- Email: [email protected]
Endosymbiotic Evolution: RNA Intermediates in Endosymbiotic Gene Transfer
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References 1. Choe, A., von Reuss, S.H., Kogan, D., Gasser, R.B., Platzer, E.G., Schroeder, F.C., and Sternberg, P.W. (2012). Ascaroside signaling is widely conserved among nematodes. Curr. Biol. 22, 772–780. 2. von Reuss, S.H., Bose, N., Srinivasan, J., Yim, J.J., Judkins, J.C., Sternberg, P.W., and Schroeder, F.C. (2012). Comparative metabolomics reveals biogenesis of ascarosides, a modular library of small-molecule signals in C. elegans. J. Am. Chem. Soc. 134, 1817–1824. 3. Butcher, R.A., Ragains, J.R., Li, W., Ruvkun, G., Clardy, J., and Mak, H.Y. (2009). Biosynthesis of the Caenorhabditis elegans dauer pheromone. Proc. Natl. Acad. Sci. USA 106, 1875–1879. 4. Pungaliya, C., Srinivasan, J., Fox, B.W., Malik, R.U., Ludewig, A.H., Sternberg, P.W., and Schroeder, F.C. (2009). A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 106, 7708–7713. 5. Golden, J.W., and Riddle, D.L. (1982). A pheromone influences larval development in the nematode Caenorhabditis elegans. Science 218, 578–580. 6. Jeong, P.Y., Jung, M., Yim, Y.H., Kim, H., Park, M., Hong, E., Lee, W., Kim, Y.H., Kim, K., and Paik, Y.K. (2005). Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone. Nature 433, 541–545. 7. Butcher, R.A., Fujita, M., Schroeder, F.C., and Clardy, J. (2007). Small-molecule pheromones that control dauer development in
8.
9.
10.
11.
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Caenorhabditis elegans. Nat. Chem. Biol. 3, 420–422. Butcher, R.A., Ragains, J.R., Kim, E., and Clardy, J. (2008). A potent dauer pheromone component in Caenorhabditis elegans that acts synergistically with other components. Proc. Natl. Acad. Sci. USA 105, 14288–14292. Kaplan, F., Srinivasan, J., Mahanti, P., Ajredini, R., Durak, O., Nimalendran, R., Sternberg, P.W., Teal, P.E., Schroeder, F.C., Edison, A.S., et al. (2011). Ascaroside expression in Caenorhabditis elegans is strongly dependent on diet and developmental stage. PLoS One 6, e17804. Srinivasan, J., Kaplan, F., Ajredini, R., Zachariah, C., Alborn, H.T., Teal, P.E., Malik, R.U., Edison, A.S., Sternberg, P.W., and Schroeder, F.C. (2008). A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature 454, 1115–1118. Yamada, K., Hirotsu, T., Matsuki, M., Butcher, R.A., Tomioka, M., Ishihara, T., Clardy, J., Kunitomo, H., and Iino, Y. (2010). Olfactory plasticity is regulated by pheromonal signaling in Caenorhabditis elegans. Science 329, 1647–1650. Macosko, E.Z., Pokala, N., Feinberg, E.H., Chalasani, S.H., Butcher, R.A., Clardy, J., and Bargmann, C.I. (2009). A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans. Nature 458, 1171–1175. Srinivasan, J., von Reuss, S.H., Bose, N., Zaslaver, A., Mahanti, P., Ho, M.C., O’Doherty, O.G., Edison, A.S., Sternberg, P.W., and Schroeder, F.C. (2012). A modular library of small molecule signals regulates social
Endosymbiotic Evolution: RNA Intermediates in Endosymbiotic Gene Transfer More than a billion years of endosymbiotic evolution has resulted in extensive gene relocation between the genetic compartments of eukaryotic cells. A new study uses chloroplast genome transformation to shed light on the mechanisms involved. Jeremy N. Timmis In the 40 years since Lynn Margulis [1,2] revived the endosymbiotic theory of eukaryotic evolution after decades of neglect, experimental results have been universally supportive of the concepts. Convincing evidence has been provided both by inference from bioinformatic analyses of nuclear genomes and by experimental recapitulation of some of the key processes [3]. Since the engulfment of the prokaryote ancestors of mitochondria and, later, plastids (chloroplasts) by a precursor of the nucleated cell, many genes have migrated to the nucleus of the host at the expense of the endosymbiont genomes. As a result, extant
cytoplasmic organellar genomes encode very few proteins, making it necessary for chloroplasts and mitochondria to import thousands of nucleus-encoded gene products required for their biogenesis and function. A new study in this issue of Current Biology [4] uncovers mechanistic detail regarding how gene transfer to the nucleus occurs. The majority of functional endosymbiotic gene transfer (EGT) probably occurred relatively soon after the formation of the first conglomerate cells, and the process appears to have gone as far as it will for mitochondrial genes in animal cells, though some protists with altered biochemistry lack a mitochondrial genome altogether. Interestingly, EGT is still ticking over
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behaviors in Caenorhabditis elegans. PLoS Biol. 10, e1001237. Kim, K., Sato, K., Shibuya, M., Zeiger, D.M., Butcher, R.A., Ragains, J.R., Clardy, J., Touhara, K., and Sengupta, P. (2009). Two chemoreceptors mediate developmental effects of dauer pheromone in C. elegans. Science 326, 994–998. McGrath, P.T., Xu, Y., Ailion, M., Garrison, J.L., Butcher, R.A., and Bargmann, C.I. (2011). Parallel evolution of domesticated Caenorhabditis species targets pheromone receptor genes. Nature 477, 321–325. Thomas, J.H., and Robertson, H.M. (2008). The Caenorhabditis chemoreceptor gene families. BMC Biol. 6, 42. Bartley, J.P., Bennett, E.A., and Darben, P.A. (1996). Structure of the ascarosides from Ascaris suum. J. Nat. Prod. 59, 921–926. Stewart, M.K., Clark, N.L., Merrihew, G., Galloway, E.M., and Thomas, J.H. (2004). High genetic diversity in the chemoreceptor superfamily of C. elegans. Genetics 169, 1985–1996. Blaxter, M.L. (2011). Nematodes: The worm and its relatives. PLoS Biol. 9, e1001050.
