ARS2 is a highly conserved metazoan protein involved in numerous aspects of nuclear RNA metabolism. As a direct partner of the nuclear cap-binding complex (CBC), it mediates interactions with diverse RNA processing and transport machineries in a transcript-dependent manner. Here, we present the human ARS2 crystal structure, which exhibits similarities and metazoan-specific differences to the plant homologue SERRATE, most notably an additional RRM domain. We present biochemical, biophysical and cellular interactome data comparing wild type and mutant ARS2 that identify regions critical for interactions with FLASH (involved in histone mRNA biogenesis), NCBP3 (a putative cap-binding protein involved in mRNA export) and single-stranded RNA. We show that FLASH and NCBP3 have overlapping binding sites on ARS2 and that CBC–ARS2–NCBP3 form a ternary complex that is mutually exclusive with CBC–ARS–PHAX (involved in snRNA export). Our results support that mutually exclusive higher-order CBC–ARS2 complexes are critical in determining Pol II transcript fate.

SummaryThe nuclear cap-binding complex (CBC) stimulates processing reactions of capped RNAs, including their splicing, 3′-end formation, degradation, and transport. CBC effects are particular for individual RNA families, but how such selectivity is achieved remains elusive. Here, we analyze three main CBC partners known to impact different RNA species. ARS2 stimulates 3′-end formation/transcription termination of several transcript types, ZC3H18 stimulates degradation of a diverse set of RNAs, and PHAX functions in pre-small nuclear RNA/small nucleolar RNA (pre-snRNA/snoRNA) transport. Surprisingly, these proteins all bind capped RNAs without strong preferences for given transcripts, and their steady-state binding correlates poorly with their function. Despite this, PHAX and ZC3H18 compete for CBC binding and we demonstrate that this competitive binding is functionally relevant. We further show that CBC-containing complexes are short lived in vivo, and we therefore suggest that RNA fate involves the transient formation of mutually exclusive CBC complexes, which may only be consequential at particular checkpoints during RNA biogenesis.

Two-component systems (TCS) serve as stimulus-response coupling mechanisms to allow organisms to adapt to a variety of environmental conditions. The opportunistic pathogen Pseudomonas aeruginosa encodes for more than 100 TCS components. To avoid unwanted cross-talk, signaling cascades are very specific, with one sensor talking to its cognate response regulator (RR). However, cross-regulation may provide means to integrate different environmental stimuli into a harmonized output response. By applying a split luciferase complementation assay, we identified a functional interaction of two RRs of the OmpR/PhoB subfamily, namely PhoB and TctD in P. aeruginosa. Transcriptional profiling, ChIP-seq analysis and a global motif scan uncovered the regulons of the two RRs as well as a quadripartite binding motif in six promoter regions. Phosphate limitation resulted in PhoB-dependent expression of the downstream genes, whereas the presence of TctD counteracted this activation. Thus, the integration of two important environmental signals e.g. phosphate availability and the carbon source are achieved by a titration of the relative amounts of two phosphorylated RRs that inversely regulate a common subset of genes. In conclusion, our results on the PhoB and TctD mediated two-component signal transduction pathways exemplify how P. aeruginosa may exploit cross-regulation to adapt bacterial behavior to complex environments.

Activation of the innate immune pattern recognition receptor NOD2 by the bacterial muramyl-dipeptide peptidoglycan fragment triggers recruitment of the downstream adaptor kinase RIP2, eventually leading to NF-κB activation and proinflammatory cytokine production. Here we show that full-length RIP2 can form long filaments mediated by its caspase recruitment domain (CARD), in common with other innate immune adaptor proteins. We further show that the NOD2 tandem CARDs bind to one end of the RIP2 CARD filament, suggesting a mechanism for polar filament nucleation by activated NOD2. We combine X-ray crystallography, solid-state NMR and high-resolution cryo-electron microscopy to determine the atomic structure of the helical RIP2 CARD filament, which reveals the intermolecular interactions that stabilize the assembly. Using structure-guided mutagenesis, we demonstrate the importance of RIP2 polymerization for the activation of NF-κB signalling by NOD2. Our results could be of use to develop new pharmacological strategies to treat inflammatory diseases characterised by aberrant NOD2 signalling.

Pol II transcribes diverse classes of RNAs that need to be directed into the appropriate nuclear maturation pathway. All nascent Pol II transcripts are 5′-capped and the cap is immediately sequestered by the nuclear cap-binding complex (CBC). Mutually exclusive interactions of CBC with different partner proteins have been implicated in transcript fate determination. Here, we characterise the direct interactions between CBC and NELF-E, a subunit of the negative elongation factor complex, ARS2 and PHAX. Our biochemical and crystal structure results show that the homologous C-terminal peptides of NELF-E and ARS2 bind identically to CBC and in each case the affinity is enhanced when CBC is bound to a cap analogue. Furthermore, whereas PHAX forms a complex with CBC and ARS2, NELF-E binding to CBC is incompatible with PHAX binding. We thus define two mutually exclusive complexes CBC–NELF–E and CBC–ARS2–PHAX, which likely act in respectively earlier and later phases of transcription.