New generation of artificial microRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis

[EN] Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are used for small RNA-based, specific gene silencing or knockdown in plants. Current methods to generate amiRNA or syn-tasiRNA constructs are not well adapted for cost-effective, large-scale production or for multiplexing to specifically suppress multiple targets. Here, we describe simple, fast, and cost-effective methods with high-throughput capability to generate amiRNA and multiplexed syn-tasiRNA constructs for efficient gene silencing in Arabidopsis (Arabidopsis thaliana) and other plant species. amiRNA or syn-tasiRNA inserts resulting from the annealing of two overlapping and partially complementary oligonucleotides are ligated directionally into a zero background BsaI/ccdB-based expression vector. BsaI/ccdB vectors for amiRNA or syn-tasiRNA cloning and expression contain a modified version of Arabidopsis MIR390a or TAS1c precursors, respectively, in which a fragment of the endogenous sequence was substituted by a ccdB cassette flanked by two BsaI sites. Several amiRNA and syn-tasiRNA sequences designed to target one or more endogenous genes were validated in transgenic plants that (1) exhibited the expected phenotypes predicted by loss of target gene function, (2) accumulated high levels of accurately processed amiRNAs or syn-tasiRNAs, and (3) had reduced levels of the corresponding target RNAs.This work was supported by the National Science Foundation (grant nos. MCB-0956526 and MCB-1231726), the National Institutes of Health (grant no. AI043288), the Japan Society for the Promotion of Science (postdoctoral fellowship to A.T.), and the National Institute of Food and Agriculture (postdoctoral fellowship no. MOW-2012-01361 to N.F.)Carbonell, A.; Takeda, A.; Fahlgren, N.; Johnson, SC.; Cuperus, JT.; Carrington, JC. (2014). New generation of artificial microRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. PLANT PHYSIOLOGY. 165(1):15-29. https://doi.org/10.1104/pp.113.234989S1529165

Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants

[EN] In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.We thank Goretti Nguyen for excellent technical assistance. A. C. was supported by a postdoctoral fellowship from the Ministerio de Ciencia e Innovacion (BMC-2008-0188). H.G.-R. was the recipient of a Helen Hay Whitney Postdoctoral fellowship (F-972). This work was supported by grants from the National Science Foundation (MCB-1231726), the National Institutes of Health (AI043288), and Monsanto Corporation.Carbonell, A.; Fahlgren, N.; García-Ruíz, H.; Gilbert, KB.; Montgomery, TA.; Nguyen, T.; Cuperus, JT.... (2012). Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. The Plant Cell. 24(9):3613-3629. https://doi.org/10.1105/tpc.112.099945S36133629249Allen, E., Xie, Z., Gustafson, A. M., & Carrington, J. C. (2005). microRNA-Directed Phasing during Trans-Acting siRNA Biogenesis in Plants. Cell, 121(2), 207-221. doi:10.1016/j.cell.2005.04.004Aukerman, M. J., & Sakai, H. (2003). Regulation of Flowering Time and Floral Organ Identity by a MicroRNA and Its APETALA2-Like Target Genes. The Plant Cell, 15(11), 2730-2741. doi:10.1105/tpc.016238Axtell, M. J., Jan, C., Rajagopalan, R., & Bartel, D. P. (2006). A Two-Hit Trigger for siRNA Biogenesis in Plants. Cell, 127(3), 565-577. doi:10.1016/j.cell.2006.09.032Baek, D., Villén, J., Shin, C., Camargo, F. D., Gygi, S. P., & Bartel, D. P. (2008). The impact of microRNAs on protein output. Nature, 455(7209), 64-71. doi:10.1038/nature07242Baumberger, N., & Baulcombe, D. C. (2005). Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proceedings of the National Academy of Sciences, 102(33), 11928-11933. doi:10.1073/pnas.0505461102Brodersen, P., Sakvarelidze-Achard, L., Bruun-Rasmussen, M., Dunoyer, P., Yamamoto, Y. Y., Sieburth, L., & Voinnet, O. (2008). Widespread Translational Inhibition by Plant miRNAs and siRNAs. Science, 320(5880), 1185-1190. doi:10.1126/science.1159151Chekanova, J. A., Gregory, B. D., Reverdatto, S. V., Chen, H., Kumar, R., Hooker, T., … Belostotsky, D. A. (2007). Genome-Wide High-Resolution Mapping of Exosome Substrates Reveals Hidden Features in the Arabidopsis Transcriptome. Cell, 131(7), 1340-1353. doi:10.1016/j.cell.2007.10.056Chen, H.-M., Chen, L.-T., Patel, K., Li, Y.-H., Baulcombe, D. C., & Wu, S.-H. (2010). 22-nucleotide RNAs trigger secondary siRNA biogenesis in plants. Proceedings of the National Academy of Sciences, 107(34), 15269-15274. doi:10.1073/pnas.1001738107Chen, X. (2004). A MicroRNA as a Translational Repressor of APETALA2 in Arabidopsis Flower Development. Science, 303(5666), 2022-2025. doi:10.1126/science.1088060Chi, S. W., Zang, J. B., Mele, A., & Darnell, R. B. (2009). Argonaute HITS-CLIP decodes microRNA–mRNA interaction maps. Nature, 460(7254), 479-486. doi:10.1038/nature08170Clough, S. J., & Bent, A. F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana. The Plant Journal, 16(6), 735-743. doi:10.1046/j.1365-313x.1998.00343.xCuperus, J. T., Carbonell, A., Fahlgren, N., Garcia-Ruiz, H., Burke, R. T., Takeda, A., … Carrington, J. C. (2010). Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis. Nature Structural & Molecular Biology, 17(8), 997-1003. doi:10.1038/nsmb.1866Curtis, M. D., & Grossniklaus, U. (2003). A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta. Plant Physiology, 133(2), 462-469. doi:10.1104/pp.103.027979Dunoyer, P., Himber, C., & Voinnet, O. (2005). DICER-LIKE 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal. Nature Genetics, 37(12), 1356-1360. doi:10.1038/ng1675Eulalio, A., Huntzinger, E., & Izaurralde, E. (2008). Getting to the Root of miRNA-Mediated Gene Silencing. Cell, 132(1), 9-14. doi:10.1016/j.cell.2007.12.024Fahlgren, N., Sullivan, C. M., Kasschau, K. D., Chapman, E. J., Cumbie, J. S., Montgomery, T. A., … Carrington, J. C. (2009). Computational and analytical framework for small RNA profiling by high-throughput sequencing. RNA, 15(5), 992-1002. doi:10.1261/rna.1473809Filipowicz, W., Bhattacharyya, S. N., & Sonenberg, N. (2008). Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nature Reviews Genetics, 9(2), 102-114. doi:10.1038/nrg2290Gandikota, M., Birkenbihl, R. P., Höhmann, S., Cardon, G. H., Saedler, H., & Huijser, P. (2007). The miRNA156/157 recognition element in the 3′ UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings. The Plant Journal, 49(4), 683-693. doi:10.1111/j.1365-313x.2006.02983.xGarcia-Ruiz, H., Takeda, A., Chapman, E. J., Sullivan, C. M., Fahlgren, N., Brempelis, K. J., & Carrington, J. C. (2010). Arabidopsis RNA-Dependent RNA Polymerases and Dicer-Like Proteins in Antiviral Defense and Small Interfering RNA Biogenesis during Turnip Mosaic Virus Infection  . The Plant Cell, 22(2), 481-496. doi:10.1105/tpc.109.073056Gasciolli, V., Mallory, A. C., Bartel, D. P., & Vaucheret, H. (2005). Partially Redundant Functions of Arabidopsis DICER-like Enzymes and a Role for DCL4 in Producing trans-Acting siRNAs. Current Biology, 15(16), 1494-1500. doi:10.1016/j.cub.2005.07.024Guo, H., Ingolia, N. T., Weissman, J. S., & Bartel, D. P. (2010). Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature, 466(7308), 835-840. doi:10.1038/nature09267Harvey, J. J. W., Lewsey, M. G., Patel, K., Westwood, J., Heimstädt, S., Carr, J. P., & Baulcombe, D. C. (2011). An Antiviral Defense Role of AGO2 in Plants. PLoS ONE, 6(1), e14639. doi:10.1371/journal.pone.0014639Havecker, E. R., Wallbridge, L. M., Hardcastle, T. J., Bush, M. S., Kelly, K. A., Dunn, R. M., … Baulcombe, D. C. (2010). TheArabidopsisRNA-Directed DNA Methylation Argonautes Functionally Diverge Based on Their Expression and Interaction with Target Loci  . The Plant Cell, 22(2), 321-334. doi:10.1105/tpc.109.072199Hendrickson, D. G., Hogan, D. J., McCullough, H. L., Myers, J. W., Herschlag, D., Ferrell, J. E., & Brown, P. O. (2009). Concordant Regulation of Translation and mRNA Abundance for Hundreds of Targets of a Human microRNA. PLoS Biology, 7(11), e1000238. doi:10.1371/journal.pbio.1000238Hunter, C., Sun, H., & Poethig, R. S. (2003). The Arabidopsis Heterochronic Gene ZIPPY Is an ARGONAUTE Family Member. Current Biology, 13(19), 1734-1739. doi:10.1016/j.cub.2003.09.004Huntzinger, E., & Izaurralde, E. (2011). Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nature Reviews Genetics, 12(2), 99-110. doi:10.1038/nrg2936Iki, T., Yoshikawa, M., Nishikiori, M., Jaudal, M. C., Matsumoto-Yokoyama, E., Mitsuhara, I., … Ishikawa, M. (2010). In Vitro Assembly of Plant RNA-Induced Silencing Complexes Facilitated by Molecular Chaperone HSP90. Molecular Cell, 39(2), 282-291. doi:10.1016/j.molcel.2010.05.014Jaubert, M., Bhattacharjee, S., Mello, A. F. S., Perry, K. L., & Moffett, P. (2011). ARGONAUTE2 Mediates RNA-Silencing Antiviral Defenses against Potato virus X in Arabidopsis    . Plant Physiology, 156(3), 1556-1564. doi:10.1104/pp.111.178012Ji, L., Liu, X., Yan, J., Wang, W., Yumul, R. E., Kim, Y. J., … Chen, X. (2011). ARGONAUTE10 and ARGONAUTE1 Regulate the Termination of Floral Stem Cells through Two MicroRNAs in Arabidopsis. PLoS Genetics, 7(3), e1001358. doi:10.1371/journal.pgen.1001358Kim, V. N., Han, J., & Siomi, M. C. (2009). Biogenesis of small RNAs in animals. Nature Reviews Molecular Cell Biology, 10(2), 126-139. doi:10.1038/nrm2632Lanet, E., Delannoy, E., Sormani, R., Floris, M., Brodersen, P., Crété, P., … Robaglia, C. (2009). Biochemical Evidence for Translational Repression by Arabidopsis MicroRNAs. The Plant Cell, 21(6), 1762-1768. doi:10.1105/tpc.108.063412Langmead, B., Trapnell, C., Pop, M., & Salzberg, S. L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology, 10(3), R25. doi:10.1186/gb-2009-10-3-r25Leung, A. K. L., Young, A. G., Bhutkar, A., Zheng, G. X., Bosson, A. D., Nielsen, C. B., & Sharp, P. A. (2011). Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs. Nature Structural & Molecular Biology, 18(2), 237-244. doi:10.1038/nsmb.1991Llave, C., Xie, Z., Kasschau, K. D., & Carrington, J. C. (2002). Cleavage of Scarecrow-like mRNA Targets Directed by a Class of Arabidopsis miRNA. Science, 297(5589), 2053-2056. doi:10.1126/science.1076311Lobbes, D., Rallapalli, G., Schmidt, D. D., Martin, C., & Clarke, J. (2006). SERRATE: a new player on the plant microRNA scene. EMBO reports, 7(10), 1052-1058. doi:10.1038/sj.embor.7400806Mallory, A., & Vaucheret, H. (2010). Form, Function, and Regulation of ARGONAUTE Proteins. The Plant Cell, 22(12), 3879-3889. doi:10.1105/tpc.110.080671Manavella, P. A., Koenig, D., & Weigel, D. (2012). Plant secondary siRNA production determined by microRNA-duplex structure. Proceedings of the National Academy of Sciences, 109(7), 2461-2466. doi:10.1073/pnas.1200169109Matranga, C., Tomari, Y., Shin, C., Bartel, D. P., & Zamore, P. D. (2005). Passenger-Strand Cleavage Facilitates Assembly of siRNA into Ago2-Containing RNAi Enzyme Complexes. Cell, 123(4), 607-620. doi:10.1016/j.cell.2005.08.044Mi, S., Cai, T., Hu, Y., Chen, Y., Hodges, E., Ni, F., … Qi, Y. (2008). Sorting of Small RNAs into Arabidopsis Argonaute Complexes Is Directed by the 5′ Terminal Nucleotide. Cell, 133(1), 116-127. doi:10.1016/j.cell.2008.02.034Montgomery, T. A., Howell, M. D., Cuperus, J. T., Li, D., Hansen, J. E., Alexander, A. L., … Carrington, J. C. (2008). Specificity of ARGONAUTE7-miR390 Interaction and Dual Functionality in TAS3 Trans-Acting siRNA Formation. Cell, 133(1), 128-141. doi:10.1016/j.cell.2008.02.033Montgomery, T. A., Yoo, S. J., Fahlgren, N., Gilbert, S. D., Howell, M. D., Sullivan, C. M., … Carrington, J. C. (2008). AGO1-miR173 complex initiates phased siRNA formation in plants. Proceedings of the National Academy of Sciences, 105(51), 20055-20062. doi:10.1073/pnas.0810241105Morel, J.-B., Godon, C., Mourrain, P., Béclin, C., Boutet, S., Feuerbach, F., … Vaucheret, H. (2002). Fertile Hypomorphic ARGONAUTE (ago1) Mutants Impaired in Post-Transcriptional Gene Silencing and Virus Resistance. The Plant Cell, 14(3), 629-639. doi:10.1105/tpc.010358Peragine, A. (2004). SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes & Development, 18(19), 2368-2379. doi:10.1101/gad.1231804Qi, Y., He, X., Wang, X.