Institut de Biologie Valrose, CNRS UMR7277 – INSERM U1091, Universite´ de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice cedex 2, France. E-mail: [email protected] DOI: 10.1016/j.cub.2012.03.035
for both mitochondrial and chloroplast genes in plant systems. Furthermore, some steps in the EGT process still occur in essentially all eukaryotes, where they continue to have a major influence on nuclear evolution [3]. Simple and shuffled tracts of genetic information from cytoplasmic organelles, resulting from DNA transfer per se, are universally seen in the nuclear genome of essentially all organisms examined [5–7]. Thus, the processes responsible for EGT and associated DNA transfer are fundamental to eukaryote evolution, the production of genetic diversity, and the emergence of multicellular organisms. A small sample of the thousands of nuclear genes that have entered plant nuclei by EGT [8] have been examined in detail, and some transfer events appear to have involved an RNA intermediate [9–11]. This suggestion is based on the observation that some nuclear genes contributing to some plant mitochondrial proteomes are more similar to spliced and edited mitochondrial mRNAs than to the equivalent gene copies that remain in the mitochondrial genome in some related plant species. There is clear
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evidence, however, that the vast majority of the genetic information that enters the nucleus is by direct DNA transfer [3,12,13] and other explanations for the apparent involvement of an RNA intermediate have been strongly advocated [3,14]. Nonetheless, the idea of RNA intermediates, and the circumstantial supporting evidence is attractive enough that controversy exists and, though attempts have been made [15], it has so far been impossible to rule RNA in or out as a component of the mechanisms involved in EGT. Ralph Bock’s group in Potsdam set out to settle the matter using chloroplast transformation [4] and, in doing so, brought to light a new aspect of endosymbiotic evolution. Aiming to search for EGT events mediated by RNA molecules, Fuentes et al. [4] designed a DNA sequence (Nt-pIF84) with which they transformed tobacco chloroplasts. The sequence was tailored as a precursor to a gene that would be functional in the nucleus only if it was transcribed, and its transcript processed, within the chloroplast prior to migration to the nucleus in RNA or cDNA form. To achieve this, a chloroplast-specific promoter drives the reporter gene (nptII), complete with a plant nucleus-specific promoter and terminator, all in antisense orientation. Chloroplast transcription was made unequivocally recognisable by adding a group II intron — one that must be spliced within the chloroplast — in the correct polarity with respect to the chloroplast promoter. The final transplastomic tobacco line contained a homogeneous population of chloroplasts that were producing nptII spliced antisense transcripts in which kanamycin resistance should result only after they were copied into double-stranded DNA and inserted into the nucleus. Next, over a million progeny of the self-fertilized, transplastomic lines were screened and, surprisingly, 91 seedlings showed strong expression of the functional reporter gene. This frequency (w1:11,000) is similar to previous experiments in which only direct DNA transfer was suspected [13,16]. Sure enough, analyses of the nuclear integrants in Fuentes et al.’s screen [4] did not reveal a single case where the chloroplast-specific intron was missing, suggesting that RNA is vanishingly rarely involved in these
DNA transfer events. This confirmed and extended previous experiments that found no evidence of RNA intermediates [15]. However, whether RNA is ever involved in functional gene transfer in a natural evolutionary EGT scenario remains an open question that now appears intractable to experimentation because of its rarity. Unexpectedly, therefore, the reporter gene was somehow able to be expressed directly in nuclear DNA without the chloroplast-specific splicing that was assumed to be a prerequisite. The explanation is that the intron used in these experiments contains cryptic sequences that are recognised by the eukaryotic mRNA splicing machinery. Fortuitous eukaryotic signals are not unknown in plastid genes and DNA. The high AT content of the plastome has been shown to provide cryptic polyadenylation signals during plastid gene activation in the nucleus [17,18] and the chloroplast psbA promoter is moderately active in the nucleus [17]. How many other chloroplast and mitochondrial sequences contain hidden nuclear signals and how much alternative splicing