Publications

2012
Wang, W, Messing J.  2012.  Analysis of ADP-glucose pyrophosphorylase expression during turion formation induced by abscisic acid in Spirodela polyrhiza (greater duckweed). BMC Plant Biology. 12(5) Abstract
Aquatic plants differ in their development from terrestrial plants in their morphology and physiology, but little is known about the molecular basis of the major phases of their life cycle. Interestingly, in place of seeds of terrestrial plants their dormant phase is represented by turions, which circumvents sexual reproduction. However, like seeds turions provide energy storage for starting the next growing season.
Li, Y, Padgett RW.  2012.  bantam is required for optic lobe development and glial cell proliferation. PLoS One. 7(3) AbstractWebsite
microRNAs (miRNAs) are small, conserved, non-coding RNAs that contribute to the control of many different cellular processes, including cell fate specification and growth control. Drosophila bantam, a conserved miRNA, is involved in several functions, such as stimulating proliferation and inhibiting apoptosis in the wing disc. Here, we reported the detailed expression pattern of bantam in the developing optic lobe, and demonstrated a new, essential role in promoting proliferation of mitotic cells in the optic lobe, including stem cells and differentiated glial cells. Changes in bantam levels autonomously affected glial cell number and distribution, and non-autonomously affected photoreceptor neuron axon projection patterns. Furthermore, we showed that bantam promotes the proliferation of mitotically active glial cells and affects their distribution, largely through down regulation of the T-box transcription factor, optomotor-blind (omb, Flybase, bifid). Expression of omb can rescue the bantam phenotype, and restore the normal glial cell number and proper glial cell positioning in most Drosophila brains. These results suggest that bantam is critical for maintaining the stem cell pools in the outer proliferation center and glial precursor cell regions of the optic lobe, and that its expression in glial cells is crucial for their proliferation and distribution.
Westra, ER, van Erp PB, Wong SP, Kunne T, Staals R, Seegers CL, Bollen S, Jore MM, de Vos WM, Dame RT et al..  2012.  CRISPR immunity relies on the conservative binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol. Cell. 46:595-605.
Djordjevic, M, Djordjevic M, Severinov K.  2012.  CRISPR transcript processing: an unusual mechanism for strong amplification of small RNAs. Biol. Direct. 7:24.
Burrows, EH, Bennette NB, Carrieri D, Dixon JL, Brinker A, Frada M, Bakdassabim S N, Falkowski PG, Dismukes GC.  2012.  Dynamics of Lipid Biosynthesis and Redistribution in the Marine Diatom Phaeodactylum tricornutum under Nitrate Deprivation. Bioenerg. Res. 5:876-885. Abstract
One approach to achieve continuous overproduction of lipids in microalgal “cell factories” relies upon depletion or removal of nutrients that act as competing electron sinks (e.g., nitrate and sulfate). However, this strategy can only be effective for bioenergy applications if lipid is synthesized primarily de novo (from CO2 fixation) rather than from the breakdown and interconversion of essential cellular components. In the marine diatom, Phaeodactylum tricornutum, it was determined, using 13C-bicarbonate, that cell growth in nitrate (NO 3 − )-deprived cultures resulted predominantly in de novo lipid synthesis (60 % over 3 days), and this new lipid consisted primarily of triacylglycerides (TAGs). Nearly complete preservation of 12C occurred in all previously existing TAGs in NO 3 − -deprived cultures and thus, further TAG accumulation would not be expected from inhibition of TAG lipolysis. In contrast, both high turnover and depletion of membrane lipids, phosphatidylcholines (PCs), were observed in NO 3 − -deprived cultures (both the headgroups and fatty acid chains), while less turnover was observed in NO 3 − replete cultures. Liquid chromatography-tandem mass spectrometry mass spectra and 13C labeling patterns of PC headgroups provided insight into lipid synthesis in marine diatoms, including suggestion of an internal pool of glycine betaine that feeds choline synthesis. It was also observed that 16C fatty acid chains incorporated into TAGs and PCs contained an average of 14 13C carbons, indicating substantial incorporation of 13C-bicarbonate into fatty acid chains under both nutrient states.
Glukhov, AS, Krutilina AI, Shlyapnikov MG, Severinov K, Lavysh D, Kochetkov VV, McGrath JW, de Leeuwe C, Shaburova OV, Krylov VN et al..  2012.  Genomic analysis of Pseudomonas putida phage tf with localized single-strand DNA interruptions. PLoS One. 7:e51163.
Berdygulova, Z, Esyunina D, Miropolskaya N, Mukhamedyarov D, Kuznedelov K, Nickels B, Severinov K, Kulbachinskiy A, Minakhin L.  2012.  The gp39 protein of phage P23-45 is a transcription antiterminator that acts by suppressing pausing by Thermus thermophilus RNA polymerase. Nucl. Acids Res. 40:4052-4063.