-J., Kohany, O., Jurka, J., & Hannon, G. J. (2006). Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature, 443(7114), 1008-1012. doi:10.1038/nature05198Rajagopalan, R., Vaucheret, H., Trejo, J., & Bartel, D. P. (2006). A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes & Development, 20(24), 3407-3425. doi:10.1101/gad.1476406Scholthof, H. B., Alvarado, V. Y., Vega-Arreguin, J. C., Ciomperlik, J., Odokonyero, D., Brosseau, C., … Moffett, P. (2011). Identification of an ARGONAUTE for Antiviral RNA Silencing in Nicotiana benthamiana        . Plant Physiology, 156(3), 1548-1555. doi:10.1104/pp.111.178764Song, J.-J., Smith, S. K., Hannon, G. J., & Joshua-Tor, L. (2004). Crystal Structure of Argonaute and Its Implications for RISC Slicer Activity. Science, 305(5689), 1434-1437. doi:10.1126/science.1102514Souret, F. F., Kastenmayer, J. P., & Green, P. J. (2004). AtXRN4 Degrades mRNA in Arabidopsis and Its Substrates Include Selected miRNA Targets. Molecular Cell, 15(2), 173-183. doi:10.1016/j.molcel.2004.06.006Wang, L., Si, Y., Dedow, L. K., Shao, Y., Liu, P., & Brutnell, T. P. (2011). A Low-Cost Library Construction Protocol and Data Analysis Pipeline for Illumina-Based Strand-Specific Multiplex RNA-Seq. PLoS ONE, 6(10), e26426. doi:10.1371/journal.pone.0026426Wang, X.-B., Jovel, J., Udomporn, P., Wang, Y., Wu, Q., Li, W.-X., … Ding, S.-W. (2011). The 21-Nucleotide, but Not 22-Nucleotide, Viral Secondary Small Interfering RNAs Direct Potent Antiviral Defense by Two Cooperative Argonautes in Arabidopsis thaliana    . The Plant Cell, 23(4), 1625-1638. doi:10.1105/tpc.110.082305Wang, Y., Juranek, S., Li, H., Sheng, G., Wardle, G. S., Tuschl, T., & Patel, D. J. (2009). Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes. Nature, 461(7265), 754-761. doi:10.1038/nature08434Wu, L., & Belasco, J. G. (2008). Let Me Count the Ways: Mechanisms of Gene Regulation by miRNAs and siRNAs. Molecular Cell, 29(1), 1-7. doi:10.1016/j.molcel.2007.12.010Xie, Z., Allen, E., Wilken, A., & Carrington, J. C. (2005). DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, 102(36), 12984-12989. doi:10.1073/pnas.0506426102Yang, L., Wu, G., & Poethig, R. S. (2011). Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis. Proceedings of the National Academy of Sciences, 109(1), 315-320. doi:10.1073/pnas.1114673109Yoshikawa, M. (2005). A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes & Development, 19(18), 2164-2175. doi:10.1101/gad.1352605Zhang, X., Zhao, H., Gao, S., Wang, W.-C., Katiyar-Agarwal, S., Huang, H.-D., … Jin, H. (2011). Arabidopsis Argonaute 2 Regulates Innate Immunity via miRNA393∗-Mediated Silencing of a Golgi-Localized SNARE Gene, MEMB12. Molecular Cell, 42(3), 356-366. doi:10.1016/j.molcel.2011.04.010Zhu, H., Hu, F., Wang, R., Zhou, X., Sze, S.-H., Liou, L. W., … Zhang, X. (2011). Arabidopsis Argonaute10 Specifically Sequesters miR166/165 to Regulate Shoot Apical Meristem Development. Cell, 145(2), 242-256. doi:10.1016/j.cell.2011.03.024Zisoulis, D. G., Lovci, M. T., Wilbert, M. L., Hutt, K. R., Liang, T. Y., Pasquinelli, A. E., & Yeo, G. W. (2010). Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nature Structural & Molecular Biology, 17(2), 173-179. doi:10.1038/nsmb.174