is explained by EGT and other nuclear genomic rearrangements remains to be seen. I have outlined only parts of this fascinating and cleverly designed research. The results suggest that RNA is rarely involved compared with direct DNA transfer. However, genomic analyses are able to look back many millions of years, and comparisons with experimental results are challenging. Also, the sheer volume of DNA bombardment of the nucleus suggests it has an overriding role in EGT, and indeed experimental prokaryotic genes that escaped to the nucleus by bulk DNA transfer, while initially inactive, are able to attain function after nuclear genomic rearrangements [17,18]. Fuentes et al. [4] examined gene transfer from a single transplastomic cell through to the sexual progeny of regenerated plants, thereby screening for EGT within the entire lineage of somatic cells within both male and female germlines. How many cells the germline includes is uncertain, so it will be interesting to see what happens when similar experiments are done in cultured somatic cells. The present screen includes events in male gametogenesis where bulk DNA transposition is at its greatest due to the programmed degradation of
plastids during pollen development [13]. The unexpected immediate expression of nptII that thwarted these carefully designed experiments highlights the enormous flexibility with which the nucleus deals with incoming genetic material. It seems that either the nucleus or the chloroplast genetic compartments, or both, are full of tricks, many of which may surprise even the best-laid plans. Therefore, these results will also serve as a warning to those who propose theoretical precautions against gene escape in biotechnological applications. References 1. Margulis, L. (1970). Origin of Eukaryotic Cells (New Haven and London: Yale University Press). 2. Archibald, J.M. (2012). Lynn Margulis (1938–2011) Obituary. Curr. Biol. 22, R4–R6. 3. Timmis, J.N., Ayliffe, M.A., Huang, C.Y., and Martin, W. (2004). Endosymbiotic gene transfer: Organelle genomes forge eukaryotic chromosomes. Nat. Rev. Genet. 5, 123–135. 4. Fuentes, I., Karcher, D., and Bock, R. (2012). Experimental reconstruction of the functional transfer of intron-containing plastid genes to the nucleus. Curr. Biol. 22, 763–771. 5. Kleine, T., Maier, U.G., and Leister, D. (2009). DNA transfer from organelles to the nucleus: the idiosyncratic genetics of endosymbiosis. Annu. Rev. Plant Biol. 60, 115–138. 6. Noutsos, C., Richly, E., and Leister, D. (2005). Generation and evolutionary fate of insertions of organelle DNA in the nuclear genomes of flowering plants. Genome Res. 15, 616–628. 7. Noutsos, C., Kleine, T., Armbruster, U., DalCorso, G., and Leister, D. (2007). Nuclear insertions of organellar DNA can create novel patches of functional exon sequences. Trends Genet. 23, 597–601. 8. Martin, W., Rujan, T., Richly, E., Hansen, A., Cornelsen, S., Lins, T., Leister, D., Stoebe, B., Hasegawa, M., and Penny, D. (2002). Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc. Natl. Acad. Sci. USA 99, 12246–12251. 9. Nugent, J.M., and Palmer, J.D. (1991). RNA-mediated transfer of the gene coxII from the mitochondrion to the nucleus during flowering plant evolution. Cell 66, 473–481. 10. Covello, P.S., and Gray, M.W. (1992). Silent mitochondrial and active nuclear genes for subunit 2 of cytochrome C oxidase (cox2) in soybean - evidence for RNA-mediated gene transfer. EMBO J. 11, 3815–3820. 11. Wischmann, C., and Schuster, W. (1995). Transfer of rps10 from the mitochondriom to the nucleus in Arabidopsis thaliana - evidence for RNA-mediated transfer and exon shuffling at the integration site. FEBS Lett. 374, 152–156. 12. Bock, R., and Timmis, J.N. (2008). Reconstructing evolution: gene transfer from plastids to the nucleus. Bioessays 30, 556–566. 13. Sheppard, A.E., Ayliffe, M.A., Blatch, L., Day, A., Delaney, S.K., Khairul-Fahmy, N., Li, Y., Madesis, P., Pryor, A.J., and Timmis, J.N. (2008). Transfer of plastid DNA to the nucleus is elevated during male gametogenesis in tobacco. Plant Physiol. 148, 328–336. 14. Henze, K., and Martin, W. (2001). How do mitochondrial genes get into the nucleus? Trends Genet. 17, 383–387. 15. Sheppard, A.E., Madesis, P., Lloyd, A.H., Day, A., Ayliffe, M.A., and Timmis, J.N. (2011).
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Introducing an RNA editing requirement into a plastid-localised transgene reduces but does not eliminate functional gene transfer to the nucleus. Plant Mol. Biol. 76, 299–309. 16. Huang, C.Y., Ayliffe, M.A., and Timmis, J.N. (2003). Direct measurement of the transfer rate of chloroplast DNA into the nucleus. Nature 422, 72–76.
