REGULATION OF THE EXPRESSION OF HETEROMERIC RECEPTOR SUBUNITS BY MODULATION OF MRNA STABILITY ¿ A CASE STUDY OF TRC40 RECEPTOR

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.