34 research outputs found
Redirecting splicing with bifunctional oligonucleotides
Abstract: Ectopic modulators of alternative splicing are
important tools to study the function of splice
variants and for correcting mis-splicing events that
cause human diseases. Such modulators can be
bifunctional oligonucleotides made of an antisense
portion that determines target specificity, and a nonhybridizing
tail that recruits proteins or RNA/protein
complexes that affect splice site selection (TOSS and
TOES, respectively, for targeted oligonucleotide
silencer of splicing and targeted oligonucleotide
enhancer of splicing). The use of TOSS and TOES
has been restricted to a handful of targets. To generalize
the applicability and demonstrate the robustness
of TOSS, we have tested this approach on more
than 50 alternative splicing events. Moreover, we
have developed an algorithm that can design active
TOSS with a success rate of 80%. To produce
bifunctional oligonucleotides capable of stimulating
splicing, we built on the observation that binding
sites for TDP-43 can stimulate splicing and improve
U1 snRNP binding when inserted downstream from
50 splice sites. A TOES designed to recruit TDP-43
improved exon 7 inclusion in SMN2. Overall, our
study shows that bifunctional oligonucleotides can
redirect splicing on a variety of genes, justifying their
inclusion in the molecular arsenal that aims to alter
the production of splice variants
Tumor microenvironmentâassociated modifications of alternative splicing
Abstract: Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we
probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified
in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected
tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also
found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing
factors that are differentially expressed in cancer tissues. Overall, âŒ20% of the alternative splicing events affected by the
down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together,
our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a
limited number of splicing factors
High-throughput quantification of splicing isoforms
Most human messenger RNAs (mRNAs) are alternatively spliced and many exhibit disease-specific splicing patterns. However, the contribution of most splicing events to the development and maintenance of human diseases remains unclear. As the contribution of alternative splicing events to diagnosis and prognosis is becoming increasingly recognized, it becomes important to develop precise methods to quantify the abundance of these isoforms in clinical samples. Here we present a pipeline for real-time PCR annotation of splicing events (RASE) that allows accurate identification of a large number of splicing isoforms in human tissues. The RASE automatically designed specific primer pairs for 81% of all alternative splicing events in the NCBI build 36 database. Experimentally, the majority of the RASE designed primers resulted in isoform-specific amplification suitable for quantification in human cell lines or in formalin-fixed, paraffin-embedded (FFPE) RNA extract. Using this pipeline it is now possible to rapidly identify splicing isoform signatures in different types of human tissues or to validate complete sets of data generated by microarray expression profiling and deep sequencing techniques
Giant Amplification of Photoswitching by a Few Photons in Fluorescent Photochromic Organic Nanoparticles
International audienceControlling or switching the optical signal from a large collection of molecules with the minimum of photons represents an extremely attractive concept. Promising fundamental and practical applications may be derived from such a photon-saving principle. With this aim in mind, we have prepared fluorescent photochromic organic nanoparticles (NPs), showing bright red emission, complete ONâOFF contrast with full reversibility, and excellent fatigue resistance. Most interestingly, upon successive UV and visible light irradiation, the NPs exhibit a complete fluorescence quenching and recovery at very low photochromic conversion levels (<5â%), leading to the fluorescence photoswitching of 420±20 molecules for only one converted photochromic molecule. This âgiant amplification of fluorescence photoswitchingâ originates from efficient intermolecular energy-transfer processes within the NPs
Increased degradation of ATP is driven by memory~regulatory T cells in kidney transplantation tolerance
International audienc
Tumor microenvironment-associated modifications of alternative splicing
Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, âŒ20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors
Dynamics of molecular transport by surfactants in emulsions
We consider the dynamics of equilibration of the chemical potential of a fluorophore in a monodisperse emulsion containing droplets with two initially different concentrations of the fluorophore. Although the exchange mechanism involves a single timescale at the droplet (microscopic) level, the organisation of the droplets determines the exchange dynamics at the population (macroscopic) level. The micelle concentration in the continuous phase and the chemistry of the fluorophore control the microscopic exchange rate while the disorder of the initial condition determines the power-law of the long timescale, recovered in a minimal analytical model. We also show here that an additive in the droplet such as Bovine Serum Albumin (BSA) acts on the microscopic exchange rate and slows down the exchange process by increasing the solubility of the fluorophore in the dispersed phase rather than by creating a viscoelastic layer at the droplet interface