Rules of engagement promote polarity in RNA trafficking

Many cell biological pathways exhibit overall polarity (net movement of molecules in one direction) even though individual molecular interactions in the pathway are freely reversible. The A2 RNA trafficking pathway exhibits polarity in moving specific RNA molecules from the nucleus to localization sites in the myelin compartment of oligodendrocytes or dendritic spines in neurons. The A2 pathway is mediated by a ubiquitously expressed trans-acting trafficking factor (hnRNP A2) that interacts with a specific 11 nucleotide cis-acting trafficking sequence termed the A2 response element (A2RE) found in several localized RNAs. Five different molecular partners for hnRNP A2 have been identified in the A2 pathway: hnRNP A2 itself, transportin, A2RE RNA, TOG (tumor overexpressed gene) and hnRNP E1, each playing a key role in one particular step of the A2 pathway. Sequential interactions of hnRNP A2 with different molecular partners at each step mediate directed movement of trafficking intermediates along the pathway. Specific "rules of engagement" (both and, either or, only if) govern sequential interactions of hnRNP A2 with each of its molecular partners. Rules of engagement are defined experimentally using three component binding assays to measure differential binding of hnRNP A2 to one partner in the presence of each of the other partners in the pathway. Here we describe rules of engagement for hnRNP A2 binding to each of its molecular partners and discuss how these rules of engagement promote polarity in the A2 RNA trafficking pathway. For molecules with multiple binding partners, specific rules of engagement govern different molecular interactions. Rules of engagement are ultimately determined by structural relationships between binding sites on individual molecules. In the A2 RNA trafficking pathway rules of engagement governing interactions of hnRNP A2 with different binding partners provide the basis for polarity of movement of intermediates along the pathway

Crystallization of the Photosystem II core complex and its chlorophyll binding subunit CP43 from transplastomic plants of Nicotianatabacum

Photosystem II from transplastomic plants of Nicotiana tabacum with a hexahistidine tag at the N-terminal end of the PsbE subunit (α-chain of the cytochrome b559) was purified according to the protocol of Fey et al. (BBA 12:1501–1509, 2008). The protein sample was then subjected to two additional gel filtration runs in order to increase its homogeneity and to standardize the amount of detergent. Large three dimensional crystals of the core complex were obtained. Crystals of one of its chlorophyll binding subunits (CP43) in isolation grew in very similar conditions that differed only in the concentration of the detergent. Diffraction of Photosystem II and CP43 crystals at various synchrotron beamlines was limited to a resolution of 7 and 14 Å, respectively. In both cases the diffraction quality was insufficient for an unambiguous assignment of the crystallographic lattice or space group

Restriction endonucleases that cleave RNA DNA heteroduplexes bind dsDNA in A like conformation

Restriction endonucleases naturally target DNA duplexes. Systematic screening has identified a small minority of these enzymes that can also cleave RNA DNA heteroduplexes and that may therefore be useful as tools for RNA biochemistry. We have chosen AvaII G amp; 8595;GWCC, where W stands for A or T as a representative of this group of restriction endonucleases for detailed characterization. Here, we report crystal structures of AvaII alone, in specific complex with partially cleaved dsDNA, and in scanning complex with an RNA DNA hybrid. The specific complex reveals a novel form of semi specific dsDNA readout by a hexa coordinated metal cation, most likely Ca2 or Mg2 . Substitutions of residues anchoring this non catalytic metal ion severely impair DNA binding and cleavage. The dsDNA in the AvaII complex is in the A like form. This creates space for 2 amp; 8242; OH groups to be accommodated without intra nucleic acid steric conflicts. PD D E XK restriction endonucleases of known structure that bind their dsDNA targets in the A like form cluster into structurally similar groups. Most such enzymes, including some not previously studied in this respect, cleave RNA DNA heteroduplexes. We conclude that A form dsDNA binding is a good predictor for RNA DNA cleavage activit

CGG Repeats in the 5'UTR of FMR1 RNA Regulate Translation of Other RNAs Localized in the Same RNA Granules.

