Stefan Ludwig Ameres

After excising an intron from pre-mRNA, the spliceosome remains trapped in a non-productive complex bound to the intron ^1–5 . Termination of this complex is critical for spliceosome recycling and intron decay ⁶ , but the mechanism remains unknown. Here we present cryo-electron microscopy structures of human spliceosomes at two sequential stages of termination. First, the RNA helicases DHX15 and Aquarius unwind the RNA active site of the spliceosome, releasing bound components including U2 snRNA and extracting the buried intron–lariat branch point. The branch point is then debranched by the spliceosome-tethered enzyme DBR1, generating the previously unknown debranched intron spliceosome. This state recruits the RNA helicase DHX35 with its co-factors GPATCH1–WDR83, assisted by YJU2B. DHX35 ejects the debranched intron from the U6 snRNA–5′ splice site duplex, driving spliceosome disassembly and intron turnover. In defective spliceosomes stalled on aberrant introns, YJU2B partners with LENG1 to guide DHX35–GPATCH1–WDR83 for termination through spliceosome quality control. Together, we reveal the mechanism of regular spliceosome termination and its parallels with spliceosome quality control, ensuring accurate and efficient pre-mRNA splicing.

Summary: Robust RNA interference in Caenorhabditis elegans requires Argonaute-targeted RNAs to be cleaved by RDE-8 and poly(UG)ylated by MUT-2, generating templates for RNA-dependent RNA polymerases that synthesize secondary small RNAs. RDE-8 and MUT-2 are physically coupled within the cleavage and poly(UG)ylation (CPUG) complex, together with the pseudonuclease NYN-1 and MUT-15, but how their activities are controlled and coordinated is unknown. Structural and biochemical analyses reveal that the RDE-8/NYN-1 nuclease module is autoinhibited, restricting cleavage outside CPUG. We identify MUT-15, the CPUG scaffold linking this nuclease module to MUT-2, as a pseudohelicase. MUT-15 remodels the nuclease module, licensing RDE-8 activity upon complex assembly. Reconstituted CPUG synthesizes long poly(UG) tails, with the substrate 3′ end determining tailing efficiency and register. Within CPUG, RNA engagement by MUT-2 limits RDE-8 access, while poly(UG) tails themselves are not cleaved by RDE-8, preventing re-cleavage. Together, CPUG couples assembly-dependent RNA cleavage to downstream processing, imposing safety and directionality. Graphical Highlights: RDE-8 forms an autoinhibited heterodimer with the pseudonuclease NYN-1 MUT-15 is a pseudohelicase that licenses RDE-8 activity upon CPUG assembly CPUG synthesizes long poly(UG) tails influenced by the substrate 3′ end RDE-8 sequence specificity protects poly(UG) tails from cleavage

Most eukaryotic proteins assemble into multisubunit complexes that coordinate essential cellular functions, yet the principles governing their assembly and proteostatic control remain largely undefined. Here, we systematically dissect the cellular assembly and functional organization of the RNA exosome, an essential ribonucleolytic complex, using an inducible dual-guide CRISPR/Cas9 system in mouse embryonic stem cells. We reveal a sequential assembly pathway where Exosc2, Exosc4, and Exosc7 initiate complex formation, facilitating the incorporation of barrel and cap subunits in a defined hierarchy. Unlike other structural subunits, the terminally incorporated cap subunit Exosc1 is dispensable for cell viability, revealing a modular, functionally resilient architecture. We demonstrate that orphan subunits are selectively degraded via the ubiquitin-proteasome system, enforcing stringent quality control over RNA exosome biogenesis. These findings define an assembly logic of of the mammalian exosome and uncover previously unrecognized plasticity in the composition and function of this essential ribonucleolytic complex.

Summary: 3′-terminal uridylation marks structured non-coding RNAs for cytoplasmic decay, yet how uridylation is quantitatively coupled to exonucleolytic degradation remains unclear. Here, we dissect the kinetic logic of uridylation-mediated RNA surveillance in Drosophila melanogaster . Using biochemical reconstitution together with high-throughput enzymology and quantitative modeling, we show that the terminal uridylyl transferase Tailor generates discrete oligo(U) intermediates through product-dependent kinetic tuning, while co-substrate promiscuity suppresses sustained processivity under physiological nucleotide conditions. Massively parallel binding and decay assays further reveal how the 3′-to-5′ exoribonuclease Dis3l2 selectively degrades Tailor-primed RNAs by integrating 3′-proximal uridine content and defined 3′-end accessibility—features encoded by short, kinetically tuned oligo(U) intermediates centered on four nucleotides—to enable productive threading of RNA substrates along an extended RNA-binding path to the catalytic site. Together, our findings establish a quantitative framework in which uridylation encodes decay competence through transient RNA 3′-end states that are matched to the mechanistic requirements for decay. Highlights: The TUTase Tailor kinetically tunes uridylation to generate short, discrete oligo(U) intermediates Mixed nucleotide availability suppresses sustained processive uridylation Dis3l2 decodes 3′-proximal uridine content and end accessibility to commit RNAs to decay Short oligo(U) tails encode RNA decay competence through transient RNA 3′-end states

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.