17. Lloyd, A.H., and Timmis, J.N. (2011). The origin and characterization of new nuclear genes originating from a cytoplasmic organellar genome. Mol. Biol. Evol. 28, 2019–2028. 18. Stegemann, S., and Bock, R. (2006). Experimental reconstruction of functional gene transfer from the tobacco plastid genome to the nucleus. Plant Cell 18, 2869–2878.
Reading Abilities: Importance of Visual-Spatial Attention Children with dyslexia may read poorly for several reasons. Recent research suggests that in addition to skills with language sounds, visual-spatial attention may be an important predictor of reading abilities. John D.E. Gabrieli1,2 and Elizabeth S. Norton1,3 You are reading these words very quickly. A typical adult has a reading vocabulary of 50,000–100,000 words, yet can identify a printed word seen for merely 1/200th of a second. Reading is essential for learning, from literature to physics, from paper to screens on e-readers and smart phones. Yet, about 10% of children have developmental dyslexia, an unexplained difficulty in learning to read [1]. Such poor reading is often associated with undesirable outcomes, such as lower educational attainment [2]. Dyslexia is likely caused by multiple factors, and the importance of those factors may vary between children [3] and across languages with different relations between spoken and written forms of language [4]. Research from Franceschini et al. [5] reported in this issue of Current Biology now reveals that a weakness in visual-spatial attention in pre-reading kindergartners is an important risk factor for becoming a poor reader. In all languages, under typical developmental conditions, children learn spoken language effortlessly and without formal instruction. In contrast, reading must be learned through explicit educational instruction over several years. Learning to read words can be conceptualized as learning to map the sound units of spoken language (phonemes) onto the written units of print (graphemes) so that meaning, initially related to spoken language, can be extended to print. Because many children with dyslexia appeared to hear and talk successfully at home before struggling to read at school, early conceptualizations of
dyslexia focused on putative visual deficits made manifest with print. Although there is evidence for visual deficits in dyslexia [6,7], the most common cause of dyslexia was reconceptualized in the 1980s as a weakness in the processing of language sounds, and especially in phonemic awareness — the ability to explicitly recognize and manipulate the sounds of language [8]. This weakness makes it difficult for beginning readers to map the sounds of language onto print and to accurately identify (decode) individual words. Additionally, weakness in rapid serial naming (even of color patches) has been associated with poor reading [9,10]. This weakness renders reading slow and laborious and impedes the comprehension, and pleasure, of reading text. Research has focused on children and adults who are well-characterized as dyslexic and have long struggled with reading. Such research has two important limitations. First, learning to read has reciprocal interactions with the basic skills that underlie reading itself. Thus, practice with reading enhances phonemic awareness and other reading-related processes [11]. Evidence that these skills are necessary precursors for learning to read, rather than simply a consequence of reading, is that pre-reading children in kindergarten who score poorly on tests of phonemic awareness and rapid naming are more likely to become poor readers over the next few years [12]. Second, remedial interventions that help children with dyslexia appear to be most potent at the youngest ages, before dyslexia is typically diagnosed. Therefore, early identification of risk factors for dyslexia helps identify
Discipline of Genetics, School of Molecular and Biomedical Science, The University of Adelaide, South Australia 5005, Australia. E-mail: [email protected] DOI: 10.1016/j.cub.2012.03.043
children who may benefit the most from early intervention. Franceschini et al. [5] addressed the cause of poor reading by behaviorally testing 96 pre-reading Italian-speaking kindergartners (five-year-olds) not only with typical tests of phonemic awareness and rapid naming, but also on two tests of visual-spatial performance. Although visual-spatial processes appear to be distant from the verbal processes associated with reading, studies in adults with dyslexia have revealed deficits in visual-spatial performance, often with nonverbal material [7]. These studies motivate the idea that a weakness of visual-spatial attention, independent of language, could cause dyslexia [13]. In the new study [5], one visual-spatial task required visual search across five lines of 31 symbols (not letters) and marking each occurrence of a target symbol. In the second task, children performed a spatial cuing task. In a control condition, children very briefly viewed, on the left or right of a central fixation point, an ellipse at one of four orientations, and then selected from among four alternatives which ellipse they had just viewed. The spatial cuing conditions built upon seminal research about visual attention from Michael Posner [14], who showed that attention is automatically or exogenously drawn to a spatial location by brief highlighting of that location. In the spatial cue condition, the left or right side of the display was very briefly highlighted (that is, cued) just before the appearance of the ellipse. Such a cue naturally attracts the participant’s visual attention to that side of the display. Then, the ellipse appeared on the just-previously highlighted side (valid cue condition) or on the opposite side (invalid cue condition). Performance is typically better on the validly cued side because attention has already been drawn to that side (and worse on the opposite side because attention has been pulled away from that side). Franceschini et al. [5] followed these pre-readers longitudinally across the
