Interplay between TPR nucleoporin and TREX‐2 complex in mRNA export

Abstract

Nuclear pore complexes (NPCs) are conduits between the cytoplasm and the nucleus. NPCs consist of roughly 35 distinct nucleoporins with filaments extending from both the nuclear and cytoplasmic faces of the pore. Nucleoporins regulate many processes, including nucleocytoplasmic trafficking, protein modification, chromatin remodeling and transcription, as well as mRNA processing and export. Our current research is focused on basket nucleoporin TPR, which works as a docking platform for other proteins, including transport receptors and subunits of the TRanscription and EXport 2 (TREX‐2) complex. We found that Auxin Inducible Degron (AID)‐mediated TPR elimination specifically disrupts TREX‐2 complex recruitment to NPCs, and results in rapid and profound changes in transcriptomic profiles of TREX‐2‐dependent RNAs and retention of those RNAs within the nucleus. The molecular mechanism through which TPR controls TREX‐2‐dependent gene expression is not clear.We are currently working in two ways to understand TREX‐2‐dependent RNA export and how TPR facilitates this pathway. First, we are examining the components of TREX‐2. GANP is a major scaffold subunit of the TREX‐2 complex, and loss of GANP disrupts the association of TREX‐2 complex subunits to the NPC. We have AID‐tagged other TREX‐2 complex members (PCID2, ENY2, CETN2) and are analyzing how their loss impacts TREX‐2 targeting, transcriptomic profiles and RNA retention. Our findings suggest that GANP or PCID2 depletion rapidly leads to polyA RNA retention inside the nucleus, but this was not the case after depletion of ENY2 or CETN2. Further comparison of TREX‐2 complex subunit profiles to each other should elucidate the sequence of their actions during mRNP maturation.Second, we are investigating the point(s) at which TPR controls TREX‐2‐dependent gene expression: TPR may help to remodel mRNP particles within the nucleus, to recruit ready‐to‐export mRNP particles to the nuclear envelope, or it may perform both of these functions. To distinguish between these possibilities, we have analyzed a TPR mutant that lacks an NPC recruitment domain, to see whether it can rescue any part of TPR’s function in this pathway without recruiting TREX‐2 to the NPC. We found that this TPR mutant resides inside the nucleus and can not restore normal export of polyA RNA. We speculate that TPR preferentially acts as a landing platform for TREX‐2‐bound mRNAs at the nuclear pore and may help with quality control of the transcripts. To explore if TPR‐dependent transcripts have unique signatures, we are analyzing mRNA length, GC content and intron context in other AID‐tagged nucleoporins and comparing transcriptomic profiles after their loss in comparison to TPR.Alltogether, these data will help us to unfold the role of TREX2 complex subunits in targeting GANP‐TPR dependent transcripts to the pore and to gain a better understanding of how TPR controls gene expression.