Tuning the critical gelation temperature of thermo-responsive diblock copolymer worm gels

Amphiphilic diblock copolymer nano-objects can be readily prepared using reversible addition–fragmentation chain transfer (RAFT) polymerization. For example, poly(glycerol monomethacrylate) (PGMA) chain transfer agents (CTA) can be chain-extended using 2-hydroxypropyl methacrylate (HPMA) via RAFT aqueous dispersion polymerization to form well-defined spheres, worms or vesicles at up to 25% solids. The worm morphology is of particular interest, since multiple inter-worm contacts lead to the formation of soft free-standing gels, which undergo reversible degelation on cooling to sub-ambient temperatures. However, the critical gelation temperature (CGT) for such thermo-responsive gels is ≤20 °C, which is relatively low for certain biomedical applications. In this work, a series of new amphiphilic diblock copolymers are prepared in which the core-forming block comprises a statistical mixture of HPMA and di(ethylene glycol) methyl ether methacrylate (DEGMA), which is a more hydrophilic monomer than HPMA. Statistical copolymerizations proceeded to high conversion and low polydispersities were achieved in all cases (Mw/Mn < 1.20). The resulting PGMA-P(HPMA-stat-DEGMA) diblock copolymers undergo polymerization-induced self-assembly at 10% w/w solids to form free-standing worm gels. SAXS studies indicate that reversible (de)gelation occurs below the CGT as a result of a worm-to-sphere transition, with further cooling to 5 °C affording weakly interacting copolymer chains with a mean aggregation number of approximately four. This corresponds to almost molecular dissolution of the copolymer spheres. The CGT can be readily tuned by varying the mean degree of polymerization and the DEGMA content of the core-forming statistical block. For example, a CGT of 31 °C was obtained for PGMA59-P(HPMA91-stat-DEGMA39). This is sufficiently close to physiological temperature (37 °C) to suggest that these new copolymer gels may offer biomedical applications as readily-sterilizable scaffolds for mammalian cells, since facile cell harvesting can be achieved after a single thermal cycle

Using gene expression profiles from peripheral blood to identify asymptomatic responses to acute respiratory viral infections

<p>Abstract</p> <p>Background</p> <p>A recent study reported that gene expression profiles from peripheral blood samples of healthy subjects prior to viral inoculation were indistinguishable from profiles of subjects who received viral challenge but remained asymptomatic and uninfected. If true, this implies that the host immune response does not have a molecular signature. Given the high sensitivity of microarray technology, we were intrigued by this result and hypothesize that it was an artifact of data analysis.</p> <p>Findings</p> <p>Using acute respiratory viral challenge microarray data, we developed a molecular signature that for the first time allowed for an accurate differentiation between uninfected subjects prior to viral inoculation and subjects who remained asymptomatic after the viral challenge.</p> <p>Conclusions</p> <p>Our findings suggest that molecular signatures can be used to characterize immune responses to viruses and may improve our understanding of susceptibility to viral infection with possible implications for vaccine development.</p

A flexible loop in yeast ribosomal protein L11 coordinates P-site tRNA binding

High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 ‘P-site loop’. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site

Modulation of gene expression in heart and liver of hibernating black bears (Ursus americanus)

