REGULATION OF THE EXPRESSION OF HETEROMERIC RECEPTOR SUBUNITS BY MODULATION OF MRNA STABILITY ¿ A CASE STUDY OF TRC40 RECEPTOR
Tesi di Dottorato
Data di Pubblicazione:
2018
Citazione:
REGULATION OF THE EXPRESSION OF HETEROMERIC RECEPTOR SUBUNITS BY MODULATION OF MRNA STABILITY ¿ A CASE STUDY OF TRC40 RECEPTOR / H.j. Fernandes Carvalho ; tutor: A. E. Panerai ; supervisori: N. Borgese, S. F. Colombo ; coordinatore: A. L. Catapano. DIPARTIMENTO DI SCIENZE FARMACOLOGICHE E BIOMOLECOLARI, 2018 Jan 25. 30. ciclo, Anno Accademico 2017. [10.13130/fernandes-carvalho-hugo-jose-_phd2018-01-25].
Abstract:
In recent years, the mechanisms of post-transcriptional regulation of gene
expression have gained increased attention, including their importance in oligomeric
protein assembly. The ER based proteins CAML and WRB have been described to form
a heteromeric protein complex that functions as a receptor for the TRC40 ATPase,
mediating the post-translational insertion of tail-anchored proteins into the ER
membrane. A recent study showed that, even if the two proteins are not in a
stoichiometric equilibrium - CAML is in ~5-fold excess over WRB -, the transient siRNA
mediated depletion of each one of the two subunits, in HeLa cells, leads to the depletion
of the second one. Interestingly, the silencing of CAML leads not only to the depletion of
WRB protein but also to the depletion and strong destabilization of WRB mRNA,
revealing a novel mechanism for the regulation of the expression of different subunits of
a protein complex. In this thesis, I have followed up on this work, trying to unveil the
mechanisms involved in the co-regulation of the expression and assembly of the TRC40
receptor complex. My results demonstrate that the interplay observed at the transcript
level is not reciprocal, i.e. CAML mRNA is not affected by WRB siRNA mediated
depletion, and that the CAML-sensitivity of the WRB transcript is attributable to a
relatively small portion (370 nt) of its 3' untranslated sequence (UTR), as shown by the
results obtained with the dual-luciferase reporter system. Moreover, co-IP experiments
show that CAML is indeed in excess over WRB and that a WRB-free population of CAML
exists. It remains however unclear, what mechanism may explain the depletion of CAML
upon WRB silencing. Finally, I propose a model according to which, on the one hand,
CAML protein either directly or indirectly interacts with the 3’UTR of WRB, promoting
is stability, which is lost when CAML is silenced. On the other hand, CAML may be
inserted in the ER membrane in an unusual way, via the TRC40 pathway, meaning that it
would require a functional TRC40 receptor complex, which is lost when WRB is silenced.
The resulting non-inserted CAML molecules would then be degraded by the ubiquitinproteasome
system. Future work is required to further understand the precise
mechanisms involved in the co-regulation of the expression of the two subunits of the
TRC40 receptor. This work could pave the way for the discovery of new posttranscriptional
co-regulatory mechanisms for the expression and assembly of oligomeric
protein subunits, which could very well be relevant to many different protein complexes.
Tipologia IRIS:
Tesi di dottorato
Keywords:
TRC40 receptor; CAML; WRB; Endoplasmic Reticulum
Elenco autori:
H.J. FERNANDES CARVALHO
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