FUS protein interacts with U7 snRNP and plays a role in replication-dependant histone genes expression

The U7 small nuclear ribonucleoprotein (U7 snRNP) is an essential factor mediating the unique 3’end processing of non-polyadenylated, replication-dependent histone mRNAs in metazoans. These histone genes expression and processing of their transcripts are cell cycle-regulated mechanisms that recruit a number of specific proteins as well as common factors required for expression and maturation of polyadenylated mRNAs. However, despite all the knowledge we have so far, there are still gaps in understanding of core histone RNA 3’ end processing, its coupling to transcription and regulation during cell cycle. To further elucidate this phenomena we used affinity chromatography based on tagged version of U7 snRNA molecule to identify proteins associated with U7 snRNP/U7 snRNA that could be potentially involved in core histone genes expression in human cells. Mass spectrometric analysis of affinity-purified fraction revealed, among others, multifunctional RNA/DNAbinding protein FUS/TLS (fused in sarcoma/translocated in liposarcoma) as a new factor interacting with U7 snRNA/RNP. Co-immunoprecipitation and RIP experiments confirmed the binding between FUS and the U7 RNA/snRNP. Interestingly, FUS:U7 snRNA interaction seems to be activated in S phase where the core histone genes are expressed. Moreover, FUS co-fractionates in 10-50% continuous glycerol gradient with other factors involved in histone premRNAs 3’end processing. However, this unique 3’end maturation was not disturbed upon FUS knockdown. Instead, we found that FUS depletion leads to a de-regulation of expression from selected histone promoters, suggesting that FUS is rather involved in regulation of core histone genes transcription. Thus, FUS bound to U7 snRNP can play a role in coupling between transcription and 3’end processing of replication dependant histone mRNAs.

Synthesis and testing of photoaffinity probes for the site-selectivechemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Synthesis and testing of photoaffinity probes for the site-selective chemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.[1] It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photolabile building blocks via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photo-labile moieties that show nanomolar binding affinities for the orthosteric binding site. Further on we developed a stable 5-HT3R overexpressing cell line and a purification method to yield the receptor in a high purity. Currently we are investigating crosslinking experiments and subsequent MS – analysis.

Synthesis and testing of photoaffinity probes for the site-selectivechemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Synthesis and testing of photoaffinity probes for the site-selective chemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Structure/affinity studies in the bicyclo-DNA series: Synthesis and properties of oligonucleotides containing bcen-T and iso-tricyclo-T nucleosides

We present the synthesis of the two novel nucleosides iso-tc-T and bcen-T, belonging to the bicyclo-/tricyclo-DNA molecular platform. In both modifications the torsion around C6’–C7’ within the carbocyclic ring is planarized by either the presence of a C6’–C7’ double bond or a cyclopropane ring. Structural analysis of these two nucleosides by X-ray analysis reveals a clear preference of torsion angle γ for the gauche orientation with the furanose ring in a near perfect 2’-endo conformation. Both modifications were incorporated into oligodeoxynucleotides and their thermal melting behavior with DNA and RNA as complements was assessed. We found that the iso-tc-T modification was significantly more destabilizing in duplex formation compared to the bcen-T modification. In addition, duplexes with complementary RNA were less stable as compared to duplexes with DNA as complement. A structure/affinity analysis, including the already known bc-T and tc-T modifications, does not lead to a clear correlation of the orientation of torsion angle γ with DNA or RNA affinity. There is, however, some correlation between furanose conformation (N- or S-type) and affinity in the sense that a preference for a 3’-endo like conformation is associated with a preference for RNA as complement. As a general rule it appears that Tm data of single modifications with nucleosides of the bicyclo-/tricyclo-DNA platform within deoxyoligonucleotides are not predictive for the stability of fully modified oligonucleotides.

Impaired genome encapsidation restricts the in vitro propagation of human parvovirus B19.

The lack of a permissive cell culture system hampers the study of human parvovirus B19 (B19V). UT7/Epo is one of the few established cell lines that can be infected with B19V but generates none or few infectious progeny. Recently, hypoxic conditions or the use of primary CD36+ erythroid progenitor cells (CD36+ EPCs) have been shown to improve the infection. These novel approaches were evaluated in infection and transfection experiments. Hypoxic conditions or the use of CD36+ EPCs resulted in a significant acceleration of the infection/transfection and a modest increase in the yield of capsid progeny. However, under all tested conditions, genome encapsidation was impaired seriously. Further analysis of the cell culture virus progeny revealed that differently to the wild-type virus, the VP1 unique region (VP1u) was exposed partially and was unable to become further externalized upon heat treatment. The fivefold axes pore, which is used for VP1u externalization and genome encapsidation, might be constricted by the atypical VP1u conformation explaining the packaging failure. Although CD36+ EPCs and hypoxia facilitate B19V infection, large quantities of infectious progeny cannot be generated due to a failure in genome encapsidation, which arises as a major limiting factor for the in vitro propagation of B19V.

The Host Nonsense-Mediated mRNA Decay Pathway Restricts Mammalian RNA Virus Replication

In addition to classically defined immune mechanisms, cell-intrinsic processes can restrict virus infection and have shaped virus evolution. The details of this virus-host interaction are still emerging. Following a genome-wide siRNA screen for host factors affecting replication of Semliki Forest virus (SFV), a positive-strand RNA (+RNA) virus, we found that depletion of nonsense-mediated mRNA decay (NMD) pathway components Upf1, Smg5, and Smg7 led to increased levels of viral proteins and RNA and higher titers of released virus. The inhibitory effect of NMD was stronger when virus replication efficiency was impaired by mutations or deletions in the replicase proteins. Consequently, depletion of NMD components resulted in a more than 20-fold increase in production of these attenuated viruses. These findings indicate that a cellular mRNA quality control mechanism serves as an intrinsic barrier to the translation of early viral proteins and the amplification of +RNA viruses in animal cells.

