Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

4.2 Target Prediction and Validation Introduction
newly synthesized proteins and the raw protein concentration is monitored.
In pSILAC, instead, the labeled amino acid is fed to the cell culture for a
short period of time (a pulse), so that only de novo protein production is
monitored [446]. Specifically, the pSILAC protocol developed by Rajwesky et
al [446] involves labelling two cell cultures (transfected and control) with a
heavy and a medium-heavy version of the same amino acid, respectively. Af-
ter 8h from transfection the cultures are pulse-labeled and the samples, united
after 32h post-transfection, are analysed by mass spectrometry. RNA from
the same samples (8h and 32h) was analyzed by Affymetrix arrays. In this
fashion, approximately 5,000 proteins were identified in HeLa cells with high
confidence. The search for six nucleotide motifs within the 3'UTRs of those
mRNAs whose protein levels decreased the most (never exceeding 4-fold) re-
vealed that the most significant motifs were exactly the seed sequences of
each respective microRNA. This showed that out of all proteins demonstrat-
ing reduced synthesis, the levels of the direct targets of microRNAs decreased
the most, and that this reduction is directly linked to the presence of the
3'UTR target site. Nevertheless, a number of repressed proteins without seed
sequences are still targeted, since their level is decreased, but prediction al-
gorithms cannot identify them because they are non-canonical and seedless.
Amongst the strongly repressed targets, a Gene Ontology search revealed that
these are mainly proteins synthesized on the Endoplasmic Reticulum. A po-
tential reason is that only mRNAs from cytosolic free ribosomes are targeted
to P-bodies for degradation [446]. Other observations were that a nine to 11
nucleotide mismatch along the mRNA-microRNA duplex was necessary for the
protein production to be repressed, being otherwise indistinguishable to that
of mRNAs lacking the seed. Although mismatches are deleterious to siRNA-
mediated mRNA cleavage, they seem to correlate with increased repression
of protein production by microRNAs. Also, the presence of multiple seeds
seemed to have multiplicative repressive effects with a higher impact when
the seeds were proximal rather than distant. On average, the repression was
found more pronounced for conserved rather than non-conserved seed sites,
indicating that there are more determinants other than the seed that mediate
efficient down-regulation of protein synthesis. Interestingly, as opposed to the
seed enrichment observed in the down-regulated genes, no seed enrichment was
observed in the up-regulated ones, strongly speaking against the microRNA-
mediated activation observed in other works [446].
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