RXR RAR
DR5
Figure 7. descriptive diagram of the construction used
in the delay on freezing and of the fixing of the RANC
The signal in 1 corresponds to the fixing of RAR and RXR on
the DR5. The complex observed is thus consisted of the nuclear receivers and is
called RANC. Signal 2 indicates that RXR in fact part. The intensity and the
delay of bands 3 and 4 indicate that TLS belonged to the RANC. From 1 ul (band
4), plus the quantity of TLS is increased, plus the complex seems destabilized
(band 5 and 6). The maximum quantity of TLS for its participation in the RANC
is 1 ul total proteinic extracts. Beyond this confirmation and by realizing
experiments of controls of the phenomena of competition or allostery, TLS could
interfere with the complex, being able to prevent RXR from binding to the
ADN.
In conclusion, these preliminary results strongly suggest that
TLS would increase the target gene transcription of AR in a way dependant on
the ligand in the cells myéloïdes and that this would be related to
a direct participation of TLS in the complex transcriptionnel RANC. The
experiments in the Cos-6 cells show that this regulation by TLS and AR could be
a general phenomenon.
Figure 8. Delay on freezing carried out with RAR and
RXR, in presence or absence of TLS.
The effect of the amount of TLS on the RANC is given.
3. Analyze in vivo activity of TLS and
rétinoïque acid on the alternate épissage of them 5 ' of
E1A
In the light of the results obtained, it is clear that TLS
acts on the way of indication to the rétinoïdes at least by its
transcriptionnelle activity.
Since TLS can play a part of factor of épissage by
supporting the selection of distal site 5 ' of épissage of the pre-m ARN
of minigene E1A in the cells érythroïdes murine IW1-32, we studied
the role of TLS on the épissage of the pre-m ARN of minigene E1A in a
human model hematopoietic, the line myéloïde K562.
ARN pre-m E1A contains sites 5 ' of épissage which lead
to the formation of several isoformes principal of ARN (of which 13S, 12S and
9S). These isoformes can be highlighted and be quantified by PCR using selected
starters spécifiquements (Figure 9).
 Figure 9. Diagrammatic representation of the alternate
épissage of transcribed E1A. the isoformes of the pre-m ARN of
E1A and the corresponding size of the products of RT-PCR are shown. The
starters for the analysis in RT-PCR are indicated by arrows.
In order to study the possible role of TLS or AR on the
épissage into 5 ' of minigene E1A, we transitorily have
Co-transfecté the K562 cells by the lipofectamine with the plamides of
expression of E1A, pCS3 (to standardize the quantities of plasmide used), of
TLS (the quantities used are indicated) and of MVC-Luc (to measure the
effectiveness of the transfection). When the luciférase is expressed,
ARNs are extracted, then rétrotranscrits. Then a PCR is carried out. The
detection of the products of PCR is made after separation on a gel Page-urea by
autoradiography (Figure 10). Lastly, the quantitative analysis of the results
is obtained by the system Molecular Analyst de Biorad (Figure 11).
After ensurehaving ensured itself of the effectiveness of the
transfection, it is advisable to obtain the adequate experimental conditions to
secure of the detection of all the isoformes. It is a question of varying
either the quantity of products of RT used for the PCR, or the number of cycles
of the PCR, or the exposure of autoradiography. Each experiment is made in
doublet and is reproduced to be ensured of the reproducibility of the results
obtained. The study is based on the choice of site 5 ' of épissage of
the minigene E1A which one expressed as a percentage deduces from the
expression of the isoformes 13S, 12S and 9S. It is, moreover, requirement to
visualize a band corresponding to the not spliced form of E1A so that the
results are interpretable.
By comparing the results without AR (1) with the results with
AR (2), it is possible to affirm, subject to additional controls (between 40
and 60% of 9S) that it would not seem that AR alone has a significant effect on
the épissage of E1A in K562.
On the other hand, the results in the presence of TLS are
reproducible and confirm that in the K562 cells, protein TLS allows a
preferential épissage isoforme 9S the detriment of the isoformes 13S and
12S. This phenomenon would be increased in the presence of AR. The results (3)
show that TLS alone supports the formation of the isoforme 9S (75% of 9S),
whereas when AR is used with TLS, the isoforme 9S is increased considerably
(90% of 9S).
These results indicate that TLS acts on the selection of
distal site 5 ' of épissage, supporting the formation of the isoforme 9S
minigene E1A, phenomenon accentuated by AR. Thus, these results suggest that
TLS acts on the épissage in the human hematopoietic cells and that AR
intervenes on the épissage through TLS.
In conclusion, these results would show for the first time
that AR would be implied directly in control post-transcriptionnel through
protein TLS.
Figure 10. The alternate épissage in
vivo of minigene E1A in the K562 cells.
The intensity of the bands corresponding to different the
isoformes from E1A for each profile is quantified by densitometry. The results
are expressed in percentage of the isoformes 13S, 12S and 9S. the studies
are made in doublets. The RT- represents the negative control of the RT, it was
carried out without enryme RT and no ADN is revealed. ARNs extracted and used
are thus free from ADN.
Figure 11. Quantification of the isoformes of the
pre-m ARN of E1A by a software Molecular Analyst de Biorad. The
percentage of the isoformes 13S, 12S and 9S is represented.
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