José Rino

Synthesis and Biological Studies of Pyrazolyl-Diamine PtII Complexes Containing Polyaromatic DNA-Binding Groups

New [PtCl(pz*NN)]n+ complexes anchored by pyrazolyl‐diamine (pz*NN) ligands incorporating anthracenyl or acridine orange DNA‐binding groups have been synthesized so as to obtain compounds that would display synergistic effects between platination and intercalation of DNA. Study of their interaction with supercoiled DNA indicated that the anthracenyl‐containing complex L2Pt displays a covalent type of binding, whereas the acridine orange counterpart L3Pt shows a combination of intercalative and covalent binding modes with a strong contribution from the former. L2Pt showed a very strong cytotoxic effect on ovarian carcinoma cell lines A2780 and A2780cisR, which are, respectively, sensitive to and resistant to cisplatin. In these cell lines, L2Pt is nine to 27 times more cytotoxic than cisplatin. In the sensitive cell line, L3Pt showed a cytotoxic activity similar to that of cisplatin, but like L2Pt was able significantly to overcome cisplatin cross‐resistance. Cell‐uptake studies showed that L2Pt accumulates preferentially in the cytoplasm, whereas L3Pt reaches the cell nucleus more easily, as clearly visualized by time‐lapse confocal imaging of live A2870 cells. Altogether, these findings seem to indicate that interaction with biological targets other than DNA might be involved in the mechanism of action of L2Pt because this compound, despite having a weaker ability to target the cell nucleus than L3Pt, as well as an inferior DNA affinity, is nevertheless more cytotoxic. Furthermore, ultrastructural studies of A2870 cells exposed to L2Pt and L3Pt revealed that these complexes induce different alterations in cell morphology, thus indicating the involvement of different modes of action in cell death.

Frontiers in fluorescence microscopy.

How we see organisms and cells depends on the tools at our disposal. For over 150 years, biologists were forced to rely on fixed, dehydrated and stained specimens in order to guess how the living cells could function. It all changed abruptly during the last two decades when the rapid development of novel imaging techniques revolutionized the way scientists look at the structures of life alive.

Pharmacological inhibition of the spliceosome subunit SF3b triggers exon junction complex-independent nonsense-mediated decay

ABSTRACT Spliceostatin A, meayamycin, and pladienolide B are small molecules that target the SF3b subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP). These compounds are attracting much attention as tools to manipulate splicing and for use as potential anti-cancer drugs. We investigated the effects of these inhibitors on mRNA transport and stability in human cells. Upon splicing inhibition, unspliced pre-mRNAs accumulated in the nucleus, particularly within enlarged nuclear speckles. However, a small fraction of the pre-mRNA molecules were exported to the cytoplasm. We identified the export adaptor ALYREF as being associated with intron-containing transcripts and show its requirement for the nucleo-cytoplasmic transport of unspliced pre-mRNA. In contrast, the exon junction complex (EJC) core protein eIF4AIII failed to form a stable complex with intron-containing transcripts. Despite the absence of EJC, unspliced transcripts in the cytoplasm were degraded by nonsense-mediated decay (NMD), suggesting that unspliced transcripts are degraded by an EJC-independent NMD pathway. Collectively, our results indicate that although blocking the function of SF3b elicits a massive accumulation of unspliced pre-mRNAs in the nucleus, intron-containing transcripts can still bind the ALYREF export factor and be transported to the cytoplasm, where they trigger an alternative NMD pathway. Highlighted Article: Upon treatment with splicing inhibitors, unspliced transcripts are exported to the cytoplasm by a mechanism dependent on ALYREF, and are degraded by an EJC-independent NMD pathway.

Single-Molecule Live-Cell Visualization of Pre-mRNA Splicing

Microscopy protocols that allow live-cell imaging of molecules and subcellular components tagged with fluorescent conjugates are indispensable in modern biological research. A breakthrough was recently introduced by the development of genetically encoded fluorescent tags that combined with fluorescence-based microscopic approaches of increasingly higher spatial and temporal resolution made it possible to detect single protein and nucleic acid molecules inside living cells. Here, we describe an approach to visualize single nascent pre-mRNA molecules and to measure in real time the dynamics of intron synthesis and excision.

Quantitative Image Analysis of Single-Molecule mRNA Dynamics in Living Cells

Single mRNA molecules can be imaged in living cells by a method that consists in genetically inserting binding sites for a bacteriophage protein in the gene of interest. The resulting reporter transgene is then integrated in the genome of cells that express the phage protein fused to a fluorescent tag. Upon transcription, binding of the fluorescent protein to its target sequence makes the RNA visible. With this approach it is possible to track, in real time, the life cycle of a precursor mRNA at the site of transcription in the nucleus and transport of mature mRNA to the cytoplasm. In order to measure the fluorescence associated with individual RNA molecules over time, we developed a semi-automated quantitative image analysis tool termed STaQTool. We describe in detail the implementation and application of the STaQTool software package, which is a generic tool able to process large 4D datasets allowing quantitative studies of different steps in gene expression.