Rupert Faraway

The nuclear export of mRNA is an important step in eukaryotic gene expression ¹ . Despite recent molecular insights into how newly transcribed mRNAs are packaged into ribonucleoprotein complexes (mRNPs) ^2,3 , the subsequent events that govern mRNA export are poorly understood. Here we uncover the molecular basis underlying key events of human mRNA export, including the remodelling of mRNP-bound transcription–export complexes (TREX), the formation of export-competent mRNPs, the docking of mRNPs at the nuclear pore complex (NPC), and the release of mRNPs at the NPC to initiate their export. Our biochemical and structural data show that the ATPase UAP56 (also known as DDX39) acts as a central molecular switch that directs nucleoplasmic mRNPs from TREX to NPC-anchored TREX-2 complexes through its ATP-gated mRNA-binding cycle. Collectively, these findings establish a mechanistic framework for a general and evolutionarily conserved mRNA export pathway.

In situ transcriptomic technologies provide a promising avenue to link gene expression, connectivity, and physiological properties of neural cell types. Commercialized methods that allow the detection of hundreds of genes in situ, however, are expensive and therefore typically used for generating unimodal reference data rather than for resource-intensive multimodal analyses. A major bottleneck is the lack of a routine means to efficiently generate cell type data. Here, we have developed hamFISH (highly amplified multiplexed fluorescence in situ hybridization), which enables the sequential detection of 32 genes using multiplexed branched DNA amplification. We used hamFISH to profile the projection, activity, and transcriptomic diversity of the medial amygdala (MeA), a critical node for innate social and defensive behaviors in mice. In total, we profiled 643,834 cells and classified neurons into 16 inhibitory and 10 excitatory types, many of which were found to be spatially clustered. We then examined the organization of outputs of these cells and activation profiles during different social contexts. Therefore, by facilitating multiplexed detection of single molecule RNAs, hamFISH provides a streamlined and versatile platform for multimodal profiling of specific brain nuclei.

The concentration of proteins containing intrinsically disordered regions must be tightly controlled to maintain cellular homeostasis ^1,2 . However, mechanisms for collective control of these proteins, which tend to localize to membraneless condensates, are less understood than pathways mediated by membrane-bound organelles ^3,4 . Here we report ‘interstasis’, a homeostatic mechanism in which increased concentration of proteins within RNA–protein condensates induces the sequestration of their own mRNAs. The selectivity of interstatic mRNA capture relies on the structure of the genetic code and conserved codon biases, which ensure that similar multivalent RNA regions encode similar low-complexity domains. For example, arginine-enriched mixed charge domains (R-MCDs) tend to be encoded by repetitive purine-rich sequences in mRNAs. Accumulation of proteins containing R-MCDs increases the cohesion of nuclear speckles, which induces selective capture of purine-rich multivalent mRNAs. The multivalent regions are bound by specific RNA-binding proteins, including TRA2 proteins, which relocalize to speckles upon interstasis to promote selective mRNA capture. CLK-mediated phosphorylation of TRA2 proteins counters their localization to speckles, thereby modulating interstasis. Thus, the condensation properties of nuclear speckles act as a sensor for interstasis, a collective negative-feedback loop that co-regulates mRNAs of highly dosage-sensitive genes, which primarily encode nuclear condensation-prone proteins.

In situ transcriptomic technologies provide a promising avenue to link gene expression, connectivity, and physiological properties of neural cell types. Commercialized methods that allow the detection of hundreds of genes in situ , however, are expensive and therefore typically used for generating unimodal reference data rather than for resource-intensive multimodal analyses. A major bottleneck is the lack of a routine means to efficiently generate cell type data. Here, we have developed hamFISH (highly amplified multiplexed fluorescence in situ hybridization), which enables the sequential detection of 32 genes using multiplexed branched DNA amplification. We used hamFISH to profile the projection, activity, and transcriptomic diversity of the medial amygdala (MeA), a critical node for innate social and defensive behaviors in mice. In total, we profiled 643,834 cells and classified neurons into 16 inhibitory and 10 excitatory types, many of which were found to be spatially clustered. We then examined the organization of outputs of these cells and activation profiles during different social contexts. Therefore, by facilitating multiplexed detection of single molecule RNAs, hamFISH provides a streamlined and versatile platform for multimodal profiling of specific brain nuclei.

Signaling pathways drive cell fate transitions largely by changing gene expression. However, the mechanisms for rapid and selective transcriptome rewiring in response to signaling cues remain elusive. Here we use deep learning to deconvolve both the sequence determinants and the trans-acting regulators that trigger extracellular signal-regulated kinase (ERK)–mitogen-activated protein kinase kinase (MEK)-induced decay of the naive pluripotency mRNAs. Timing of decay is coupled to embryo implantation through ERK–MEK phosphorylation of LIN28A, which repositions pLIN28A to the highly A+U-rich 3′ untranslated region (3′UTR) termini of naive pluripotency mRNAs. Interestingly, these A+U-rich 3′UTR termini serve as poly(A)-binding protein (PABP)-binding hubs, poised for signal-induced convergence with LIN28A. The multivalency of AUU motifs determines the efficacy of pLIN28A–PABP convergence, which enhances PABP 3′UTR binding, decreases the protection of poly(A) tails and activates mRNA decay to enable progression toward primed pluripotency. Thus, the signal-induced convergence of LIN28A with PABP–RNA hubs drives the rapid selection of naive mRNAs for decay, enabling the transcriptome remodeling that ensures swift developmental progression.

Ever-increasing volumes of sequencing data offer potential for large-scale meta-analyses to address significant biological questions. However, challenges such as insufficient data processing information, data quality concerns, and issues related to accessibility and curation often present obstacles. Additionally, most experimental biologists lack the time and expertise needed to independently analyse, manage and share their own data. To overcome these hurdles, we present Flow, a web-based platform that links bioinformatic analysis and database solutions with a user-friendly interface and web API. Flow currently accommodates a range of genomics methods and further DSL2-compliant Nextflow pipelines can be added via a simple JSON schema file. Deployable on local systems or cloud services, an instance is freely accessible to academic researchers at https://flow.bio .

Protein dosage is regulated to maintain cellular homeostasis and health. The dosage of proteins containing disordered low complexity domains (LCDs) must be particularly well-controlled to prevent aberrant disease, yet no mechanism to maintain homeostasis has been identified ^{1, 2} . Here we report a mutual homeostatic mechanism that controls the concentration of such proteins, termed ’interstasis’, in which proteins with similar LCDs co-regulate their combined dosage through collective negative feedback. We focused on the mechanism that exploits the fundamental multivalency of GA-rich RNA regions that encode charged LCDs, including those with arginine-enriched mixed charge domains (R-MCDs). Modest variations in the abundance of an R-MCD protein change the properties of nuclear speckles, a protein-RNA condensate, selectively trapping multivalent GA-rich mRNAs to promote their nuclear retention. This interstasis depends on conserved codon biases, shared by amniotes, which enhance the multivalency of GA-rich regions encoding charged LCDs. The threshold of interstasis is modulated by CLK kinases, which affect the nuclear speckle localisation of proteins such as TRA2B, key binder of GA-rich RNAs. Notably, many classes of LCDs are encoded by RNA regions containing multivalency-enhancing codon biases, each preferentially bound by specific proteins, suggesting that interstasis might co-regulate many classes of functionally related LCD-containing proteins through dose-sensitivity of various types of protein-RNA condensates.