Nuclear pore complexes (NPCs) form aqueous conduits in the nuclear envelope and gate the diffusion of large proteins between the cytoplasm and nucleoplasm. NPC proteins (nucleoporins) that contain phenylalanine-glycine motifs in filamentous, natively unfolded domains (FG domains) line the diffusion conduit of the NPC, but their role in the size-selective barrier is unclear. We show that deletion of individual FG domains in yeast relaxes the NPC permeability barrier. At the molecular level, the FG domains of five nucleoporins anchored at the NPC center form a cohesive meshwork of filaments through hydrophobic interactions, which involve phenylalanines in FG motifs and are dispersed by aliphatic alcohols. In contrast, the FG domains of four peripherally anchored nucleoporins are generally noncohesive. The results support a two-gate model of NPC architecture featuring a central diffusion gate formed by a meshwork of cohesive FG nucleoporin filaments and a peripheral gate formed by repulsive FG nucleoporin filaments.

Sequence-specific RNA–protein interactions, though commonly used in biological systems to regulate translation, are challenging to selectively modulate. Here, we demonstrate the use of a chemically-inducible RNA–protein interaction to regulate eukaryotic translation. By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5′-untranslated region (5′-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5′-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA–TetR interactions. Using a reverse TetR variant, we illustrate the potential for expanding the regulatory properties of the system through protein engineering strategies.

Tumor-infiltrating lymphocytes (TILs) are critical to anti-cancer immune responses, but their diverse phenotypes and functions remain poorly understood and challenging to study. We therefore developed a single-cell barcoding technology for deep characterization of TILs without the need for cell-sorting or culture. Our emulsion-based method captures full-length, natively paired B-cell and T-cell receptor (BCR and TCR) sequences from lymphocytes among millions of input cells. We validated the method with 3 million B-cells from healthy human blood and 350,000 B-cells from an HIV elite controller, before processing 400,000 cells from an unsorted dissociated ovarian adenocarcinoma and recovering paired BCRs and TCRs from over 11,000 TILs. We then extended the barcoding method to detect DNA-labeled antibodies, allowing ultra-high throughput, simultaneous protein detection and RNA sequencing from single cells.

SNS-314 is a potent small-molecule inhibitor of Aurora kinases developed as a novel anti-cancer therapeutic agent for the treatment of diverse human malignancies.

The importance and pervasiveness of naturally occurring regulation of RNA function in biology is increasingly being recognized. A common mechanism uses inducible protein-RNA interactions to shape diverse aspects of cellular RNA fate. Recapitulating this regulatory mode in cells using a novel set of protein-RNA interactions is appealing given the potential to subsequently modulate RNA biology in a manner decoupled from endogenous cellular physiology. Achieving this outcome, however, has previously proven challenging. Here, we describe a ligand-responsive protein-RNA interaction module, which can be used to target a specific RNA for subsequent regulation. Using the Systematic Evolution of Ligands by Exponential Enrichment method, RNA aptamers binding to the bacterial Tet Repressor protein (TetR) with low-to sub-nanomolar affinities were obtained. This interaction is reversibly controlled by tetracycline in a manner analogous to the interaction of TetR with its cognate DNA operator. Aptamer minimization and mutational analyses support a functional role for two conserved sequence motifs in TetR binding. As an initial illustration of using this system to achieve protein-based regulation of RNA function in living cells, insertion of a TetR aptamer into the 5'-UTR of a reporter mRNA confers post-transcriptionally regulated, ligandinducible protein synthesis in E. coli. Altogether, these results define and validate an inducible protein-RNA interaction module that incorporates desirable aspects of a ubiquitous mechanism for regulating RNA function in Nature, and which can be used as a foundational interaction for functionally and reversibly controlling the multiple fates of RNA in cells.3