Type II interferon (IFNγ) signaling is essential for innate immunity and critical for effective immunological checkpoint blockade in cancer immunotherapy. Genetic screen identification of post-transcriptional regulators of this pathway has been challenging since such factors are often essential for cell viability. Here, we utilize our inducible CRISPR/Cas9 approach to screen for key post-transcriptional regulators of IFNγ signaling, and in this way, we identify ERH and the ERH-associated splicing and RNA export factors MAGOH, SRSF1, and ALYREF. Loss of these factors impairs post-transcriptional mRNA maturation of JAK2, a crucial kinase for IFNγ signaling, resulting in abrogated JAK2 protein levels and diminished IFNγ signaling. Further analysis highlights a critical role for ERH in preventing intron retention in AU-rich regions in specific transcripts, such as JAK2. This regulation is markedly different from previously described retention of GC-rich introns. Overall, these findings reveal that post-transcriptional JAK2 processing is a critical rate-limiting step for the IFNγ-driven innate immune response.

Summary: Most eukaryotic proteins assemble into multisubunit complexes that coordinate essential cellular functions, yet the principles governing their assembly and proteostatic control remain largely undefined. Here, we systematically dissect the cellular assembly and functional organization of the RNA exosome, an essential ribonucleolytic complex, using an inducible dual-guide CRISPR/Cas9 system in mouse embryonic stem cells. We reveal a sequential assembly pathway where Exosc2, Exosc4, and Exosc7 initiate complex formation, facilitating the incorporation of barrel and cap subunits in a defined hierarchy. Unlike other structural subunits, the terminally incorporated cap subunit Exosc1 is dispensable for cell viability, revealing a modular, functionally resilient architecture. We demonstrate that orphan subunits are selectively degraded via the ubiquitin-proteasome system, enforcing stringent quality control over RNA exosome biogenesis. These findings establish a framework for decoding the assembly logic of essential macromolecular machines and uncover previously unrecognized plasticity in the composition and function of the RNA exosome.

Motivation: The efficient and reproducible analysis of high-throughput sequencing datasets necessitates the development of methodical and robust computational pipelines that integrate established and bespoke bioinformatics analysis tools, often written in high-level programming languages such as Python. Despite the increasing availability of programming libraries for genomics, there is a noticeable lack of tools specifically focused on transcriptomics. Key tasks in this area include the association of gene features (e.g. transcript isoforms, introns or untranslated regions) with relevant subsections of (large) genomics datasets across diverse data formats, as well as efficient querying of these data based on genomic locations and annotation attributes. Results: To address the needs of transcriptomics data analyses, we developed rnalib, a Python library designed for creating custom bioinformatics analysis methods. Built on existing Python libraries like pysam and pyBigWig, rnalib offers random access support, enabling efficient access to relevant subregions of large, genome-wide datasets. Rnalib extends the filtering and access capabilities of these libraries and includes additional checks to prevent common errors when integrating genomics datasets. The library is centred on an object-oriented Transcriptome class that provides methods for stepwise annotation of gene features with both, local and remote data sources. The rnalib Application Programming Interface cleanly separates immutable genomic locations from associated, mutable data, and offers a wide range of methods for iterating, querying, and exporting collated datasets. Rnalib establishes a fast, readable, reproducible, and robust framework for developing novel transcriptomics data analysis tools and methods. Availability and implementation: Source code, documentation, and tutorials are available at https://github.com/popitsch/rnalib.

Type II interferon (IFNγ) signaling is essential for innate immunity and critical for effective immunological checkpoint blockade in cancer immunotherapy. Genetic screen identification of post-transcriptional regulators of this pathway has been challenging since such factors are often essential for cell viability. Here, we utilize our inducible CRISPR/Cas9 approach to screen for key post-transcriptional regulators of IFNγ signaling, and in this way identify ERH and the ERH-associated splicing and RNA export factors MAGOH, SRSF1, and ALYREF. Loss of these factors impairs post-transcriptional mRNA maturation of JAK2 , a crucial kinase for IFNγ signaling, resulting in abrogated JAK2 protein levels and diminished IFNγ signaling. Further analysis highlights a critical role for ERH in preventing intron retention in AU-rich regions in specific transcripts, such as JAK2 . This regulation is markedly different from previously described retention of GC-rich introns. Overall, these findings reveal that post-transcriptional JAK2 processing is a critical rate-limiting step for the IFNγ-driven innate immune response.

The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs). We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and posttranscriptional regulators in mouse embryonic stem cells. Each screen recovers known and identifies new regulators, including long ORFs inaccessible by other methods. Among other hits, we find that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.