<p>Abstract</p> <p>Background</p> <p>Hibernation is an adaptive strategy to survive in highly seasonal or unpredictable environments. The molecular and genetic basis of hibernation physiology in mammals has only recently been studied using large scale genomic approaches. We analyzed gene expression in the American black bear, <it>Ursus americanus</it>, using a custom 12,800 cDNA probe microarray to detect differences in expression that occur in heart and liver during winter hibernation in comparison to summer active animals.</p> <p>Results</p> <p>We identified 245 genes in heart and 319 genes in liver that were differentially expressed between winter and summer. The expression of 24 genes was significantly elevated during hibernation in both heart and liver. These genes are mostly involved in lipid catabolism and protein biosynthesis and include RNA binding protein motif 3 (<it>Rbm3</it>), which enhances protein synthesis at mildly hypothermic temperatures. Elevated expression of protein biosynthesis genes suggests induction of translation that may be related to adaptive mechanisms reducing cardiac and muscle atrophies over extended periods of low metabolism and immobility during hibernation in bears. Coordinated reduction of transcription of genes involved in amino acid catabolism suggests redirection of amino acids from catabolic pathways to protein biosynthesis. We identify common for black bears and small mammalian hibernators transcriptional changes in the liver that include induction of genes responsible for fatty acid β oxidation and carbohydrate synthesis and depression of genes involved in lipid biosynthesis, carbohydrate catabolism, cellular respiration and detoxification pathways.</p> <p>Conclusions</p> <p>Our findings show that modulation of gene expression during winter hibernation represents molecular mechanism of adaptation to extreme environments.</p

Widespread Regulation of miRNA Biogenesis at the Dicer Step by the Cold-Inducible RNA-Binding Protein, RBM3

MicroRNAs (miRNAs) play critical roles in diverse cellular events through their effects on translation. Emerging data suggest that modulation of miRNA biogenesis at post-transcriptional steps by RNA-binding proteins is a key point of regulatory control over the expression of some miRNAs and the cellular processes they influence. However, the extent and conditions under which the miRNA pathway is amenable to regulation at posttranscriptional steps are poorly understood. Here we show that RBM3, a cold-inducible, developmentally regulated RNA-binding protein and putative protooncogene, is an essential regulator of miRNA biogenesis. Utilizing miRNA array, Northern blot, and PCR methods, we observed that over 60% of miRNAs detectable in a neuronal cell line were significantly downregulated by knockdown of RBM3. Conversely, for select miRNAs assayed by Northern blot, induction of RBM3 by overexpression or mild hypothermia increased their levels. Changes in miRNA expression were accompanied by changes in the levels of their ∼70 nt precursors, whereas primary transcript levels were unaffected. Mechanistic studies revealed that knockdown of RBM3 does not reduce Dicer activity or impede transport of pre-miRNAs into the cytoplasm. Rather, we find that RBM3 binds directly to ∼70 nt pre-miRNA intermediates and promotes / de-represses their ability as larger ribonucleoproteins (pre-miRNPs) to associate with active Dicer complexes. Our findings suggest that the processing of a majority of pre-miRNPs by Dicer is subject to an intrinsic inhibitory influence that is overcome by RBM3 expression. RBM3 may thus orchestrate changes in miRNA expression during hypothermia and other cellular stresses, and in the euthermic contexts of early development, differentiation, and oncogenesis where RBM3 expression is highly elevated. Additionally, our data suggest that temperature-dependent changes in miRNA expression mediated by RBM3 may contribute to the therapeutic effects of hypothermia, and are an important variable to consider in in vitro studies of translation-dependent cellular events

RBM3 mediates structural plasticity and protective effects of cooling in neurodegeneration.

In the healthy adult brain synapses are continuously remodelled through a process of elimination and formation known as structural plasticity. Reduction in synapse number is a consistent early feature of neurodegenerative diseases, suggesting deficient compensatory mechanisms. Although much is known about toxic processes leading to synaptic dysfunction and loss in these disorders, how synaptic regeneration is affected is unknown. In hibernating mammals, cooling induces loss of synaptic contacts, which are reformed on rewarming, a form of structural plasticity. We have found that similar changes occur in artificially cooled laboratory rodents. Cooling and hibernation also induce a number of cold-shock proteins in the brain, including the RNA binding protein, RBM3 (ref. 6). The relationship of such proteins to structural plasticity is unknown. Here we show that synapse regeneration is impaired in mouse models of neurodegenerative disease, in association with the failure to induce RBM3. In both prion-infected and 5XFAD (Alzheimer-type) mice, the capacity to regenerate synapses after cooling declined in parallel with the loss of induction of RBM3. Enhanced expression of RBM3 in the hippocampus prevented this deficit and restored the capacity for synapse reassembly after cooling. RBM3 overexpression, achieved either by boosting endogenous levels through hypothermia before the loss of the RBM3 response or by lentiviral delivery, resulted in sustained synaptic protection in 5XFAD mice and throughout the course of prion disease, preventing behavioural deficits and neuronal loss and significantly prolonging survival. In contrast, knockdown of RBM3 exacerbated synapse loss in both models and accelerated disease and prevented the neuroprotective effects of cooling. Thus, deficient synapse regeneration, mediated at least in part by failure of the RBM3 stress response, contributes to synapse loss throughout the course of neurodegenerative disease. The data support enhancing cold-shock pathways as potential protective therapies in neurodegenerative disorders

