RNA-RNA recombination is one of the strongest forces shaping the genomes of plant RNA viruses. The detection of recombination is a challenging task that prompted the development of both in vitro and in vivo experimental systems. In the divided genome of Brome mosaic virus system, both inter- and intrasegmental crossovers are described. Other systems utilize satellite or defective interfering RNAs (DI-RNAs) of Turnip crinkle virus, Tomato bushy stunt virus, Cucumber necrosis virus, and Potato virus X. These assays identified the mechanistic details of the recombination process, revealing the role of RNA structure and proteins in the replicase-mediated copy-choice mechanism. In copy choice, the polymerase and the nascent RNA chain from which it is synthesized switch from one RNA template to another. RNA recombination was found to mediate the rearrangement of viral genes, the repair of deleterious mutations, and the acquisition of nonself sequences influencing the phylogenetics of viral taxa. The evidence for recombination, not only between related viruses but also among distantly related viruses, and even with host RNAs, suggests that plant viruses unabashedly test recombination with any genetic material at hand.

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.

Previously we demonstrated frequent homologous crossovers among molecules of the RNA3 segment in the tripartite brome mosaic bromovirus (BMV) RNA genome (A. Bruyere, M. Wantroba, S. Flasinski, A. Dzianott, and J. J. Bujarski, J. Virol. 74:4214-4219, 2000). To further our knowledge about mechanisms of viral RNA genome variability, in this paper we have studied homologous recombination in BMV RNA1 and RNA2 components during infection. We have found that basal RNA-RNA crossovers could occur within coding regions of both RNAs, although recombination frequencies slightly varied at different RNA sections. In all cases, the frequencies were much lower than the rate observed for the intercistronic recombination hot spot in BMV RNA3. Probability calculations accounted for at least one homologous crossover per RNA molecule per replication cycle. In addition, we have demonstrated an efficient repair of mutations within the conserved 3 and 5 noncoding regions, most likely due to error-prone BMV RNA replication. Overall, our data verify that homologous crossovers are common events a during virus life cycle, and we discuss their importance for viral RNA genetics.Enormous genetic variability is one of the unusual features of RNA viruses. Numerous experiments reveal two main sources of genetic polymorphism that contribute to the rapid evolution of RNA viruses: error-prone replication and RNA recombination (25). The former introduces into the viral RNA genome a wide spectrum of point mutations at the rate of 10 Ϫ4 to 10 Ϫ5 per nucleotide per replication cycle (33, 34). The latter is a widespread phenomenon described in many groups of RNA viruses, including picornaviruses (3,16,20), coronaviruses (5, 41), and alphaviruses (15) and in plant viruses: plum pox potyvirus (9), bromoviruses (4, 7, 8, 13), alfalfa mosaic virus (36), cucumber mosaic virus (CMV) (1, 10), tobacco mosaic virus (2), turnip crinkle virus (29, 30), and tomato bushy stunt tombusvirus (38). RNA recombination is also seen in bacteriophage Q␤ (31), in negative RNA viruses (11), in double-stranded RNA viruses (35), and in retroviruses (17, 24) as well as during the formation of defective interfering RNAs (38).In spite of intensive studies, the mechanism of RNA recombination is not well understood. The copy choice mechanism, which is the most widely accepted (21), assumes that RNA recombinants result from template switching by viral RNA polymerase (RdRp) during RNA replication. Depending on the primary structure of the recombining molecules and on the location of junction sites, two types of recombination events have been recognized: homologous and nonhomologous (21), with the former being 10-fold higher than the latter in the case of brome mosaic bromovirus (BMV) (26).There is little information about homologous recombination in natural virus populations, because recombination products do not differ from parental RNAs. The crossovers in poliovirus RNA tended to occur within potential inter-and intramolecular heteroduplex regions (3, 20), whereas in mouse hepatitis ...

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-016-1936-8) contains supplementary material, which is available to authorized users.

Lyme disease (also called borreliosis) is a prevalent chronic disease transmitted by ticks and caused by Borrelia burgdorferi s. l. spirochete. At least one tick protein, namely TROSPA from I. scapularis, commonly occurring in the USA, was shown to be required for colonization of the vector by bacteria. Located in the tick gut, TROSPA interacts with the spirochete outer surface protein A (OspA) and initiates the tick colonization. Ixodes ricinus is a primary vector involved in B. burgdorferi s. l. transmission in most European countries. In this study, we characterized the capacities of recombinant TROSPA protein from I. ricinus to interact with OspA from different Borrelia species and to induce an immune response in animals. We also showed that the N-terminal part of TROSPA (a putative transmembrane domain) is not involved in the interaction with OspA and that reduction of the total negative charge on the TROSPA protein impaired TROSPA-OspA binding. In general, the data presented in this paper indicate that recombinant TROSPA protein retains the capacity to form a complex with OspA and induces a significant level of IgG in orally immunized rats. Thus, I. ricinus TROSPA may be considered a good candidate component for an animal vaccine against Borrelia.