References 1. Choe, A., von Reuss, S.H., Kogan, D., Gasser, R.B., Platzer, E.G., Schroeder, F.C., and Sternberg, P.W. (2012). Ascaroside signaling is widely conserved among nematodes. Curr. Biol. 22, 772–780. 2. von Reuss, S.H., Bose, N., Srinivasan, J., Yim, J.J., Judkins, J.C., Sternberg, P.W., and Schroeder, F.C. (2012). Comparative metabolomics reveals biogenesis of ascarosides, a modular library of small-molecule signals in C. elegans. J. Am. Chem. Soc. 134, 1817–1824. 3. Butcher, R.A., Ragains, J.R., Li, W., Ruvkun, G., Clardy, J., and Mak, H.Y. (2009). Biosynthesis of the Caenorhabditis elegans dauer pheromone. Proc. Natl. Acad. Sci. USA 106, 1875–1879. 4. Pungaliya, C., Srinivasan, J., Fox, B.W., Malik, R.U., Ludewig, A.H., Sternberg, P.W., and Schroeder, F.C. (2009). A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 106, 7708–7713. 5. Golden, J.W., and Riddle, D.L. (1982). A pheromone influences larval development in the nematode Caenorhabditis elegans. Science 218, 578–580. 6. Jeong, P.Y., Jung, M., Yim, Y.H., Kim, H., Park, M., Hong, E., Lee, W., Kim, Y.H., Kim, K., and Paik, Y.K. (2005). Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone. Nature 433, 541–545. 7. Butcher, R.A., Fujita, M., Schroeder, F.C., and Clardy, J. (2007). Small-molecule pheromones that control dauer development in
8.
9.
10.
11.
12.
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Caenorhabditis elegans. Nat. Chem. Biol. 3, 420–422. Butcher, R.A., Ragains, J.R., Kim, E., and Clardy, J. (2008). A potent dauer pheromone component in Caenorhabditis elegans that acts synergistically with other components. Proc. Natl. Acad. Sci. USA 105, 14288–14292. Kaplan, F., Srinivasan, J., Mahanti, P., Ajredini, R., Durak, O., Nimalendran, R., Sternberg, P.W., Teal, P.E., Schroeder, F.C., Edison, A.S., et al. (2011). Ascaroside expression in Caenorhabditis elegans is strongly dependent on diet and developmental stage. PLoS One 6, e17804. Srinivasan, J., Kaplan, F., Ajredini, R., Zachariah, C., Alborn, H.T., Teal, P.E., Malik, R.U., Edison, A.S., Sternberg, P.W., and Schroeder, F.C. (2008). A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature 454, 1115–1118. Yamada, K., Hirotsu, T., Matsuki, M., Butcher, R.A., Tomioka, M., Ishihara, T., Clardy, J., Kunitomo, H., and Iino, Y. (2010). Olfactory plasticity is regulated by pheromonal signaling in Caenorhabditis elegans. Science 329, 1647–1650. Macosko, E.Z., Pokala, N., Feinberg, E.H., Chalasani, S.H., Butcher, R.A., Clardy, J., and Bargmann, C.I. (2009). A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans. Nature 458, 1171–1175. Srinivasan, J., von Reuss, S.H., Bose, N., Zaslaver, A., Mahanti, P., Ho, M.C., O’Doherty, O.G., Edison, A.S., Sternberg, P.W., and Schroeder, F.C. (2012). A modular library of small molecule signals regulates social
Endosymbiotic Evolution: RNA Intermediates in Endosymbiotic Gene Transfer More than a billion years of endosymbiotic evolution has resulted in extensive gene relocation between the genetic compartments of eukaryotic cells. A new study uses chloroplast genome transformation to shed light on the mechanisms involved. Jeremy N. Timmis In the 40 years since Lynn Margulis [1,2] revived the endosymbiotic theory of eukaryotic evolution after decades of neglect, experimental results have been universally supportive of the concepts. Convincing evidence has been provided both by inference from bioinformatic analyses of nuclear genomes and by experimental recapitulation of some of the key processes [3]. Since the engulfment of the prokaryote ancestors of mitochondria and, later, plastids (chloroplasts) by a precursor of the nucleated cell, many genes have migrated to the nucleus of the host at the expense of the endosymbiont genomes. As a result, extant
cytoplasmic organellar genomes encode very few proteins, making it necessary for chloroplasts and mitochondria to import thousands of nucleus-encoded gene products required for their biogenesis and function. A new study in this issue of Current Biology [4] uncovers mechanistic detail regarding how gene transfer to the nucleus occurs. The majority of functional endosymbiotic gene transfer (EGT) probably occurred relatively soon after the formation of the first conglomerate cells, and the process appears to have gone as far as it will for mitochondrial genes in animal cells, though some protists with altered biochemistry lack a mitochondrial genome altogether. Interestingly, EGT is still ticking over
14.
