Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Retroviruses have evolved sophisticated mechanisms to ensure timely export of incompletely spliced viral messenger ribonucleic acids (mRNAs) for gene expression and for viral packaging. For example, the Human Immunodeficiency Virus type 1 (HIV-1) encodes the Rev regulatory protein, a sequence-specific RNA-binding protein that is responsible for the cytoplasmic accumulation of intron-containing viral mRNAs. The HIV-1 Rev protein contains an amino terminal (N-terminal) Arginine-Rich Motif (ARM) RNA-binding domain (RBD) and a carboxy terminal (C-terminal) leucine-rich activation domain which functions as a Nuclear Export Signal (NES). The Rev ARM interacts in a sequence-specific manner with a cis-acting viral RNA stem-loop structure, the Rev Responsive Element (RRE), located in all incompletely spliced viral mRNAs. This initial interaction is followed by the recruitment of additional Rev molecules to form a RiboNucleoProtein (RNP) complex involving the RRE and Rev molecules. The cytoplasmic accumulation of the Rev:RRE RNP complex is dependent on the interaction of Rev with key cellular cofactors. Rev activation domain mutants exhibit a trans-dominant negative phenotype, suggesting that this domain of Rev interacts with cellular proteins required for Rev function. Biochemical and genetic studies have identified several cellular proteins that bind to the activation domain of Rev and are therefore candidate cofactors for Rev function. Amongst these is the human Rev Interacting Protein [hRIP, 79], which is also known as the Rev/Rex activation domain-binding protein [Rab, 18]. hRIP was identified in a yeast two-hybrid assay with the HIV-1 Rev and its functionally equivalent Human T-cell Leukemia Virus type-1 (HTLV-1) Rex protein as baits. The interaction between hRIP and HIV-1 Rev is dependent on a functional Rev NES, as predicted for a bona fide Rev cellular cofactor, and the Nucleoporin-like (Nup-like) repeats in the C-terminus of hRIP (18, 79]. Additional genetic studies indicated that the interaction between hRIP and Rev is indirect and is most likely mediated by the cellular export receptor CRM1 (Chromosomal Region Maintenance 1) [1, 153]. A role for hRIP in Rev function or HIV-1 replication has remained elusive. The goal of this dissertation was to determine whether hRIP is required for Rev function and HIV-1 replication. We used two approaches, a dominant-negative mutant and RNA interference (RNAi), to ablate hRIP activity and analyzed Rev function and HIV-1 replication using standard assays. The results of this dissertation demonstrate that hRIP is required for Rev function and HIV-1 replication. We show that Rev function is inhibited upon ablation of hRIP activity by either a trans-dominant negative mutant or RNAL Furthermore, we find that depletion of endogenous hRIP by RNAi results in the loss of viral replication in human cell lines and primary human macrophages. Unexpectedly, in the absence of functional hRIP, RRE-containing viral RNAs accumulate in the nuclear periphery where hRIP is localized. Comparable ablation of hRIP activity did not affect the intracellular localization or trafficking of a variety of proteins or cellular poly (A+ mRNA, suggesting that the inhibition of Rev-directed RNA export is specific. In conclusion, the results of this dissertation demonstrate that hRIP is involved in the movement of Rev-directed RNAs from the nuclear periphery to the cytoplasm. Therefore, hRIP is required for Rev function and HIV-1 replication. The hRIP protein is not essential for the maintenance of cell viability and thus might represent a novel target for the development of antiviral agents for HIV-1.

A monoclonal antibody, mAb 203.37, raised against purified nuclear matrix proteins identified a single ~270 kDa protein that localized to the nuclear envelope. Double-label immunofluorescent microscopy using differential permeabilization protocols showed that this protein was present exclusively on the nucleoplasmic side of the nuclear envelope and that it co-localized with components of the nuclear pore complex. The nucleotide sequence of clones isolated using mAb 203.37 identified this protein as Tpr, a protein previously shown to be involved in oncogenic fusions with a number of protein kinases. Sequence analysis showed Tpr to be a 2348 amino acid protein with a predicted molecular weight of 265 kDa protein and a bipartite structure consisting of an ~1600 amino acid N-terminal domain that is almost entirely an α-helical coiled-coil followed by a highly acidic non-coiled carboxy-terminus. Ectopic expression of epitope-tagged Tpr constructs revealed two functional domains for Tpr: a nuclear pore complex binding domain and a nuclear localization sequence. The amino-terminus of Tpr, the portion of the protein shown to activate protein kinase oncogenes, did not localize to the nuclear pore complex indicating that the transforming activity of Tpr-protein kinase chimeras did not involve interactions with the nuclear pore complex. Ectopic expression of Tpr and a number of Tpr constructs resulted in the accumulation of poly (A)+ RNA in the nuclear interior but did not effect the import of a reporter protein into the nucleus indicating a role for Tpr in the export of mRNA from the nucleus.

Signal transduction by transforming growth factor β (TGF-β) cytokines is mediated by an evolutionarily conserved mechanism that depends on the Smad proteins to transduce an extracellular stimulus into the nucleus. In the unstimulated state, Smads spontaneously shuttle across the nuclear envelope and distribute throughout the cell. Upon TGF-β or bone morphogenetic protein (BMP) stimulation, the receptor-activated Smads are phosphorylated, assemble into complexes with Smad4, and become mostly localized in the nucleus. Such signal-induced nuclear translocation of activated Smads is essential for TGF-β–dependent gene regulation that is critical for embryonic development and homeostasis. The molecular machinery responsible for this process, especially how the activated Smads are imported as complexes, is not entirely clear. Thus, I became interested in investigating the molecular requirements for nuclear targeting of Smads upon stimulation. Recently, whole-genome RNAi screening offers a complementary cell-based approach to functionally identify molecules that mediate nuclear accumulation of Smads in response to TGF-β. In the first part of this dissertation, I performed a genome-wide RNAi screen that uncovered the importin moleskin (Msk) required in nuclear import of Dpp-activated MAD. Both genetic and biochemical studies further confirmed this finding. I also investigated Smad interactions with the Msk mammalian orthologues, Importin7 and 8 and validated that Smads are bona fide cargos of Imp7/8. Besides the importin Msk, the screen also uncovered a subset of nucleoporins as required factors in signal-induced nuclear accumulation of MAD. Thus in the second part of this thesis, I focused on how the NPC mediates this Msk-dependent nuclear import of activated MAD. Most of these nucleoporins, including Sec13, Nup75, Nup93 and Nup205, were thought to be structural nucleoporins without known cargo-specific functions. We, however, demonstrated that this subset of nucleoporins was specifically used in the Msk-dependent nuclear import of activated MAD but not the constitutive import of cargos containing a classic nuclear localization signal (cNLS). I also uncovered novel pathway-specific functions of Sec13 and Nup93. Regulation of TGF-β signaling can be achieved not only by modulating Smad nuclear translocation but also by modifying Smad phosphorylation status. Previously we identified a kinase, Misshapen (Msn), that caused the linker phosphorylation of MAD, resulting in negative regulation of Dpp signaling (Drosophila BMP). In the third part of this thesis, I investigated the biological relevance of Msn kinase to Dpp signaling in Drosophila wings. Both over-expression and RNAi studies suggest that Msn is a negative regulator of the Dpp/MAD pathway in vivo. As a whole, my findings delineated two critical requirements for MAD nuclear import: the importin Msk and a unique subset of nucleoporins. For the first time, structural Nups are implicated in the direct involvement of cargo import, providing a unique trans-NPC mechanism.