Specificity of ARGONAUTE7-miR390 Interaction and Dual Functionality in TAS3 Trans-Acting siRNA Formation

SummaryTrans-acting siRNA form through a refined RNAi mechanism in plants. miRNA-guided cleavage triggers entry of precursor transcripts into an RNA-DEPENDENT RNA POLYMERASE6 pathway, and sets the register for phased tasiRNA formation by DICER-LIKE4. Here, we show that miR390-ARGONAUTE7 complexes function in distinct cleavage or noncleavage modes at two target sites in TAS3a transcripts. The AGO7 cleavage, but not the noncleavage, function could be provided by AGO1, the dominant miRNA-associated AGO, but only when AGO1 was guided to a modified target site through an alternate miRNA. AGO7 was highly selective for interaction with miR390, and miR390 in turn was excluded from association with AGO1 due entirely to an incompatible 5′ adenosine. Analysis of AGO1, AGO2, and AGO7 revealed a potent 5′ nucleotide discrimination function for some, although not all, ARGONAUTEs. miR390 and AGO7, therefore, evolved as a highly specific miRNA guide/effector protein pair to function at two distinct tasiRNA biogenesis steps

Measuring burden of disease in both asthma and COPD by merging the ACQ and CCQ:less is more?

Symptoms of asthma and COPD often overlap, and both diseases can co-exist in one patient. The asthma control questionnaire (ACQ) and clinical COPD questionnaire (CCQ) were developed to assess disease burden in respectively asthma or COPD. This study explores the possibility of creating a new questionnaire to assess disease burden in all obstructive lung diseases by integrating and reducing questions of the ACQ and CCQ. Data of patients with asthma, COPD and asthma-COPD overlap (ACO) were collected from a primary and secondary care center. Patients completed ACQ and CCQ on the same day. Linear regression tested correlations. Principal Component Analysis (PCA) was used for item reduction. The secondary cohort with asthma and COPD patients was used for initial question selection (development cohort). These results were reproduced in the primary care cohort and secondary cohort of patients with ACO. The development cohort comprised 252 patients with asthma and 96 with COPD. Correlation between ACQ and CCQ in asthma was R = 0.82, and in COPD R = 0.83. PCA determined a selection of 9 questions. Reproduction in primary care data (asthma n = 1110, COPD n = 1041, ACO = 355) and secondary care data of ACO patients (n = 53) resulted in similar correlations and PCA-derived selection of questions. In conclusion, PCA determined a selection of nine questions of the ACQ and CCQ: working title ‘the Obstructive Lung Disease Questionnaire’. These results suggest that this pragmatic set of questions might be sufficient to assess disease burden in obstructive lung disease in both primary as secondary care.</p

Quantitative principles of cis-translational control by general mRNA sequence features in eukaryotes.

BackgroundGeneral translational cis-elements are present in the mRNAs of all genes and affect the recruitment, assembly, and progress of preinitiation complexes and the ribosome under many physiological states. These elements include mRNA folding, upstream open reading frames, specific nucleotides flanking the initiating AUG codon, protein coding sequence length, and codon usage. The quantitative contributions of these sequence features and how and why they coordinate to control translation rates are not well understood.ResultsHere, we show that these sequence features specify 42-81% of the variance in translation rates in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Mus musculus, and Homo sapiens. We establish that control by RNA secondary structure is chiefly mediated by highly folded 25-60 nucleotide segments within mRNA 5' regions, that changes in tri-nucleotide frequencies between highly and poorly translated 5' regions are correlated between all species, and that control by distinct biochemical processes is extensively correlated as is regulation by a single process acting in different parts of the same mRNA.ConclusionsOur work shows that general features control a much larger fraction of the variance in translation rates than previously realized. We provide a more detailed and accurate understanding of the aspects of RNA structure that directs translation in diverse eukaryotes. In addition, we note that the strongly correlated regulation between and within cis-control features will cause more even densities of translational complexes along each mRNA and therefore more efficient use of the translation machinery by the cell

Identification of MIR390a precursor processing defective mutants in Arabidopsis by direct genome sequencing

Transacting siRNA (tasiRNA) biogenesis in Arabidopsis is initiated by microRNA (miRNA) –guided cleavage of primary transcripts. In the case of TAS3 tasiRNA formation, ARGONAUTE7 (AGO7)– miR390 complexes interact with primary transcripts at two sites, resulting in recruitment of RNA-DEPENDENT RNA POLYMERASE6 for dsRNA biosynthesis. An extensive screen for Arabidopsis mutants with specific defects in TAS3 tasiRNA biogenesis or function was done. This yielded numerous ago7 mutants, one dcl4 mutant, and two mutants that accumulated low levels of miR390. A direct genome sequencing-based approach to both map and rapidly identify one of the latter mutant alleles was developed. This revealed a G-to-A point mutation (mir390a-1) that was calculated to stabilize a relatively nonpaired region near the base of the MIR390a foldback, resulting in misprocessing of the miR390/ miR390* duplex and subsequent reduced TAS3 tasiRNA levels. Directed substitutions, as well as analysis of variation at paralogous miR390-generating loci (MIR390a and MIR390b), indicated that base pair properties and nucleotide identity within a region 4–6 bases below the miR390/miR390* duplex region contributed to the efficiency and accuracy of precursor processing