Vvedenskaya, IO, Sharp JS, Goldman SR, Kanabar PN, Livny J, Dove SL, Nickels BE.  2012.  Growth phase-dependent control of transcription start site selection and gene expression by nanoRNAs. Genes Dev. 26(13):1498-1507. Abstract
Prokaryotic and eukaryotic RNA polymerases can use 2- to ~4-nt RNAs, ‘‘nanoRNAs,’’ to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.
Li, Y., Dooner HK.  2012.  Helitron Proliferation and Gene-Fragment Capture. Topics in Current Genetics, 24: Plant Transposable Elements- Impact on Genome Structure and Function. :193-227.
Park, EC, Ghose P, Shao Z, Ye Q, Kang L, Xu XZ, Powell-Coffman JA, Rongo C.  2012.  Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism.. EMBO Journal. Epub ahead of print AbstractWebsite
Oxygen influences behaviour in many organisms, with low levels (hypoxia) having devastating consequences for neuron survival. How neurons respond physiologically to counter the effects of hypoxia is not fully understood. Here, we show that hypoxia regulates the trafficking of the glutamate receptor GLR-1 in C. elegans neurons. Either hypoxia or mutations in egl-9, a prolyl hydroxylase cellular oxygen sensor, result in the internalization of GLR-1, the reduction of glutamate-activated currents, and the depression of GLR-1-mediated behaviours. Surprisingly, hypoxia-inducible factor (HIF)-1, the canonical substrate of EGL-9, is not required for this effect. Instead, EGL-9 interacts with the Mint orthologue LIN-10, a mediator of GLR-1 membrane recycling, to promote LIN-10 subcellular localization in an oxygen-dependent manner. The observed effects of hypoxia and egl-9 mutations require the activity of the proline-directed CDK-5 kinase and the CDK-5 phosphorylation sites on LIN-10, suggesting that EGL-9 and CDK-5 compete in an oxygen-dependent manner to regulate LIN-10 activity and thus GLR-1 trafficking. Our findings demonstrate a novel mechanism by which neurons sense and respond to hypoxia.
Shadrin, A, Sheppard C, Matthews S, Severinov K, Wigneshweraraj S.  2012.  Inhibition of transcription initiation by T7 Gp2 occurs through at least two different sets of interactions with the E. coli RNA polymerase. Microbiology SGM. 158:2753-2764.
Wang, W, Wu Y, Messing J.  2012.  The mitochondrial genome of an aquatic plant, Spirodela polyrhiza. PLoS ONE. 7(10) Abstract
Spirodela polyrhiza is a species of the order Alismatales, which represent the basal lineage of monocots with more ancestral features than the Poales. Its complete sequence of the mitochondrial (mt) genome could provide clues for the understanding of the evolution of mt genomes in plant.
Nickels, BE.  2012.  A new way to start: nanoRNA-mediated priming of transcription initiation.. Transcription. 3(6):300-304. Abstract
A recent study provides evidence that RNA polymerase uses 2- to ~4-nt RNAs, species termed "nanoRNAs," to prime transcription initiation in Escherichia coli. Priming of transcription initiation with nanoRNAs represents a previously undocumented component of transcription start site selection and gene expression.
Datsenko, KA, Pougach K, Tikhonov A, Wanner BL, Severinov K, Semenova E.  2012.  Prior encounters dramatically stimulate adaptive bacterial CRISPR immune response to viruses. Nature Commun. 3:945.
Zhang, D, Isack NR, Glodowski DR, Liu J, Chen CC, Xu XZ, Grant BD, Rongo C.  2012.  RAB-6.2 and the retromer regulate glutamate receptor recycling through a retrograde pathway.. The Journal of Cell Biology. 196:85-101. AbstractWebsite
Regulated membrane trafficking of AMPA-type glutamate receptors (AMPARs) is a key mechanism underlying synaptic plasticity, yet the pathways used by AMPARs are not well understood. In this paper, we show that the AMPAR subunit GLR-1 in Caenorhabditis elegans utilizes the retrograde transport pathway to regulate AMPAR synaptic abundance. Mutants for rab-6.2, the retromer genes vps-35 and snx-1, and rme-8 failed to recycle GLR-1 receptors, resulting in GLR-1 turnover and behavioral defects indicative of diminished GLR-1 function. In contrast, expression of constitutively active RAB-6.2 drove the retrograde transport of GLR-1 from dendrites back to cell body Golgi. We also find that activated RAB-6.2 bound to and colocalized with the PDZ/phosphotyrosine binding domain protein LIN-10. RAB-6.2 recruited LIN-10. Moreover, the regulation of GLR-1 transport by RAB-6.2 required LIN-10 activity. Our results demonstrate a novel role for RAB-6.2, its effector LIN-10, and the retromer complex in maintaining synaptic strength by recycling AMPARs along the retrograde transport pathway.
