Wednesday, September 28, 2011

Story behind the paper: small RNAs in diatom (interview w/ Andrew Allen)


Here is another "Story behind the paper".  This one focuses on the following paper: Norden-Krichmar, T.M., Allen, A.E., Gaasterland, T., Hildebrand, M.  (2011) Characterization of the small RNA transcriptome of the diatom, Thalassiosira pseudonana. PLoS ONE 6(8): e22870. doi:10.1371/journal.pone.002870

I wrote some questions up for Andrew Allen, one of the authors.  I note I did this before my "new" system of inviting authors to write guest posts directly themselves.  Not sure which approach is better but guest posts are certainly easier for me so I will probably do that more.

1.     What is the history behind this work?  How did it start?  Why did you do it?

These studies on small RNA in diatoms are the result of collaboration between my group at the J. Craig Venter Institute (JCVI) and Mark Hildebrand’s group at Scripps Institute of Oceanography (SIO). Each lab group is interested in the ecology, evolution, and physiology of diatoms. More specifically we would like to know more about how diatoms sense and respond to environmental signals. Therefore we are interested in mechanisms of transcriptional regulation in diatoms and other microalgae. An earlier study suggested that cytosine methylation is an important mechanism for repression of transcriptional activity of retrotransposons, and associated mobility, in diatoms. In response to stress, nitrogen stress especially, long terminal repeat retrotransposons (LTR-RTs) display decreased levels of cytosine methylation (hypomethylation) and elevated transcriptional activity.


Mamus, F., Allen, A.E., Mhiri, C., Hu, H., Jabbari, K., Vardi, A., Grandbastien, M.A., Bowler, C. (2009). Potential impact of stress activated retrotransposons on genome evolution in a marine diatom. BMC Genomics 10:624.

Classically small RNAs are known to play a key role in triggering gene silencing by DNA methylation. Also short interfering small RNAs (siRNAs) have been found to play a role in silencing retrotransposons and other repeat elements


Therefore we were interested to investigate the small RNA repertoire of diatoms. Our first experiments were based on 454 sequencing of libraries constructed from small RNA purified from the diatom Thalassiosira pseudonana. It was clear to us that, despite promising results, much deeper sequencing would be required for a meaningful characterization of the small RNA transcriptome. We used ABI SOLiD sequencing to further explore the diversity and expression of small RNAs in T. pseudonana. Although deep sequencing was ultimately necessary to obtain sufficient coverage and resolution for statistically sound analyses the SOLiD and 454 data were remarkably congruent.

At the time these studies were being conducted, 2009, there were some specific challenges associated with analyses of the SOLiD small RNA data. Extraction all types of small RNAs for a non-standard organism was not straightforward.

Initial processing of the SOLiD data using commercial products, such as ABI’s Small RNA Pipeline and CLCbio’s CLC NGS Cell reference assembly software, yielded an average of approximately 6% reads aligned to the T. pseudonana genome. For ABI’s Small RNA Pipeline, even when omitting the filtering step by known miRNAs from the Sanger miRBase, the software gave a higher priority to matching the adapter sequences rather than matching to the genome, in order to produce small RNAs in the miRNA size range. Similarly, because CLCbio’s CLC NGS Cell program was not able to align any sequence less than 27 nucleotides in length, and many small RNAs are in this size range, it also had to be abandoned in this study.

The methodology presented in this study provides the steps necessary to discover all types of small RNA genes in next generation sequence data, and to perform a comparative analysis of different libraries of sequence data. Briefly, an approach was necessary to extract the small RNA sequences from the constant 35 nucleotide colorspace format SOLiD data, convert the colorspace data to its basespace equivalent, and map the sequences to the reference genome.  The colorspace data, which is a numerical representation of the color produced during sequencing for each successive two-nucleotide pair, was first converted to its basespace equivalent using CLCbio’s tofasta software.  The basespace format sequences were then aligned to the T. pseudonana reference genome with BLAST, acting to simultaneously determine the alignment locations and trim the spurious adapter nucleotides from the ends of the small RNA sequences.  This method yielded a recovery rate of 22% of the reads aligned to the genome, which is two or three times more reads than the ABI SOLiD Small RNA pipeline and CLCbio’s NGS Cell program, thereby producing a large data set for further analysis.

