtDCL4 comprising Platform, PAZ, Connector helix and two RNase III domains. To assemble the AtDCL4-dsRNA complex two modeling approaches were used. In the first method, to establish conformations that allow building a consistent model of the complex, we used Normal Mode Analysis for both dsRNA and AtDCL4. The second strategy involved template-based approach for positioning of the PAZ domain and manual arrangement of the Connector helix. Our results suggest that the spatial orientation of the Connector helix, Platform and PAZ relative to the RNase III domains is crucial for measuring dsRNA of defined length. The modeled complexes provide information about interactions that may contribute to the relative orientations of these domains and to dsRNA binding. All these information can be helpful for understanding the mechanism of AtDCL4-mediated dsRNA recognition and binding, to produce small RNA of specific size.

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzymes activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

EvOligo: A Novel Software to Design and Group Libraries of Oligonucleotides Applicable for Nucleic Acid-Based Experiments

Experimental methods based on DNA and RNA hybridization, such as multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, or microarray analysis, require the use of mixtures of multiple oligonucleotides (primers or probes) in a single test tube. To provide an optimal reaction environment, minimal self- and cross-hybridization must be achieved among these oligonucleotides. To address this problem, we developed EvOligo, which is a software package that provides the means to design and group DNA and RNA molecules with defined lengths. EvOligo combines two modules. The first module performs oligonucleotide design, and the second module performs oligonucleotide grouping. The software applies a nearest-neighbor model of nucleic acid interactions coupled with a parallel evolutionary algorithm to construct individual oligonucleotides, and to group the molecules that are characterized by the weakest possible cross-interactions. To provide optimal solutions, the evolutionary algorithm sorts oligonucleotides into sets, preserves preselected parts of the oligonucleotides, and shapes their remaining parts. In addition, the oligonucleotide sets can be designed and grouped based on their melting temperatures. For the users convenience, EvOligo is provided with a user-friendly graphical interface. EvOligo was used to design individual oligonucleotides, oligonucleotide pairs, and groups of oligonucleotide pairs that are characterized by the following parameters: (1) weaker cross-interactions between the non-complementary oligonucleotides and (2) more uniform ranges of the oligonucleotide pair melting temperatures than other available software products. In addition, in contrast to other grouping algorithms, EvOligo offers time-efficient sorting of paired and unpaired oligonucleotides based on various parameters defined by the user.