CGG repeats in the 5'UTR of Fragile X Mental Retardation 1 (FMR1) RNA mediate RNA localization and translation in granules. Large expansions of CGG repeats (> 200 repeats) in FMR1, referred to as full mutations, are associated with fragile X syndrome (FXS). Smaller expansions (55-200 repeats), referred to as premutations, are associated with fragile X tremor ataxia syndrome (FXTAS) and fragile X premature ovarian insufficiency (FXPOI). TMPyP4 is a porphyrin ring compound that destabilizes CGG repeat RNA secondary structure. Here we show that exogenous CGG repeat RNA by itself, lacking the FMRP ORF, microinjected into hippocampal neurons is localized in RNA granules and inhibits translation of ARC RNA, which is localized in the same granules. TMPyP4 rescues translation of ARC RNA in granules. We also show that in human premutation fibroblasts with endogenous CGG repeat expansions in the FMR1 gene, translation of ARC RNA is inhibited and calcium homeostasis is disrupted and both phenotypes are rescued by TMPyP4. Inhibition of granule translation by expanded CGG repeats and rescue of granule translation by TMPy4, represent potential pathogenic mechanism and therapeutic strategy, respectively, for FXTAS and FXPOI

Crystallization of the photosystem II core complex and its chlorophyll binding subunit CP43 from transplastomic plants of Nicotiana tabacum.

Photosystem II from transplastomic plants of Nicotiana tabacum with a hexahistidine tag at the N-terminal end of the PsbE subunit (α-chain of the cytochrome b(559)) was purified according to the protocol of Fey et al. (BBA 12:1501-1509, 2008). The protein sample was then subjected to two additional gel filtration runs in order to increase its homogeneity and to standardize the amount of detergent. Large three dimensional crystals of the core complex were obtained. Crystals of one of its chlorophyll binding subunits (CP43) in isolation grew in very similar conditions that differed only in the concentration of the detergent. Diffraction of Photosystem II and CP43 crystals at various synchrotron beamlines was limited to a resolution of 7 and 14 Å, respectively. In both cases the diffraction quality was insufficient for an unambiguous assignment of the crystallographic lattice or space group

Herpes Simplex Virus Type 1 Helicase-Primase: DNA Binding and Consequent Protein Oligomerization and Primase Activation▿ †

The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of UL5, UL8, and UL52, possesses 5′ to 3′ helicase, single-stranded DNA (ssDNA)-dependent ATPase, primase, and DNA binding activities. In this study we confirm that the UL5-UL8-UL52 complex has higher affinity for forked DNA than for ssDNA and fails to bind to fully annealed double-stranded DNA substrates. In addition, we show that a single-stranded overhang of greater than 6 nucleotides is required for efficient enzyme loading and unwinding. Electrophoretic mobility shift assays and surface plasmon resonance analysis provide additional quantitative information about how the UL5-UL8-UL52 complex associates with the replication fork. Although it has previously been reported that in the absence of DNA and nucleoside triphosphates the UL5-UL8-UL52 complex exists as a monomer in solution, we now present evidence that in the presence of forked DNA and AMP-PNP, higher-order complexes can form. Electrophoretic mobility shift assays reveal two discrete complexes with different mobilities only when helicase-primase is bound to DNA containing a single-stranded region, and surface plasmon resonance analysis confirms larger amounts of the complex bound to forked substrates than to single-overhang substrates. Furthermore, we show that primase activity exhibits a cooperative dependence on protein concentration while ATPase and helicase activities do not. Taken together, these data suggest that the primase activity of the helicase-primase requires formation of a dimer or higher-order structure while ATPase activity does not. Importantly, this provides a simple mechanism for generating a two-polymerase replisome at the replication fork

CGG repeat expansions in the FMR1 gene inhibit translation of Venus-ARC RNA in human fibroblasts.

