The Mkk3/6-p38–Signaling Cascade Alters the Subcellular Distribution of Hnrnp A1 and Modulates Alternative Splicing Regulation

Individual members of the serine-arginine (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) A/B families of proteins have antagonistic effects in regulating alternative splicing. Although hnRNP A1 accumulates predominantly in the nucleus, it shuttles continuously between the nucleus and the cytoplasm. Some but not all SR proteins also undergo nucleo-cytoplasmic shuttling, which is affected by phosphorylation of their serine/arginine (RS)–rich domain. The signaling mechanisms that control the subcellular localization of these proteins are unknown. We show that exposure of NIH-3T3 and SV-40 transformed green monkey kidney (COS) cells to stress stimuli such as osmotic shock or UVC irradiation, but not to mitogenic activators such as PDGF or EGF, results in a marked cytoplasmic accumulation of hnRNP A1, concomitant with an increase in its phosphorylation. These effects are mediated by the MKK3/6-p38 pathway, and moreover, p38 activation is necessary and sufficient for the induction of hnRNP A1 cytoplasmic accumulation. The stress-induced increase in the cytoplasmic levels of hnRNP A/B proteins and the concomitant decrease in their nuclear abundance are paralleled by changes in the alternative splicing pattern of an adenovirus E1A pre-mRNA splicing reporter. These results suggest the intriguing possibility that signaling mechanisms regulate pre-mRNA splicing in vivo by influencing the subcellular distribution of splicing factors

Herpes simplex virus ICP27 protein directly interacts with the nuclear pore complex through NUP62, inhibiting host nucleocytoplasmic transport pathways

The herpes simplex virus ICP27 protein is important for the expression and nuclear export of viral mRNAs. Although several binding sites have been mapped along the ICP27 sequence for various RNA and protein partners including the transport receptor TAP of the host cell nuclear transport machinery, several aspects of ICP27 trafficking through the nuclear pore complex remain unclear. We investigated if ICP27 could interact directly with the nuclear pore complex itself, finding that ICP27 directly binds the core nucleoporin Nup62. This is confirmed through co-immunoprecipitation and in vitro binding assays with purified components. Mapping with ICP27 deletion and point mutants further shows that the interaction requires sequences in both the N and C-termini of ICP27. Expression of wildtype ICP27 protein inhibited both classical, importin α/β-dependent and transportin dependent nuclear import. In contrast, an ICP27 point mutant that does not interact with Nup62 had no such inhibitory effect. We suggest that ICP27 association with Nup62 provides additional binding sites at the nuclear pore for ICP27 shuttling thus supporting ICP27-mediated transport. We propose that ICP27 competes with some host cell transport receptors for binding, resulting in inhibition of those host transport pathways

Unconstraining the Unhiggs

We investigate whether or not perturbative unitarity is preserved in the Unhiggs model for the scattering process of heavy quarks and longitudinal gauge bosons $\bar q q \to V_L^+ V_L^-$. With the Yukawa coupling given in the original formulation of the Unhiggs model, the model preserves unitarity for Unhiggs scaling dimensions $d\leq 1.5$. We examine the LHC phenomenology that is implied by the Unhiggs model in this parameter range in detail and discuss to what extent the LHC can test $d$ if an excess is measured in the phenomenologically clean $ZZ$ channel in the future or if the LHC measurement remains consistent with the background. We then make use of the AdS/CFT correspondence to derive a new Yukawa coupling that is conformally invariant at high energies, and show that with this Yukawa coupling the theory is unitary for $1 \leq d < 2$.Comment: 19 pages, 10 figures; typos corrected, version published by PR

Specific Y14 domains mediate its nucleo-cytoplasmic shuttling and association with spliced mRNA

Pre-mRNA splicing deposits multi-protein complexes, termed exon junction complexes (EJCs), on mRNAs near exon-exon junctions. The core of EJC consists of four proteins, eIF4AIII, MLN51, Y14 and Magoh. Y14 is a nuclear protein that can shuttle between the nucleus and the cytoplasm, and binds specifically to Magoh. Here we delineate a Y14 nuclear localization signal that also confers its nuclear export, which we name YNS. We further identified a 12-amino-acid peptide near Y14's carboxyl terminus that is required for its association with spliced mRNAs, as well as for Magoh binding. Furthermore, the Y14 mutants, which are deficient in binding to Magoh, could still be localized to the nucleus, suggesting the existence of both the nuclear import pathway and function for Y14 unaccompanied by Magoh

The RNA Helicase DHX34 Activates NMD by Promoting a Transition from the Surveillance to the Decay-Inducing Complex

SummaryNonsense-mediated decay (NMD) is a surveillance mechanism that degrades aberrant mRNAs. A complex comprising SMG1, UPF1, and the translation termination factors eRF1 and eRF3 (SURF) is assembled in the vicinity of a premature termination codon. Subsequently, an interaction with UPF2, UPF3b, and the exon junction complex induces the formation of the decay-inducing complex (DECID) and triggers NMD. We previously identified the RNA helicase DHX34 as an NMD factor in C. elegans and in vertebrates. Here, we investigate the mechanism by which DHX34 activates NMD in human cells. We show that DHX34 is recruited to the SURF complex via its preferential interaction with hypophosphorylated UPF1. A series of molecular transitions induced by DHX34 include enhanced recruitment of UPF2, increased UPF1 phosphorylation, and dissociation of eRF3 from UPF1. Thus, DHX34 promotes mRNP remodeling and triggers the conversion from the SURF complex to the DECID complex resulting in NMD activation

Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20-30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150-300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides