A seven-transmembrane protein-TM7SF3, resides in nuclear speckles and regulates alternative splicing.

The seven-transmembrane superfamily member 3 protein (TM7SF3) is a p53-regulated homeostatic factor that attenuates cellular stress and the unfolded protein response. Here we show that TM7SF3 localizes to nuclear speckles; eukaryotic nuclear bodies enriched in splicing factors. This unexpected location for a trans -membranal protein enables formation of stable complexes between TM7SF3 and pre-mRNA splicing factors including DHX15, LARP7, HNRNPU, RBM14, and HNRNPK. Indeed, TM7SF3 regulates alternative splicing of >330 genes, mainly at the 3'end of introns by directly modulating the activity of splicing factors such as HNRNPK. These effects are observed both in cell lines and primary human pancreatic islets. Accordingly, silencing of TM7SF3 results in differential expression of 1465 genes (about 7% of the human genome); with 844 and 621 genes being up- or down-regulated, respectively. Our findings implicate TM7SF3, as a resident protein of nuclear speckles and suggest a role for seven-transmembrane proteins as regulators of alternative splicing.

Nuclear speckles - a driving force in gene expression.

Nuclear speckles are dynamic membraneless bodies located in the cell nucleus. They harbor RNAs and proteins, many of which are splicing factors, that together display complex biophysical properties dictating nuclear speckle formation and maintenance. Although these nuclear bodies were discovered decades ago, only recently has in-depth genomic analysis begun to unravel their essential functions in modulation of gene activity. Major advancements in genomic mapping techniques combined with microscopy approaches have enabled insights into the roles nuclear speckles may play in enhancing gene expression, and how gene positioning to specific nuclear landmarks can regulate gene expression and RNA processing. Some studies have drawn a link between nuclear speckles and disease. Certain maladies either involve nuclear speckles directly or dictate the localization and reorganization of many nuclear speckle factors. This is most striking during viral infection, as viruses alter the entire nuclear architecture and highjack host machinery. As discussed in this Review, nuclear speckles represent a fascinating target of study not only to reveal the links between gene positioning, genome subcompartments and gene activity, but also as a potential target for therapeutics.

Availability of splicing factors in the nucleoplasm can regulate the release of mRNA from the gene after transcription.

Gene expression dynamics can be measured in single living cells. Using a detectable transcriptionally active gene in living cells, we previously found that an mRNA undergoing several splicing events was retained at this gene after transcription until completion of mRNA processing. To determine the reason for this delay in release and whether mRNA retention on the gene might depend on splicing factor availability, we modulated the levels of splicing factors in the nucleus. Increasing the abundance of the diffusing fraction of splicing factors by their overexpression or by Clk1 kinase overexpression to disassemble nuclear speckles, led to a reduction in splicing factor residence times on the active gene, and the retained mRNA was rapidly released from the gene. Other treatments such as overexpression of a mutant inactive Clk1, the downregulation of MALAT1 lncRNA or of the Son protein, or the overexpression of the splicing factor import factor TNPO3, did not affect the dynamics of mRNA release from the gene. We found that the faster release of the mRNA from the gene mediated by increased availability of splicing factors, was dependent on the RS domain of the splicing factors and its phosphorylation state. We propose that the relative abundancies of splicing factors in the nucleoplasm can affect their availability for the splicing events taking place, and regulate the kinetics of mRNA release from the gene after processing.

Triple-color FRET analysis reveals conformational changes in the WIP-WASp actin-regulating complex.

Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeletal machinery. Binding of WASp-interacting protein (WIP) to WASp modulates WASp activity and protects it from degradation. Formation of the WIP-WASp complex is crucial for the adaptive immune response. We found that WIP and WASp interacted in cells through two distinct molecular interfaces. One interaction occurred between the WASp-homology-1 (WH1) domain of WASp and the carboxyl-terminal domain of WIP that depended on the phosphorylation status of WIP, which is phosphorylated by protein kinase C θ (PKCθ) in response to T cell receptor activation. The other interaction occurred between the verprolin homology, central hydrophobic region, and acidic region (VCA) domain of WASp and the amino-terminal domain of WIP. This latter interaction required actin, because it was inhibited by latrunculin A, which sequesters actin monomers. With triple-color fluorescence resonance energy transfer (3FRET) technology, we demonstrated that the WASp activation mechanism involved dissociation of the first interaction, while leaving the second interaction intact. This conformation exposed the ubiquitylation site on WASp, leading to degradation of WASp. Together, these data suggest that the activation and degradation of WASp are delicately balanced and depend on the phosphorylation state of WIP. Our molecular analysis of the WIP-WASp interaction provides insight into the regulation of actin-dependent processes.

Polymer mediated assembly of fullerenes into non-closed packed two-dimensional arrays.

Polymer-mediated depletion interactions were found to induce the assembly of fullerene derivates at the water-air interface into non-closed-packed 2-dimensional arrays of nanometric thickness.