Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs.

Diapause survival and post‐diapause performance (i.e., if a queen starts to lay eggs) of in total 2210 bumblebee queens (Bombus terrestris) were measured under different diapause regimes: 5 temperatures (−5, 0, 5, 10 and 15 °C) in combination with 5 durations of exposure (1, 2, 4, 6 and 8 months). The results show that weight at the start of diapause determines to a large extent whether a queen will be able to survive diapause. Queens with a wet weight below 0.6 g prior to diapause did not survive, but for those queens exceeding this threshold a higher pre‐ diapause weight did not increase their post‐diapause performance. There was no effect of temperature on diapause survival; 76% of the variance in survival could be explained by the duration of the treatment. Neither temperature nor duration of exposure had an effect on post‐diapause performance. The preoviposition period of the queens that laid eggs was also determined. The preoviposition period was affected by both temperature and duration of exposure: the preoviposition period decreased with decreasing temperature but also with increasing duration of the treatment.

Drosha-processed microRNAs (miRNAs) have been shown to be exported from the nucleus to the cytoplasm by Exportin 5, where they are processed a second time to generate mature miRNAs. In this work we show that miRNAs also use CRM1 for nuclearcytoplasmic shuttling. Inhibition of CRM1 by Leptomycin B results in nuclear accumulation of miRNA guide sequences. Nuclear to cytoplasmic transport can be actively competed by synthetic small interfering RNAs, indicating that this pathway is shared by different classes of processed small RNAs. We also find that CRM1 coimmunoprecipitates with Ago-1, Ago-2, Topo2␣, EzH2, and Mta, consistent with a role of Argonautes and small RNAs in chromatin remodeling.competition ͉ nucleus ͉ cytoplasm ͉ transport M icroRNAs (miRNAs) are implicated in numerous biological processes. Through partial or full complementarities with their targets, these short RNAs can modulate cellular gene expression and have a critical role in development as well as in some diseases. Studies on their mechanism of action have exposed unforeseen complexities, so the roles of miRNAs in the regulation of gene repression are not yet completely understood. miRNAs are transcribed in primary miRNA containing (primiRNA) transcripts that can be several kilobases long. With the exception of a minor class of miRNAs called mirtrons, which are processed by the RNA splicing machinery (1), the majority of miRNAs are processed from the primary transcripts into precursor miRNA (pre-miRNAs) within the nucleus by a complex containing the RNase III enzyme Drosha and its partner DGCR8 (2). The pre-miRNAs are transported to the cytoplasm by the nuclear karyopherin Exportin-5 in a Ran-GTP-dependent manner and processed a second time by the RNase III family member Dicer to generate an approximately 20-to 25-nt duplex, one strand of which is incorporated into the RNA-induced silencing complex (RISC) (2). A member of the Argonaute family of proteins, Ago-2, has been identified as the catalytic core of this complex. The miRNA guide sequence directs base pairing to the 3ЈUTR of its mRNA target and guides target specific repression of protein synthesis (2). It is conventionally accepted that once the premiRNA is processed in the cytoplasm, the guide sequence remains in this cellular compartment. In this work, we show that in addition to Exportin 5, another karyopherin, CRM1 (XPO-1, Exportin-1), is part of the RNA interference pathway. CRM1 allows the nuclear-cytoplasmic shuttling of the miRNA guide sequences in a complex containing RNA Helicase A (RHA) and Argonaute proteins. Synthetic small interfering RNAs (siRNAs) also use this shuttling pathway and compete with endogenous miRNAs for CRM1. Although the function of endogenous miRNAs in the nucleus is poorly understood, we find that CRM1 coimmunoprecipitates with nuclear proteins such as Topo2␣, EzH2, as well as Ago1 and Ago2, suggesting chromatin remodeling as a possible miRNA function. Results and DiscussionIt is known that nuclear Drosha-processed pre-miRNAs depend on the karyopherin Exp...

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Metapopulation dynamics of a persisting predator-prey system in the laboratory: time series analysis Janssen, A.R.M.; van Gool, F.T.J.; Lingeman, R.; Jacas, J.A.; van de Klashorst, G. Published in:Experimental and Applied Acarology DOI:10.1023/A:1018479828913 Link to publicationCitation for published version (APA): Janssen, A., van Gool, F. T. J., Lingeman, R., Jacas, J. A., & van de Klashorst, G. (1997). Metapopulation dynamics of a persisting predator-prey system in the laboratory: time series analysis. Experimental and Applied Acarology, 21, 415-430. https://doi.org/10.1023/A:1018479828913 General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. ABSTRACTThe scarcity of experimental evidence for the persistence of predator-prey systems at the metapopulation level inspired us to develop a simple predator-prey experiment that could be used for testing several theoretical predictions concerning persistence and its causes. The experimental system used consisted of one or several islands with small bean plants, the phytophagous mite Tetranychus urticae and the predatory mite Phytoseiulus persimilis. In the first experiment, one large system was used consisting of 90 small bean plants, prey and predators. The system persisted for only 120 days. Second, a system was used consisting of eight islands with ten plants each where the islands were connected by bridges. Two replicate experiments showed persistence for at least 393 days. The difference between the first and the second experiments suggests that the longer persistence is caused by a limited migration between the eight islands. Despite efforts to start both replicates of the second experiment with similar initial conditions, the dynamics of both replicates varied substantially. In one replicate the prey and predator numbers showed a trend through time, whereas the numbers fluctuated around a fixed value in the other replicate. A time series analysis of the data of the prey and predators showed the presence of periodicity with a lag of 8.5 weeks in one replicate, whereas such cyclic behaviour was not found in the other replicate. The differences between the two replicates suggest that it is difficult to perform experiments where one replicate is perturbed and the other serves as an undisturbed control. We suggest using a...