4 research outputs found
Probing ribosomal RNA structural rearrangements: a time lapse of ribosome assembly dynamics
Ribosome synthesis is a very complex and energy consuming process
in which pre-ribosomal RNA (pre-rRNA) processing and folding events,
sequential binding of ribosomal proteins and the input of approximately 200
trans-acting ribosome assembly factors need to be tightly coordinated. In the
yeast Saccharomyces cerevisiae, ribosome assembly starts in the nucleolus
with the formation of a very large 90S-sized complex. This ~2.2MDa pre-ribosomal
complex is subsequently processed into the 40S and 60S assembly
intermediates (pre-40S and pre-60S), which subsequently mature largely
independently. Although we have a fairly complete picture of the protein
composition of these pre-ribosomes, still very little is known about the rRNA
structural rearrangements that take place during the assembly of the 40S and
60S subunits and the role of the ribosome assembly factors in this process. To
address this, the Granneman lab developed a method called ChemModSeq,
which made it possible to generate nucleotide resolution maps of RNA flexibility
in ribonucleoprotein complexes by combining SHAPE chemical probing, high-throughput
sequencing and statistical modelling. By applying ChemModSeq to
ribosome assembly intermediates, we were able to obtain nucleotide resolution
insights into rRNA structural rearrangements during late (cytoplasmic) stages
of 40S assembly and for the early (nucleolar) stages of 60S assembly.
The results revealed structurally distinct cytoplasmic pre-40S particles
in which rRNA restructuring events coincide with the hierarchical dissociation
of assembly factors. These rearrangements are required to trigger stable
incorporation of a number of ribosomal proteins and the completion of the
head domain. Rps17, one of the ribosomal proteins that fully assembled into
pre-40S complexes only at a later assembly stage, was further characterized.
Surprisingly, my ChemModSeq analyses of nucleolar pre-60S complexes
indicated that most of the rRNA folding steps take place at a very specific stage
of maturation. One of the most striking observations was the stabilization of
5.8S pre-rRNA region, which coincided with the dissociation of the assembly
factor Rrp5 and stable incorporation of a number of ribosomal proteins
Inhibition of Tumor VEGFR2 Induces Serine 897 EphA2-Dependent Tumor Cell Invasion and Metastasis in NSCLC
Anti-angiogenic treatment targeting vascular endothelial growth factor (VEGF)-VEGFR2 signaling has shown limited efficacy in lung cancer patients. Here, we demonstrate that inhibition of VEGFR2 in tumor cells, expressed in similar to 20% of non-squamous non-small cell lung cancer (NSCLC) patients, leads to a pro-invasive phenotype. Drug-induced inhibition of tumor VEGFR2 interferes with the formation of the EphA2/VEGFR2 heterocomplex, thereby allowing RSK to interact with Serine 897 of EphA2. Inhibition of RSK decreases phosphorylation of Serine 897 EphA2. Selective genetic modeling of Serine 897 of EphA2 or inhibition of EphA2 abrogates the formation of metastases in vivo upon VEGFR2 inhibition. In summary, these findings demonstrate that VEGFR2-targeted therapy conditions VEGFR2-positive NSCLC to Serine 897 EphA2-dependent aggressive tumor growth and metastasis. These data shed light on the molecular mechanisms explaining the limited efficacy of VEGFR2-targeted anti-angiogenic treatment in lung cancer patients