2. What is next?

We would like to establish improved conceptual integration for the role of small RNAs in various aspects of diatom evolution, metabolism, and biochemistry. More highly resolved expression patterns of small RNAs in response to specific environmental conditions will be required to make associations between specific small RNA loci and specific cellular processes. It seems likely that copia type retrotransposons play a major role in diatom genome evolution through promoting genome rearrangements and modification of gene expression levels through displacement and insertion of various promoter binding sites. We would like to attain a better understanding of the role small RNAs in mediating transposon occurrence and transcriptional and insertional activity.  For example, in relation to retrotransposons, is the role of small RNAs strictly relegated to defense and silencing or do small RNAs also play a role in fostering establishment of transposons that ultimately have a positive impact on fitness?


3. Any interesting stories about the project like fights among authors (OK, maybe not that) - but anything more on the personal side of things?

The lead author of the study Trina Norden-Krichmar, a bioinformaticist, did a lot of the lab work for this project.  Diatom culturing, RNA purification, running gels,454 small RNA library construction, PCR, TOPO cloning, Northern blots, etc. are somewhat unusual activity for most bioinformaticians.  Interestingly, prior to earning a PhD Trina was a computer programmer who enjoyed open ocean swimming at the La Jolla Cove.  As a result of this recreational activity she was motivated to go back to school for a PhD in Marine Biology. Trina also authored a paper on small RNAs in the marine invertebrate Ciona.



4. Can you send links to any other information of value including Authors web sites


My JCVI

My Mendeley (which has all PDFs mentioned here)

Mark H.

Terry G.

Other papers of interest (e.g., some recent Nature paper by you)

Other recent studies of interest include a publication in Nature earlier this year, Evolution and metabolic significance of the urea cycle in photosynthetic diatoms.

Evolution of intracellular urea synthesis by the ornithine-urea cycle (OUC) is classically known to have facilitated a wide range of physiological innovations and life history adaptations in vertebrates. For example, urea synthesis enables rapid osmoregulation in elasmobranchs (sharks, skates, rays) and bony fish, and ammonia detoxification in amphibians and mammals, which was likely a prerequisite for life on land. Ruminants and some hibernating mammals recycle nitrogen between the liver and gut through urea.

Evolutionarily it was unusual and highly unexpected to find a gene encoding the OUC form of the gene carbamoyl phosphate synthetase (CPS) in diatoms. CPS evolution is evolution is a fascinating story and with many chapters of gene duplication and fusion. Origin of the ornithine-urea cycle can be traced to ancient duplication and subsequent neofunctionalization of ancestral eukaryotic carbamoyl phosphate synthase (CPS); CPSII. CPSII, renamed pgCPS in this study, to reflect function and substrate (pyrmidine metabolism and glutamine) is an ancient eukaryotic enzyme that resulted from fusion bacterial amidotransferase and synthetase subunits. Interestingly there is significant internal similarity within the synthetase domain which is the result of ancient duplication of a kinase domain. It has long been held that pgCPS duplicated in early diverging metazoans to form ugCPS (urea cycle, glutamine) which is targeted to mitochondria. Subsequently, in vertebrates, unCPS (urea cycle, ammonium) appeared and provided foundation for the modern vertebrate urea cycle. Therefore, discovery of unCPS in unicellular stramenopile and haptophyte algae was highly unexpected. Also, physiologically, in animals, the urea cycle is a catabolic pathway that ultimately serves to export fixed nitrogen (in the form of urea) from cells. It was somewhat puzzling and conceptually challenging to imagine a role for the urea within the context of photosynthetic cells. In addition to either glutamine or ammonium CPS utilizes inorganic carbon in the form of HCO3- and therefore represents a form of carbon fixation as well. In diatoms, it appears that the urea cycle is the basis for a distribution and repackaging hub for inorganic carbon and nitrogen and is particularly important for redistribution and turnover of cellular nitrogen following episodic pulses of nitrate; which occur during oceanic upwelling events. Although chloroplast and bacterial derived transfer of genes to the diatom nuclear genome have been described, very little is known about the contribution of the secondary endosymbiotic host (exosymbiont) to diatom metabolism. Results of this study indicate that the secondary endosymbiotic host genome made important physiological and biochemical contributions to the diatom nuclear genome sufficient to significantly distinguish secondary endosymbiotic algae from plants and green algae.