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behaviors in Caenorhabditis elegans. PLoS Biol. 10, e1001237. Kim, K., Sato, K., Shibuya, M., Zeiger, D.M., Butcher, R.A., Ragains, J.R., Clardy, J., Touhara, K., and Sengupta, P. (2009). Two chemoreceptors mediate developmental effects of dauer pheromone in C. elegans. Science 326, 994–998. McGrath, P.T., Xu, Y., Ailion, M., Garrison, J.L., Butcher, R.A., and Bargmann, C.I. (2011). Parallel evolution of domesticated Caenorhabditis species targets pheromone receptor genes. Nature 477, 321–325. Thomas, J.H., and Robertson, H.M. (2008). The Caenorhabditis chemoreceptor gene families. BMC Biol. 6, 42. Bartley, J.P., Bennett, E.A., and Darben, P.A. (1996). Structure of the ascarosides from Ascaris suum. J. Nat. Prod. 59, 921–926. Stewart, M.K., Clark, N.L., Merrihew, G., Galloway, E.M., and Thomas, J.H. (2004). High genetic diversity in the chemoreceptor superfamily of C. elegans. Genetics 169, 1985–1996. Blaxter, M.L. (2011). Nematodes: The worm and its relatives. PLoS Biol. 9, e1001050.
Institut de Biologie Valrose, CNRS UMR7277 – INSERM U1091, Universite´ de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice cedex 2, France. E-mail: [email protected] DOI: 10.1016/j.cub.2012.03.035
for both mitochondrial and chloroplast genes in plant systems. Furthermore, some steps in the EGT process still occur in essentially all eukaryotes, where they continue to have a major influence on nuclear evolution [3]. Simple and shuffled tracts of genetic information from cytoplasmic organelles, resulting from DNA transfer per se, are universally seen in the nuclear genome of essentially all organisms examined [5–7]. Thus, the processes responsible for EGT and associated DNA transfer are fundamental to eukaryote evolution, the production of genetic diversity, and the emergence of multicellular organisms. A small sample of the thousands of nuclear genes that have entered plant nuclei by EGT [8] have been examined in detail, and some transfer events appear to have involved an RNA intermediate [9–11]. This suggestion is based on the observation that some nuclear genes contributing to some plant mitochondrial proteomes are more similar to spliced and edited mitochondrial mRNAs than to the equivalent gene copies that remain in the mitochondrial genome in some related plant species. There is clear
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evidence, however, that the vast majority of the genetic information that enters the nucleus is by direct DNA transfer [3,12,13] and other explanations for the apparent involvement of an RNA intermediate have been strongly advocated [3,14]. Nonetheless, the idea of RNA intermediates, and the circumstantial supporting evidence is attractive enough that controversy exists and, though attempts have been made [15], it has so far been impossible to rule RNA in or out as a component of the mechanisms involved in EGT. Ralph Bock’s group in Potsdam set out to settle the matter using chloroplast transformation [4] and, in doing so, brought to light a new aspect of endosymbiotic evolution. Aiming to search for EGT events mediated by RNA molecules, Fuentes et al. [4] designed a DNA sequence (Nt-pIF84) with which they transformed tobacco chloroplasts. The sequence was tailored as a precursor to a gene that would be functional in the nucleus only if it was transcribed, and its transcript processed, within the chloroplast prior to migration to the nucleus in RNA or cDNA form. To achieve this, a chloroplast-specific promoter drives the reporter gene (nptII), complete with a plant nucleus-specific promoter and terminator, all in antisense orientation. Chloroplast transcription was made unequivocally recognisable by adding a group II intron — one that must be spliced within the chloroplast — in the correct polarity with respect to the chloroplast promoter. The final transplastomic tobacco line contained a homogeneous population of chloroplasts that were producing nptII spliced antisense transcripts in which kanamycin resistance should result only after they were copied into double-stranded DNA and inserted into the nucleus. Next, over a million progeny of the self-fertilized, transplastomic lines were screened and, surprisingly, 91 seedlings showed strong expression of the functional reporter gene. This frequency (w1:11,000) is similar to previous experiments in which only direct DNA transfer was suspected [13,16]. Sure enough, analyses of the nuclear integrants in Fuentes et al.’s screen [4] did not reveal a single case where the chloroplast-specific intron was missing, suggesting that RNA is vanishingly rarely involved in these