Expression Patterns of Protein Kinases Correlate with Gene Architecture and Evolutionary Rates

Protein kinase (PK) genes comprise the third largest superfamily that occupy ∼2% of the human genome. They encode regulatory enzymes that control a vast variety of cellular processes through phosphorylation of their protein substrates. Expression of PK genes is subject to complex transcriptional regulation which is not fully understood.Our comparative analysis demonstrates that genomic organization of regulatory PK genes differs from organization of other protein coding genes. PK genes occupy larger genomic loci, have longer introns, spacer regions, and encode larger proteins. The primary transcript length of PK genes, similar to other protein coding genes, inversely correlates with gene expression level and expression breadth, which is likely due to the necessity to reduce metabolic costs of transcription for abundant messages. On average, PK genes evolve slower than other protein coding genes. Breadth of PK expression negatively correlates with rate of non-synonymous substitutions in protein coding regions. This rate is lower for high expression and ubiquitous PKs, relative to low expression PKs, and correlates with divergence in untranslated regions. Conversely, rate of silent mutations is uniform in different PK groups, indicating that differing rates of non-synonymous substitutions reflect variations in selective pressure. Brain and testis employ a considerable number of tissue-specific PKs, indicating high complexity of phosphorylation-dependent regulatory network in these organs. There are considerable differences in genomic organization between PKs up-regulated in the testis and brain. PK genes up-regulated in the highly proliferative testicular tissue are fast evolving and small, with short introns and transcribed regions. In contrast, genes up-regulated in the minimally proliferative nervous tissue carry long introns, extended transcribed regions, and evolve slowly.PK genomic architecture, the size of gene functional domains and evolutionary rates correlate with the pattern of gene expression. Structure and evolutionary divergence of tissue-specific PK genes is related to the proliferative activity of the tissue where these genes are predominantly expressed. Our data provide evidence that physiological requirements for transcription intensity, ubiquitous expression, and tissue-specific regulation shape gene structure and affect rates of evolution

An Internal Ribosome Entry Site Directs Translation of the 39-Gene from Pelargonium Flower Break Virus Genomic RNA: Implications for Infectivity