Also three studies have been published this year related carbon metabolism and the carbon concentrating mechanism (CCM) of diatoms. The occurrence of efficient CCM(s) in diatoms has long been hypothesized as a result of the relatively high affinity of diatom cells for inorganic carbon compared to much lower affinity of the enzyme RubisCO for CO2. In other words, in order to overcome RubisCO inefficiencies, such as slow turnover and a propensity to fix O2 (i.e., photorespiration), there has been strong evolutionary selection for cellular adaptations that enable elevated CO2 at the site of fixation by RubisCO. Also over geological time, atmospheric concentrations of CO2 have decreased while O2 has increased; presumably strengthening selection for CCMs in productive modern microalgae.

A manuscript by Hokinson et al published in PNAS is based on mass spectrometric measurements of passive and active cellular inorganic carbon fluxes in wild type and chloroplast carbon anhydrase (CA) over expression cell lines of the diatom Phaeodactylum tricornutum. Carbonic anhydrases (or carbonate dehydratases) are metalloenzymes that catalyze the rapid interconversion of carbon dioxide and water to bicarbonate and protons. Model simulations of these fluxes suggest that, due to membrane permeability to CO2, only around one-third of the inorganic carbon transported from the cytoplasm into the chloroplast is fixed photsynthetically; and the rest is lost by CO2 diffusion back to the cytoplasm. Therefore in order to achieve the CO2 concentration necessary to saturate carbon fixation it is hypothesized that CO2 is most likely concentrated within the pyrenoid; a specialized non-membrane bound proteinaceous structure within the chloroplast that contains high levels of RuisCO.

Hopkinson, B.M., Dupont, C.L., Allen, A.E., Moreal, F.M.M. (2011). Efficiency of the CO2-concentrating mechanisms of diatoms. Proceedings of the National Academy of Sciences of the United States of America, USA. 108(10):3830-7.


In a paper by Tachibana et al. published in Photosynthesis Research nine and thirteen carbonic anhydrase (CAs) were identified and experimentally localized in the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana respectively. Immunostaining experiments show that PtCA1, a β-CA, is localized to the central part of the pyrenoid in the chloroplast.  Other CAs are shown to be localized to the periplastidal compartment, chloroplast endoplasmic reticulum, and mitochondria in P. tricornutum and the stroma and periplasm of T. pseudonana.

Tachibana, M., Allen, A.E., Kikutani, S., Endo, Y., Bowler, C., Matsuda. (2011). Localization of putative carbonic anhydrases in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana. Photosynthesis Research. Advance Access published March 2 2011, doi:10.1007/s11120-011-9634-4

A paper published by Allen et al. in Molecular Biology and Evolution (open access) examines  the functional diversification of fructose bisphosphate aldolase (FBA) genes in diatoms. Class I and class II FBAs are involved in Calvin-Bensen cycle reaction and glycolysis. Patterns of FBA evolution have been useful for questions related to chloroplast acquisition and evolution in primary and secondary endosymbiotic algae. The universal occurrence of class II FBAs in chromalveolate (diatoms, dinoflagellates, haptophytes and crytophytes) plastids has been interpreted as evidence for chromalveolate monophyly and a single origin for secondary plastid of red algal descent. In this new paper, Allen et al., demonstrate that class I and class II FBAs are localized to the diatom pyrenoid. Class II pyrenoid localized FBA appears to be the result of a chromalveolate specific gene duplication event. The significance of FBA localization in diatom pyrenoids in not fully understood but enzymatic activity and gene transcription appears significantly enhanced under periods of iron (Fe) limitation; when photosynthesis is somewhat down regulated. The authors suggest that pyrenoid localization of some Calvin cycle components might provide a regulatory link between CCM and Calvin cycle activity.

Allen, A.E., Moustafa, A., Montsant, A., Eckert, A., Kroth, P., Bowler, C. (2011). Evolution and functional diversification of fructose bisphosphate aldolase genes in photosynthetic marine diatoms. Molecular Biology and Evolution. Advance Access published September 8, 2011, doi:10.1093/molbev/msr223

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