DNA transfer events. This confirmed and extended previous experiments that found no evidence of RNA intermediates [15]. However, whether RNA is ever involved in functional gene transfer in a natural evolutionary EGT scenario remains an open question that now appears intractable to experimentation because of its rarity. Unexpectedly, therefore, the reporter gene was somehow able to be expressed directly in nuclear DNA without the chloroplast-specific splicing that was assumed to be a prerequisite. The explanation is that the intron used in these experiments contains cryptic sequences that are recognised by the eukaryotic mRNA splicing machinery. Fortuitous eukaryotic signals are not unknown in plastid genes and DNA. The high AT content of the plastome has been shown to provide cryptic polyadenylation signals during plastid gene activation in the nucleus [17,18] and the chloroplast psbA promoter is moderately active in the nucleus [17]. How many other chloroplast and mitochondrial sequences contain hidden nuclear signals and how much alternative splicing is explained by EGT and other nuclear genomic rearrangements remains to be seen. I have outlined only parts of this fascinating and cleverly designed research. The results suggest that RNA is rarely involved compared with direct DNA transfer. However, genomic analyses are able to look back many millions of years, and comparisons with experimental results are challenging. Also, the sheer volume of DNA bombardment of the nucleus suggests it has an overriding role in EGT, and indeed experimental prokaryotic genes that escaped to the nucleus by bulk DNA transfer, while initially inactive, are able to attain function after nuclear genomic rearrangements [17,18]. Fuentes et al. [4] examined gene transfer from a single transplastomic cell through to the sexual progeny of regenerated plants, thereby screening for EGT within the entire lineage of somatic cells within both male and female germlines. How many cells the germline includes is uncertain, so it will be interesting to see what happens when similar experiments are done in cultured somatic cells. The present screen includes events in male gametogenesis where bulk DNA transposition is at its greatest due to the programmed degradation of
plastids during pollen development [13]. The unexpected immediate expression of nptII that thwarted these carefully designed experiments highlights the enormous flexibility with which the nucleus deals with incoming genetic material. It seems that either the nucleus or the chloroplast genetic compartments, or both, are full of tricks, many of which may surprise even the best-laid plans. Therefore, these results will also serve as a warning to those who propose theoretical precautions against gene escape in biotechnological applications. References 1. Margulis, L. (1970). Origin of Eukaryotic Cells (New Haven and London: Yale University Press). 2. Archibald, J.M. (2012). Lynn Margulis (1938–2011) Obituary. Curr. Biol. 22, R4–R6. 3. Timmis, J.N., Ayliffe, M.A., Huang, C.Y., and Martin, W. (2004). Endosymbiotic gene transfer: Organelle genomes forge eukaryotic chromosomes. Nat. Rev. Genet. 5, 123–135. 4. Fuentes, I., Karcher, D., and Bock, R. (2012). Experimental reconstruction of the functional transfer of intron-containing plastid genes to the nucleus. Curr. Biol. 22, 763–771. 5. Kleine, T., Maier, U.G., and Leister, D. (2009). DNA transfer from organelles to the nucleus: the idiosyncratic genetics of endosymbiosis. Annu. Rev. Plant Biol. 60, 115–138. 6. Noutsos, C., Richly, E., and Leister, D. (2005). Generation and evolutionary fate of insertions of organelle DNA in the nuclear genomes of flowering plants. Genome Res. 15, 616–628. 7. Noutsos, C., Kleine, T., Armbruster, U., DalCorso, G., and Leister, D. (2007). Nuclear insertions of organellar DNA can create novel patches of functional exon sequences. Trends Genet. 23, 597–601. 8. Martin, W., Rujan, T., Richly, E., Hansen, A., Cornelsen, S., Lins, T., Leister, D., Stoebe, B., Hasegawa, M., and Penny, D. (2002). Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc. Natl. Acad. Sci. USA 99, 12246–12251. 9. Nugent, J.M., and Palmer, J.D. (1991). RNA-mediated transfer of the gene coxII from the mitochondrion to the nucleus during flowering plant evolution. Cell 66, 473–481. 10. Covello, P.S., and Gray, M.W. (1992). Silent mitochondrial and active nuclear genes for subunit 2 of cytochrome C oxidase (cox2) in soybean - evidence for RNA-mediated gene transfer. EMBO J. 11, 3815–3820. 11. Wischmann, C., and Schuster, W. (1995). Transfer of rps10 from the mitochondriom to the nucleus in Arabidopsis thaliana - evidence for RNA-mediated transfer and exon shuffling at the integration site. FEBS Lett. 374, 152–156. 12. Bock, R., and Timmis, J.N. (2008). Reconstructing evolution: gene transfer from plastids to the nucleus. Bioessays 30, 556–566. 13. Sheppard, A.E., Ayliffe, M.A., Blatch, L., Day, A., Delaney, S.K., Khairul-Fahmy, N., Li, Y., Madesis, P., Pryor, A.J., and Timmis, J.N. (2008). Transfer of plastid DNA to the nucleus is elevated during male gametogenesis in tobacco. Plant Physiol. 148, 328–336. 14. Henze, K., and Martin, W. (2001). How do mitochondrial genes get into the nucleus? Trends Genet. 17, 383–387. 15. Sheppard, A.E., Madesis, P., Lloyd, A.H., Day, A., Ayliffe, M.A., and Timmis, J.N. (2011).
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Introducing an RNA editing requirement into a plastid-localised transgene reduces but does not eliminate functional gene transfer to the nucleus. Plant Mol. Biol. 76, 299–309. 16. Huang, C.Y., Ayliffe, M.A., and Timmis, J.N. (2003). Direct measurement of the transfer rate of chloroplast DNA into the nucleus. Nature 422, 72–76.