[EN] Pelargonium flower break virus (PFBV, genus Carmovirus) has a single-stranded positive-sense genomic RNA (gRNA) which contains five ORFs. The two 59-proximal ORFs encode the replicases, two internal ORFs encode movement proteins, and the 39-proximal ORF encodes a polypeptide (p37) which plays a dual role as capsid protein and as suppressor of RNA silencing. Like other members of family Tombusviridae, carmoviruses express ORFs that are not 59-proximal from subgenomic RNAs. However, in one case, corresponding to Hisbiscus chlorotic ringspot virus, it has been reported that the 39-proximal gene can be translated from the gRNA through an internal ribosome entry site (IRES). Here we show that PFBV also holds an IRES that mediates production of p37 from the gRNA, raising the question of whether this translation strategy may be conserved in the genus. The PFBV IRES was functional both in vitro and in vivo and either in the viral context or when inserted into synthetic bicistronic constructs. Through deletion and mutagenesis studies we have found that the IRES is contained within a 80 nt segment and have identified some structural traits that influence IRES function. Interestingly, mutations that diminish IRES activity strongly reduced the infectivity of the virus while the progress of the infection was favoured by mutations potentiating such activity. These results support the biological significance of the IRES-driven p37 translation and suggest that production of the silencing suppressor from the gRNA might allow the virus to early counteract the defence response of the host, thus facilitating pathogen multiplication and spread.This research was supported by grants BFU2006-11230 and BFU2009-11699 from the Spanish Ministerio de Ciencia e Innovacion (MICINN) and by grants ACOM/2006/210 and ACOMP/2009/040 (to CH) and GVPRE/2008/121 (to OF-M) from the Generalitat Valenciana. The latter was the recipient of an I3P postdoctoral contract from the Spanish Consejo Superior de Investigaciones Cientificas and an additional contract from MICINN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Fernandez Miragall, O.; Hernandez Fort, C. (2011). An Internal Ribosome Entry Site Directs Translation of the 39-Gene from Pelargonium Flower Break Virus Genomic RNA: Implications for Infectivity. PLoS ONE. 6(7):22617-22617. https://doi.org/10.1371/journal.pone.0022617S226172261767Gallie, D. R. (1996). Translational control of cellular and viral mRNAs. Plant Molecular Biology, 32(1-2), 145-158. doi:10.1007/bf00039381Kozak, M. (2002). Pushing the limits of the scanning mechanism for initiation of translation. Gene, 299(1-2), 1-34. doi:10.1016/s0378-1119(02)01056-9Sachs, A. B., Sarnow, P., & Hentze, M. W. (1997). Starting at the Beginning, Middle, and End: Translation Initiation in Eukaryotes. Cell, 89(6), 831-838. doi:10.1016/s0092-8674(00)80268-8Kozak, M. (1992). Regulation of Translation in Eukaryotic Systems. Annual Review of Cell Biology, 8(1), 197-225. doi:10.1146/annurev.cb.08.110192.001213Sonenberg, N., & Hinnebusch, A. G. (2009). Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets. Cell, 136(4), 731-745. doi:10.1016/j.cell.2009.01.042F�tterer, J., & Hohn, T. (1996). Translation in plants-rules and exceptions. Plant Molecular Biology, 32(1-2), 159-189. doi:10.1007/bf00039382Gale, M., Tan, S.-L., & Katze, M. G. (2000). Translational Control of Viral Gene Expression in Eukaryotes. Microbiology and Molecular Biology Reviews, 64(2), 239-280. doi:10.1128/mmbr.64.2.239-280.2000Kozak, M. (2001). Constraints on reinitiation of translation in mammals. Nucleic Acids Research, 29(24), 5226-5232. doi:10.1093/nar/29.24.5226Pelletier, J., & Sonenberg, N. (1988). Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature, 334(6180), 320-325. doi:10.1038/334320a0Mokrejš, M., Mašek, T., Vopálenský, V., Hlubuček, P., Delbos, P., & Pospíšek, M. (2009). IRESite—a tool for the examination of viral and cellular internal ribosome entry sites. Nucleic Acids Research, 38(suppl_1), D131-D136. doi:10.1093/nar/gkp981Basso, J., Dallaire, P., Charest, P. J., Devantier, Y., & Laliberte, J.-F. (1994). Evidence for an Internal Ribosome Entry Site Within the 5’ Non-translated Region of Turnip Mosaic Potyvirus RNA. Journal of General Virology, 75(11), 3157-3165. doi:10.1099/0022-1317-75-11-3157Levis, C., & Astier-Manifacier, S. (1993). The 5′ untranslated region of PVY RNA, even located in an internal position, enables initiation of translation. Virus Genes, 7(4), 367-379. doi:10.1007/bf01703392Karetnikov, A., & Lehto, K. (2007). The RNA2 5’ leader of Blackcurrant reversion virus mediates efficient in vivo translation through an internal ribosomal entry site mechanism. Journal of General Virology, 88(1), 286-297. doi:10.1099/vir.0.82307-0Ivanov, P. A., Karpova, O. V., Skulachev, M. V., Tomashevskaya, O. L., Rodionova, N. P., Dorokhov, Y. L., & Atabekov, J. G. (1997). A Tobamovirus Genome That Contains an Internal Ribosome Entry Site Functionalin Vitro. Virology, 232(1), 32-43. doi:10.1006/viro.1997.8525Skulachev, M. V., Ivanov, P. A., Karpova, O. V., Korpela, T., Rodionova, N. P., Dorokhov, Y. L., & Atabekov, J. G. (1999). Internal Initiation of Translation Directed by the 5′-Untranslated Region of the Tobamovirus Subgenomic RNA I2. Virology, 263(1), 139-154. doi:10.1006/viro.1999.9928Jaag, H. M., Kawchuk, L., Rohde, W., Fischer, R., Emans, N., & Prufer, D. (2003). An unusual internal ribosomal entry site of inverted symmetry directs expression of a potato leafroll polerovirus replication-associated protein. Proceedings of the National Academy of Sciences, 100(15), 8939-8944. doi:10.1073/pnas.1332697100Balvay, L., Rifo, R. S., Ricci, E. P., Decimo, D., & Ohlmann, T. (2009). Structural and functional diversity of viral IRESes. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1789(9-10), 542-557. doi:10.1016/j.bbagrm.2009.07.005Kneller, E. L. P., Rakotondrafara, A. M., & Miller, W. A. (2006). Cap-independent translation of plant viral RNAs. Virus Research, 119(1), 63-75. doi:10.1016/j.virusres.2005.10.010Rico, P., & Hern�ndez, C. (2004). Complete nucleotide sequence and genome organization of Pelargonium flower break virus. Archives of Virology, 149(3), 641-651. doi:10.1007/s00705-003-0231-5Martinez-Turino, S., & Hernandez, C. (2010). Identification and characterization of RNA-binding activity in the ORF1-encoded replicase protein of Pelargonium flower break virus. Journal of General Virology, 91(12), 3075-3084. doi:10.1099/vir.0.023093-0Martínez-Turiño, S., & Hernández, C. (2011). A membrane-associated movement protein of Pelargonium flower break virus shows RNA-binding activity and contains a biologically relevant leucine zipper-like motif. Virology, 413(2), 310-319. doi:10.1016/j.virol.2011.03.001Martinez-Turino, S., & Hernandez, C. (2009). Inhibition of RNA silencing by the coat protein of Pelargonium flower break virus: distinctions from closely related suppressors. Journal of General Virology, 90(2), 519-525. doi:10.1099/vir.0.006098-0Rico, P., & Hernández, C. (2009). Characterization of the subgenomic RNAs produced by Pelargonium flower break virus: Identification of two novel RNAs species. Virus Research, 142(1-2), 100-107. doi:10.1016/j.virusres.2009.01.018Koh, D. C.-Y., Wong, S.