Diversity of morphological patterns in supramolecular polymers formed from the amphiphilic oligomers of pyrenes

Very important aspects of the modern nanotechnology are control and prediction of arraying patterns of opto- and electroactive molecules in discrete objects on nanoscale level both on surface and solution. Consequqntly, a self-assembly of small molucules provides such an opportunity.For example, oligopyrenotides (OPs, short amphiphilic pyrene oligomers) represent a novel class of amphiphilic molecules which tend to aggegate in aqueous phase. As has been already shown, OPs are able to form 1D supramolecular polymer only under high salt concentration. Since programmed arraying of polyaromatic hydrocarbons in structurally defined objects could offer enhanced performance over the individual components, prediction and controlling of their spatial arrangement remains challenging. Herein we demonstrate that substitution type of the pyrene is crutial, and it determines a morphology of the assemblies. Thus, a 1.6-linkage causes a formation of large, free-standing 2D supromolecular polymers with a thickness 2 nm. These assemblies possess a high degree of an internal order: the interior consists of hydrophobic pyrenes and alkyl chains, whereas the exterior exists as a net of hydrophilic negatively charged phosphates. Contrary, a 1.8-linkage exclusiveley leads to a formation of long (up to a few micrometer), nanometer thick helical supramolecular polymers. These structures tend to form even more complex structures (bundles, superhelixes). Moreover for both molecules, the polymerizations occurs via a nucleation-elongation mechanism. To study Py3 self-assembly, we carried out whole set of spectroscopic (UV/vis, fluorescence, DLS) and microscopic experiments (AFM).

Hierarchical self-assembly of nucleotide-appended oligopyrenotides into defined supramolecular objects

Supramolecular DNA assembly blends DNA building blocks with synthetic organic and inorganic molecules giving structural and functional advantages both to the initial self-assembly process and to the final construct. Synthetic molecules can bring a number of additional interactions into DNA nanotechnology. Incorporating extended aromatic molecules as connectors of DNA strands allows folding of these strands through π-π stacking (DNA “foldamers”). In previous work it was shown that short oligopyrenotides (phosphodiester-linked pyrene oligomers) behave as staircase-like foldamers, which cooperatively self-assemble into two-dimensional supramolecular polymers in aqueous medium. Herein, we demonstrate that a 10-mer DNA-sequence modified with 7 pyrene units (see illustration) forms dimensionally-defined supramolecular polymers under thermodynamic conditions in water. We present the self-assembly behavior, morphological studies, and the spectroscopic properties of the investigated DNA-sequences (illustrative AFM picture shown below).

Procerenone: a Fatty Acid Triterpenoid from the Fruit Pericarp of Omphalocarpum procerum (Sapotaceae)

Phytochemical investigation of a dichloromethane-methanol (1:1) extract of the fruit pericarp of Omphalocarpum procerum which exhibited antiplasmodial activity during preliminary screening led to the isolation of the new fatty ester triterpenoid 3β-hexadecanoyloxy-28-hydroxyolean-12-en-11-one (1), together with five known compounds 2-6. The structure of the new compound as well as those of the known compounds was established by means of spectroscopic methods and by comparison with previously reported data. Compounds 1- 4 were evaluated in-vitro for their cytotoxicity against L6 cell lines and antiprotozoal activities against Plasmodium falciparum, Leishmania donovani, Trypanosoma brucei rhodesiense and Trypanosoma cruzi (species responsible for human malaria, visceral leishmaniasis, African trypanosomiasis and Chagas disease, respectively). The tested compounds showed weak to moderate antiprotozoal activity and, no significant effect was detected regarding their cytotoxic potency.

Probing interactions of NMD factors in a distance-dependent manner by BioID

The understanding of molecular mechanisms requires the elucidation of protein-protein interaction in vivo. For large multi-factor complexes like those assembling on mRNA, co-immunoprecipitation assays often identify many peripheral interactors that complicate the interpretation of such results and that might conceal other insightful mechanistic connections. Here we address the protein-protein interaction network for key factors in the nonsense-mediated mRNA decay (NMD) pathway in a distant-dependent manner using BioID1,2. In this novel approach, the mutant E. coli biotin-protein ligase BirAR118G is fused to the bait protein and biotinylates proximal proteins promiscuously. Hence, interactors positioned close to the bait in vivo are enriched by streptavidin purification and identified by mass spectrometry or western blotting. We present a validation of the BioID assay and preliminary results for close interactors of UPF1 and other key players in NMD.

Probing interactions of NMD factors in a distance-dependent manner by BioID

The understanding of molecular mechanisms requires the elucidation of protein-protein interaction in vivo. For large multi-factor complexes like those assembling on mRNA, co-immunoprecipitation assays often identify many peripheral interactors that complicate the interpretation of such results and that might conceal other insightful mechanistic connections. Here we address the protein-protein interaction network for key factors in the nonsense-mediated mRNA decay (NMD) pathway in a distant-dependent manner using BioID1,2. In this novel approach, the mutant E. coli biotin-protein ligase BirAR118G is fused to the bait protein and biotinylates proximal proteins promiscuously. Hence, interactors positioned close to the bait in vivo are enriched by streptavidin purification and identified by mass spectrometry or western blotting. We present a validation of the BioID assay and preliminary results for close interactors of UPF1 and other key players in NMD.