17. Lloyd, A.H., and Timmis, J.N. (2011). The origin and characterization of new nuclear genes originating from a cytoplasmic organellar genome. Mol. Biol. Evol. 28, 2019–2028. 18. Stegemann, S., and Bock, R. (2006). Experimental reconstruction of functional gene transfer from the tobacco plastid genome to the nucleus. Plant Cell 18, 2869–2878.
Reading Abilities: Importance of Visual-Spatial Attention Children with dyslexia may read poorly for several reasons. Recent research suggests that in addition to skills with language sounds, visual-spatial attention may be an important predictor of reading abilities. John D.E. Gabrieli1,2 and Elizabeth S. Norton1,3 You are reading these words very quickly. A typical adult has a reading vocabulary of 50,000–100,000 words, yet can identify a printed word seen for merely 1/200th of a second. Reading is essential for learning, from literature to physics, from paper to screens on e-readers and smart phones. Yet, about 10% of children have developmental dyslexia, an unexplained difficulty in learning to read [1]. Such poor reading is often associated with undesirable outcomes, such as lower educational attainment [2]. Dyslexia is likely caused by multiple factors, and the importance of those factors may vary between children [3] and across languages with different relations between spoken and written forms of language [4]. Research from Franceschini et al. [5] reported in this issue of Current Biology now reveals that a weakness in visual-spatial attention in pre-reading kindergartners is an important risk factor for becoming a poor reader. In all languages, under typical developmental conditions, children learn spoken language effortlessly and without formal instruction. In contrast, reading must be learned through explicit educational instruction over several years. Learning to read words can be conceptualized as learning to map the sound units of spoken language (phonemes) onto the written units of print (graphemes) so that meaning, initially related to spoken language, can be extended to print. Because many children with dyslexia appeared to hear and talk successfully at home before struggling to read at school, early conceptualizations of
dyslexia focused on putative visual deficits made manifest with print. Although there is evidence for visual deficits in dyslexia [6,7], the most common cause of dyslexia was reconceptualized in the 1980s as a weakness in the processing of language sounds, and especially in phonemic awareness — the ability to explicitly recognize and manipulate the sounds of language [8]. This weakness makes it difficult for beginning readers to map the sounds of language onto print and to accurately identify (decode) individual words. Additionally, weakness in rapid serial naming (even of color patches) has been associated with poor reading [9,10]. This weakness renders reading slow and laborious and impedes the comprehension, and pleasure, of reading text. Research has focused on children and adults who are well-characterized as dyslexic and have long struggled with reading. Such research has two important limitations. First, learning to read has reciprocal interactions with the basic skills that underlie reading itself. Thus, practice with reading enhances phonemic awareness and other reading-related processes [11]. Evidence that these skills are necessary precursors for learning to read, rather than simply a consequence of reading, is that pre-reading children in kindergarten who score poorly on tests of phonemic awareness and rapid naming are more likely to become poor readers over the next few years [12]. Second, remedial interventions that help children with dyslexia appear to be most potent at the youngest ages, before dyslexia is typically diagnosed. Therefore, early identification of risk factors for dyslexia helps identify
Discipline of Genetics, School of Molecular and Biomedical Science, The University of Adelaide, South Australia 5005, Australia. E-mail: [email protected] DOI: 10.1016/j.cub.2012.03.043
children who may benefit the most from early intervention. Franceschini et al. [5] addressed the cause of poor reading by behaviorally testing 96 pre-reading Italian-speaking kindergartners (five-year-olds) not only with typical tests of phonemic awareness and rapid naming, but also on two tests of visual-spatial performance. Although visual-spatial processes appear to be distant from the verbal processes associated with reading, studies in adults with dyslexia have revealed deficits in visual-spatial performance, often with nonverbal material [7]. These studies motivate the idea that a weakness of visual-spatial attention, independent of language, could cause dyslexia [13]. In the new study [5], one visual-spatial task required visual search across five lines of 31 symbols (not letters) and marking each occurrence of a target symbol. In the second task, children performed a spatial cuing task. In a control condition, children very briefly viewed, on the left or right of a central fixation point, an ellipse at one of four orientations, and then selected from among four alternatives which ellipse they had just viewed. The spatial cuing conditions built upon seminal research about visual attention from Michael Posner [14], who showed that attention is automatically or exogenously drawn to a spatial location by brief highlighting of that location. In the spatial cue condition, the left or right side of the display was very briefly highlighted (that is, cued) just before the appearance of the ellipse. Such a cue naturally attracts the participant’s visual attention to that side of the display. Then, the ellipse appeared on the just-previously highlighted side (valid cue condition) or on the opposite side (invalid cue condition). Performance is typically better on the validly cued side because attention has already been drawn to that side (and worse on the opposite side because attention has been pulled away from that side). Franceschini et al. [5] followed these pre-readers longitudinally across the