-M., & Liu, D. X. (2003). Synergism of the 3′-Untranslated Region and an Internal Ribosome Entry Site Differentially Enhances the Translation of a Plant Virus Coat Protein. Journal of Biological Chemistry, 278(23), 20565-20573. doi:10.1074/jbc.m210212200Hellen, C. U. T. (2001). Internal ribosome entry sites in eukaryotic mRNA molecules. Genes & Development, 15(13), 1593-1612. doi:10.1101/gad.891101Martínez-Salas, E. (1999). Internal ribosome entry site biology and its use in expression vectors. Current Opinion in Biotechnology, 10(5), 458-464. doi:10.1016/s0958-1669(99)00010-5Dobrikova, E., Florez, P., Bradrick, S., & Gromeier, M. (2003). Activity of a type 1 picornavirus internal ribosomal entry site is determined by sequences within the 3’ nontranslated region. Proceedings of the National Academy of Sciences, 100(25), 15125-15130. doi:10.1073/pnas.2436464100Belsham, G. J. (2009). Divergent picornavirus IRES elements. Virus Research, 139(2), 183-192. doi:10.1016/j.virusres.2008.07.001Fernández-Miragall, O., Quinto, S. L. de, & Martínez-Salas, E. (2009). Relevance of RNA structure for the activity of picornavirus IRES elements. Virus Research, 139(2), 172-182. doi:10.1016/j.virusres.2008.07.009Pestova, T. V., Kolupaeva, V. G., Lomakin, I. B., Pilipenko, E. V., Shatsky, I. N., Agol, V. I., & Hellen, C. U. T. (2001). Molecular mechanisms of translation initiation in eukaryotes. Proceedings of the National Academy of Sciences, 98(13), 7029-7036. doi:10.1073/pnas.111145798FERNANDEZ-MIRAGALL, O. (2003). Structural organization of a viral IRES depends on the integrity of the GNRA motif. RNA, 9(11), 1333-1344. doi:10.1261/rna.5950603ROBERTSON, M. E. M., SEAMONS, R. A., & BELSHAM, G. J. (1999). A selection system for functional internal ribosome entry site (IRES) elements: Analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES. RNA, 5(9), 1167-1179. doi:10.1017/s1355838299990301Dorokhov, Y. L., Skulachev, M. V., Ivanov, P. A., Zvereva, S. D., Tjulkina, L. G., Merits, A., … Atabekov, J. G. (2002). Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry. Proceedings of the National Academy of Sciences, 99(8), 5301-5306. doi:10.1073/pnas.082107599Xia, X., & Holcik, M. (2009). Strong Eukaryotic IRESs Have Weak Secondary Structure. PLoS ONE, 4(1), e4136. doi:10.1371/journal.pone.0004136Lu, J., Zhang, J., Wang, X., Jiang, H., Liu, C., & Hu, Y. (2006). In vitro and in vivo identification of structural and sequence elements in the 5’ untranslated region of Ectropis obliqua picorna-like virus required for internal initiation. Journal of General Virology, 87(12), 3667-3677. doi:10.1099/vir.0.82090-0Yang, L. J., Hidaka, M., Sonoda, J., Masaki, H., & Uozumi, T. (1997). Mutational Analysis of the Potato Virus Y 5′ Untranslated Region for Alteration in Translational Enhancement in Tobacco Protoplasts. Bioscience, Biotechnology, and Biochemistry, 61(12), 2131-2133. doi:10.1271/bbb.61.2131BERGAMINI, G., PREISS, T., & HENTZE, M. W. (2000). Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system. RNA, 6(12), 1781-1790. doi:10.1017/s1355838200001679Bradrick, S. S. (2006). The hepatitis C virus 3’-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase. Nucleic Acids Research, 34(4), 1293-1303. doi:10.1093/nar/gkl019Lopez de Quinto, S. (2002). IRES-driven translation is stimulated separately by the FMDV 3’-NCR and poly(A) sequences. Nucleic Acids Research, 30(20), 4398-4405. doi:10.1093/nar/gkf569Song, Y., Friebe, P., Tzima, E., Junemann, C., Bartenschlager, R., & Niepmann, M. (2006). The Hepatitis C Virus RNA 3’-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site. Journal of Virology, 80(23), 11579-11588. doi:10.1128/jvi.00675-06Koh, D. C.-Y., Liu, D. X., & Wong, S.