<p>Panel A—Human fibroblasts from control individuals C0603 (control male, 31 repeats), GM00497 (control male, unknown repeat number), GM00498 (control male, unknown repeat number) and FMR1 premutation carriers FX08-2 (female, 31, 105 repeats), FX11-2 (female, 20, 79 repeats), FX13-2 (female, 33, 85 repeats), and WC26 (female, two premutation alleles, 60, 90 repeats) were microinjected with Venus-ARC RNA and after 2 hours Venus-ARC RNA and newly-synthesized Venus-ARC protein were imaged by dual channel confocal microscopy. Representative images are shown for untreated control and premutation cells and for premutation cells treated with TMPyP4. Scale bars indicate 5 micrometers. Panel B—Cumulative Kolmogorov Smirnov plots for specific translational activities (newly-synthesized Venus-ARC protein/microinjected Venus-ARC RNA) in individual cells for 3 control and 4 premutation fibroblast cell lines as described for Panel A, untreated (top) and treated with TMPyP4 (bottom). Panel C—Frequency distribution plots for specific translational activities (newly-synthesized Venus-ARC protein/microinjected Venus-ARC RNA) in cells for 3 control and 4 premutation fibroblast cell lines as described for Panel B, untreated (top) and treated with TMPyP4 (bottom). Panel D—PCR analysis of CGG repeat numbers in FX08-02, FX11-02, FX13-02 and C0603 fibroblasts with a 100 bp DNA ladder (left panel) and in WC26 and C0603 fibroblasts with a 1 kb ladder (right panel). In the panel on the left, in the FX08-2, FX11-2, and FX13-2 lanes, the band at the bottom of the gel represents female gender specific PCR product, the bands immediately above the gender specific band represent PCR products from CGG repeat alleles in the normal range and the bands above represent PCR products from expanded CGG repeat alleles. The fainter products near the top of the gel are of unknown origin. In the C0603 lane, the two bands near the bottom of the gel represent male and female gender specific PCR products, and the band above the gender specific bands represents PCR product from the normal CGG repeat allele. In the panel on right, the two bands in the WC26 lane both represent PCR products from expanded CGG repeat alleles and the single band in the C0603 lane represents PCR product from the CGG repeat allele in the normal range. Panel E—Table showing ID, gender, FMR1 alleles and CGG repeat numbers (based on panel D) for each cell line. Panel F—Western blotting of FMRP expression in full mutation (FX08-1, FX11-01, FX13-01) and premutation (FX08-2, FX11-02 and FX13-02) cell lines with actin loading controls.</p

CGG repeat RNA and ARC RNA are co-localized in granules and CGG repeat RNA inhibits translation of ARC RNA in hippocampal neurons.

<p>Panel A shows a schematic outline of the experimental protocol. Differentially labeled CGG repeat RNA and Venus-ARC RNA were co-injected into the cytoplasm of hippocampal neurons where they are localized in granules in dendrites. Venus-ARC RNA is translated in granules near dendritic spines. In some cases hippocampal neurons were incubated with membrane-permeant TMPyP4 before injecting RNA. Panel B shows the distribution of Venus-ARC RNA (red), CGG99 RNA (blue) and newly-synthesized Venus-ARC protein (green) in discrete granules in a three channel confocal images of a dendritic segment. Seven granules are visible in this dendritic segment, all of which contain Venus-ARC RNA, two of which also contain CGG99 RNA, and five of which lack detectable CGG99 RNA. Newly synthesized Venus-ARC protein is detected in the five granules lacking CGG99 RNA but not in the two granules containing CGG99 RNA. Scale bar indicates 1 micrometer. Panel C shows 3-dimensional graphs of fluorescence intensities for Venus-ARC RNA, Venus-ARC protein and CGG99 (right panel) or CGG0 (left panel) RNAs in a population of individual granules in hippocampal neurons. Panel D shows Kolmogorov-Smirnov cumulative frequency plots of specific translational activities (Venus-ARC protein/Venus-ARC RNA) for granules containing detectable (defined as integrated fluorescence intensity > 10 arbitrary units) CGG99 RNA (red), or in granules where CGG99 RNA was undetectable (defined as integrated fluorescence intensity < 10 arbitrary units) (black),. Specific translational activities were measured for granules in untreated (left panel) or in TMPyP4-treated (right panel) hippocampal neurons.</p