A systematic review assessing non-pharmacological conservative treatment studies for people with non-inflammatory multi-joint pain: clinical outcomes and research design considerations

To systematically review the evidence to determine the clinical outcomes and the important methodological quality features of interventional studies on adults with non-inflammatory multi-joint pain (MJP). Systematic search of published and unpublished literature using the databases: AMED, CINAHL, MEDLINE, EMBASE, psycINFO, SPORTDiscus, PEDro, OpenGrey, the EU Clinical Trials Register, World Health Organization International Clinical Trial Registry Platform, ClinicalTrials.gov and the ISRCTN registry (search: inception to 19th October 2017). All papers reporting the clinical outcomes of non-pharmacological interventions for people with non-inflammatory MJP were included. Studies were critically appraised using the Downs and Black Critical Appraisal and the TIDieR reporting checklists. Data were analysed using a Best Evidence Synthesis approach. From 3824 citations, four papers satisfied the eligibility criteria. Three studies reported outcomes from multidisciplinary rehabilitation programmes and one study reported the findings of a spa therapy intervention. All interventions significantly improved pain, function and quality of life in the short-term. There was limited reporting of measures for absenteeism, presenteeism and psychosocial outcomes. The evidence was ‘weak’, and due to a lack of controlled trials, there is limited evidence to ascertain treatment effectiveness. Design consideration for future trials surround improved reporting of participant characteristics, interventions and the standardisation of core outcome measures. There is insufficient high-quality trial data to determine the effectiveness of treatments for non-inflammatory MJP. Given the significant health burden which this condition presents on both individuals and wider society, developing and testing interventions and accurately reporting these, should be a research priority

Functional Analysis of Three Arabidopsis

In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses

Cost of hospitalised patients due to complicated urinary tract infections: a retrospective observational study in countries with high prevalence of multidrug-resistant Gram-negative bacteria: the COMBACTE-MAGNET, RESCUING study

OBJECTIVE: Complicated urinary tract infections (cUTIs) impose a high burden on healthcare systems and are a frequent cause of hospitalisation. The aims of this paper are to estimate the cost per episode of patients hospitalised due to cUTI and to explore the factors associated with cUTI-related healthcare costs in eight countries with high prevalence of multidrug resistance (MDR). DESIGN: This is a multinational observational, retrospective study. The mean cost per episode was computed by multiplying the volume of healthcare use for each patient by the unit cost of each item of care and summing across all components. Costs were measured from the hospital perspective. Patient-level regression analyses were used to identify the factors explaining variation in cUTI-related costs. SETTING: The study was conducted in 20 hospitals in eight countries with high prevalence of multidrug resistant Gram-negative bacteria (Bulgaria, Greece, Hungary, Israel, Italy, Romania, Spain and Turkey). PARTICIPANTS: Data were obtained from 644 episodes of patients hospitalised due to cUTI. RESULTS: The mean cost per case was €5700, with considerable variation between countries (largest value €7740 in Turkey; lowest value €4028 in Israel), mainly due to differences in length of hospital stay. Factors associated with higher costs per patient were: type of admission, infection source, infection severity, the Charlson comorbidity index and presence of MDR. CONCLUSIONS: The mean cost per hospitalised case of cUTI was substantial and varied significantly between countries. A better knowledge of the reasons for variations in length of stays could facilitate a better standardised quality of care for patients with cUTI and allow a more efficient allocation of healthcare resources. Urgent admissions, infections due to an indwelling urinary catheterisation, resulting in septic shock or severe sepsis, in patients with comorbidities and presenting MDR were related to a higher cost