-M. (2002). A Six-Nucleotide Segment within the 3’ Untranslated Region of Hibiscus Chlorotic Ringspot Virus Plays an Essential Role in Translational Enhancement. Journal of Virology, 76(3), 1144-1153. doi:10.1128/jvi.76.3.1144-1153.2002Stupina, V. A., Meskauskas, A., McCormack, J. C., Yingling, Y. G., Shapiro, B. A., Dinman, J. D., & Simon, A. E. (2008). The 3’ proximal translational enhancer of Turnip crinkle virus binds to 60S ribosomal subunits. RNA, 14(11), 2379-2393. doi:10.1261/rna.1227808Truniger, V., Nieto, C., González-Ibeas, D., & Aranda, M. (2008). Mechanism of plant eIF4E-mediated resistance against a Carmovirus (Tombusviridae): cap-independent translation of a viral RNA controlledin cisby an (a)virulence determinant. The Plant Journal, 56(5), 716-727. doi:10.1111/j.1365-313x.2008.03630.xMiller, W. A., Wang, Z., & Treder, K. (2007). The amazing diversity of cap-independent translation elements in the 3′-untranslated regions of plant viral RNAs. Biochemical Society Transactions, 35(6), 1629-1633. doi:10.1042/bst0351629Miller, W. A., & White, K. A. (2006). Long-Distance RNA-RNA Interactions in Plant Virus Gene Expression and Replication. Annual Review of Phytopathology, 44(1), 447-467. doi:10.1146/annurev.phyto.44.070505.143353Koh, D. C.-Y., Wang, X., Wong, S.-M., & Liu, D. X. (2006). Translation initiation at an upstream CUG codon regulates the expression of Hibiscus chlorotic ringspot virus coat protein. Virus Research, 122(1-2), 35-44. doi:10.1016/j.virusres.2006.06.008Castaño, A., Ruiz, L., & Hernández, C. (2009). Insights into the translational regulation of biologically active open reading frames of Pelargonium line pattern virus. Virology, 386(2), 417-426. doi:10.1016/j.virol.2009.01.017Fraser, C. S., & Doudna, J. A. (2006). Structural and mechanistic insights into hepatitis C viral translation initiation. Nature Reviews Microbiology, 5(1), 29-38. doi:10.1038/nrmicro1558LÓPEZ-LASTRA, M., RIVAS, A., & BARRÍA, M. I. (2005). Protein synthesis in eukaryotes: The growing biological relevance of cap-independent translation initiation. Biological Research, 38(2-3). doi:10.4067/s0716-97602005000200003Pacheco, A., & Martinez-Salas, E. (2010). Insights into the Biology of IRES Elements through Riboproteomic Approaches. Journal of Biomedicine and Biotechnology, 2010, 1-12. doi:10.1155/2010/458927Bernstein, J., Sella, O., Le, S.-Y., & Elroy-Stein, O. (1997). PDGF2/c-sismRNA Leader Contains a Differentiation-linked Internal Ribosomal Entry Site (D-IRES). Journal of Biological Chemistry, 272(14), 9356-9362. doi:10.1074/jbc.272.14.9356Scheper, G. C., Voorma, H. O., & Thomas, A. A. M. (1994). Basepairing with 18S ribosomal RNA in internal initiation of translation. FEBS Letters, 352(3), 271-275. doi:10.1016/0014-5793(94)00975-9Dresios, J., Chappell, S. A., Zhou, W., & Mauro, V. P. (2005). An mRNA-rRNA base-pairing mechanism for translation initiation in eukaryotes. Nature Structural & Molecular Biology, 13(1), 30-34. doi:10.1038/nsmb1031Reigadas, S., Pacheco, A., Ramajo, J., de Quinto, S. L., & Martinez-Salas, E. (2005). Specific interference between two unrelated internal ribosome entry site elements impairs translation efficiency. FEBS Letters, 579(30), 6803-6808. doi:10.1016/j.febslet.2005.11.015Ishitani, M., Xiong, L., Stevenson, B., & Zhu, J. K. (1997). Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways. The Plant Cell, 9(11), 1935-1949. doi:10.1105/tpc.9.11.1935Knoester, M., van Loon, L. C., van den Heuvel, J., Hennig, J., Bol, J. F., & Linthorst, H. J. M. (1998). Ethylene-insensitive tobacco lacks nonhost resistance against soil-borne fungi. Proceedings of the National Academy of Sciences, 95(4), 1933-1937. doi:10.1073/pnas.95.4.1933Mathews, D. H., Sabina, J., Zuker, M., & Turner, D. H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. Journal of Molecular Biology, 288(5), 911-940. doi:10.1006/jmbi.1999.2700Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research, 31(13), 3406-3415. doi:10.1093/nar/gkg59