Ishikawa, HO, Xu A, Ogura E, Manning G, Irvine KD.  2012.  The Raine Syndrome Protein FAM20C Is a Golgi Kinase That Phosphorylates Bio-Mineralization Proteins.. PLoS One. 7:e42988. AbstractWebsite
Raine syndrome is caused by mutations in FAM20C, which had been reported to encode a secreted component of bone and teeth. We found that FAM20C encodes a Golgi-localized protein kinase, distantly related to the Golgi-localized kinase Four-jointed. Drosophila also encode a Golgi-localized protein kinase closely related to FAM20C. We show that FAM20C can phosphorylate secreted phosphoproteins, including both Casein and members of the SIBLING protein family, which modulate biomineralization, and we find that FAM20C phosphorylates a biologically active peptide at amino acids essential for inhibition of biomineralization. We also identify autophosphorylation of FAM20C, and characterize parameters of FAM20C's kinase activity, including its Km, pH and cation dependence, and substrate specificity. The biochemical properties of FAM20C match those of an enzymatic activity known as Golgi casein kinase. Introduction of point mutations identified in Raine syndrome patients into recombinant FAM20C impairs its normal localization and kinase activity. Our results identify FAM20C as a kinase for secreted phosphoproteins and establish a biochemical basis for Raine syndrome.
Wu, Y, Messing J.  2012.  RNA Interference Can Rebalance the Nitrogen Sink of Maize Seeds without Losing Hard Endosperm. PLoS One. 7:e32850. AbstractWebsite
BACKGROUND: One of the goals of plant breeding is to create crops to provide better nutrition for humans and livestock. Insufficient intake of protein is one of the most severe factors affecting the growth and development of children in developing countries. More than a century ago, in 1896, Hopkins initiated the well-known Illinois long-term selection for maize seed protein concentration, yielding four protein strains. By continuously accumulating QTLs, Illinois High Protein (IHP) reached a protein level 2.5-fold higher than normal maize, with the most increased fraction being the zein protein, which was shown to contain no lysine soon after the long-term selection program initiated. Therefore, IHP is of little value for feeding humans and monogastric animals. Although high-lysine lines of non-vitreous mutants were based on reduced zeins, the kernel soft texture precluded their practical use. Kernel hardness in opaque 2 (o2) could be restored in quality protein maize (QPM) with quantitative trait loci called o2 modifiers (Mo2s), but those did not increase total protein levels. METHODS: The most predominant zeins are the 22- and 19-kDa alpha-zeins. To achieve a combination of desired traits, we used RNA interference (RNAi) against both alpha-zeins in IHP and evaluated the silencing effect by SDS-PAGE. Total protein, amino acid composition and kernel texture were analyzed. CONCLUSIONS: The alpha-zeins were dramatically reduced, but the high total seed protein level remained unchanged by complementary increase of non-zein proteins. Moreover, the residual zein levels still allowed for a vitreous hard seed. Such dramatic rebalancing of the nitrogen sink could have a major impact in world food supply.
Mekler, V, Minakhin L, Kuznedelov K, Mukhamedyarov D, Severinov K.  2012.  Species-specific interactions determining different stability of promoter complexes formed by RNA polymerases from Escherichia coli and Thermus aquaticus. Nucl. Acids Res. 40:11352-11362.
Nocek, B, Tikhonov A, Babnigg G, Gu M, Zhou M, Makarova KS, Vondehhoff G, Van Aershot A, Anderson W, Severinov K et al..  2012.  Structural and functional characterization of microcin C resistance peptidase MccF from Bacillus anthracis. J. Mol. Biol. 420:366-383.
James, E, Liu M, Sheppard C, Mekler V, Camara B, Liu B, Simpson P, Cota E, Severinov K, Matthews S et al..  2012.  Structural basis for the inhibition of the Escherichia coli RNA polymerase by T7 Gp2. Molecular Cell. 47:755-776.
Agarwal, V, Tikhonov A, Metlytskaya A, Severinov K, Nair S.  2012.  Structure and function of a serine carboxypeptidase adapted for degradation of the protein synthesis inhibitor Microcin C7. Proc. Natl. Acad. Sci. USA. 109:4425-4430.
Pavlova, O, Lavysh D, Klimuk E, Djordjevic M, Ravcheev DA, Gelfand MS, Severinov K, Akulenko N.  2012.  Temporal regulation of gene expression of the Escherichia coli bacteriophage phiEco32. J. Mol. Biol. 416:389-399.
Pougach, K, Severinov K.  2012.  Use of semi-quantitative Northern blot analysis to determine relative quantities of bacterial CRISPR transcripts. Methods in Molecular Biology on Bacterial Regulatory RNA . :73-86.