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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