In human cells, the Nuclear EXosome Targeting (NEXT) and Poly(A) tail eXosome Targeting (PAXT) adaptors direct the nuclear exosome to degrade prematurely terminated RNA Polymerase II (Pol II) transcripts, ensuring nuclear RNA quality control. How these adaptors interact with transcription termination machineries remains largely unclear. Here, we leveraged in silico structure predictions of protein complexes to identify and model previously unreported interactions of NEXT- and PAXT-associated components with two transcription termination and processing machineries, the Integrator and Cleavage and Polyadenylation (CPA) complexes. Our computational models were validated through complementary in vitro biochemical approaches and single-particle cryo-EM analyses. We show that the ZC3H18 protein uses two different domains to directly recognize the INTS9/11 endonuclease module of Integrator and the mammalian Polyadenylation Specificity Factor (mPSF), a core CPA component. In turn, ZC3H18 can directly bind the scaffolding subunits of NEXT and PAXT via mutually exclusive interactions. Furthermore, we provide evidence that accessory PAXT components can be directly integrated with the mPSF core, establishing configurations that are mutually exclusive with those of canonical CPA subunits. These findings reveal a versatile interaction network capable of forming alternative structural frameworks linking transcription termination with nuclear RNA quality control.

Intron-containing mRNAs are cotranscriptionally spliced and assembled into messenger ribonucleoprotein (mRNP) particles, a process monitored by surveillance pathways. Here, we combined biochemical and structural approaches to elucidate the mechanisms by which mRNPs are sorted between two opposing fates: nuclear degradation and cytoplasmic export. While the human GANP-PCID2 complex is known to connect mRNPs to nuclear export, our data indicate that the LENG8-PCID2 complex operates as an mRNP decay connector, coupling nuclear mRNPs to the RNA-degrading exosome via the PAXT adaptor complex. Both recognize the mRNP component UAP56, but LENG8-PCID2 uniquely associates with early splicing factors through a direct interaction with U1A and RRP1B. Similarly, the Thp3-Csn12 ortholog in budding yeast couples the early splicing factors Mud2-Bbp with the nuclear exosome. The spliceosome-exosome mRNP decay pathway we uncovered reveals molecular principles that remain strikingly conserved across evolution, despite the fundamental differences in splicing and decay between humans and budding yeast.

Evolution reflects a balance between innovation and constraint, often repurposing existing components in new contexts. Convergent evolution exemplifies this interplay, with similar traits evolving independently in different species, yet the genomic mechanisms enabling this repeatability remain poorly understood. Here, by analyzing 10 chromosome-scale genome assemblies, including seven newly generated, we found that the S-locus supergene (a cluster of tightly linked genes controlling a floral dimorphism called distyly) arose independently multiple times within the primrose family, Darwin's iconic system for studying distyly. In each case, the same gene was independently duplicated and co-opted. However, the resulting genomic architectures differed, ranging from hemizygous (present on one chromosome copy) to heterozygous (on both copies), challenging the prevailing view that hemizygosity is intrinsic to S-loci and suggesting alternative evolutionary routes to distyly supergene formation. By uncovering multiple mechanisms for supergene origins, our work shows how convergent evolution can produce similar phenotypes by reusing the same genetic building blocks while exploring distinct genomic configurations.

Nonsense-mediated mRNA decay (NMD) relies on the coordinated assembly and action of multiple protein factors. Degradation of target mRNAs begins with endonucleolytic cleavage near premature stop codons, but the mechanisms of endonuclease activation and regulation remain unclear. Using structural predictions, biochemical in vitro assays, and cell-based NMD analysis, we show that SMG5 and SMG6 interact via their PIN domains to form a composite interface (cPIN) with full endonuclease activity. In vitro reconstituted SMG5-SMG6 cPIN heterodimers show high activity, as SMG5 completes the SMG6 active site and substrate binding site. Mutations in residues at their predicted interaction surfaces, RNA-binding sites, or active site attenuate or abolish cPIN activity in vitro and impair cellular NMD. Our findings demonstrate how paralogous PIN domains complement each other to assemble a highly active endonuclease in NMD, providing a structural and mechanistic explanation for efficient NMD substrate degradation.

Anatomical (scape, pedicel, leaf lamina, and leaf midrib) and pollen morphological features of 11 taxa belonging to the genus Primula subgen. Primula (P. vulgaris subsp. vulgaris, P. vulgaris subsp. rubra, and P. megaseifolia, P. meyeri, P. elatior subsp. pallasii, P. pseudoelatior, P. veris subsp. columnae, P. veris subsp. macrocalyx) and subgen. Aleurita (P. longipes, P. algida, and P. auriculata) were examined using light and scanning electron microscopy to shed light on the existing taxonomic and phylogenetic relationships. The anatomical and palynological features of the majority of the taxa studied were revealed for the first time in this study. In anatomical studies, the very large intercellular space under the epidermis, the number of sclerenchyma layers in the scape, and the midrib shape in the leaf lamina were found to be of taxonomic importance at the subgenus level. In palynological studies, pollen grain size, pollen surface ornamentation patterns, and pollen aperture types could be used as important characters to distinguish species, sections, and subgenera. Significant differences were found between subgenera and between the pollen grains of pin and thrum flowers of the same taxon. Mostly, the subgen. Aleurita taxa had significantly smaller pollen grains than the subgen. Primula taxa, and pollen grains from flowers with pin morphology were smaller than those from flowers with thrum morphology. Our studies showed that both anatomical and palynological features have not only taxonomic but also phylogenetic importance.

Upf1 is a master regulator of nonsense-mediated mRNA decay (NMD), an mRNA surveillance and degradation pathway conserved from yeast to human. In Saccharomyces cerevisiae, Upf1 exists in two distinct complexes with factors that mediate NMD activation or 5'-3' mRNA degradation. We combined endogenous purifications and biochemical reconstitutions of yeast Upf1 complexes with structural analyses and biochemical assays to elucidate the molecular mechanisms driving the organization of the Upf1-5'-3' and Upf1-2-3 complexes. We show that yeast Upf1 is in a constitutive complex, whereby its CH, RecA, and C-terminal domains interact with the mRNA decapping factor Dcp2, NMD-associated proteins Nmd4 and Ebs1, and the 5'-3' exoribonuclease Xrn1, respectively. Together, the interacting surfaces and closed conformation of Upf1 in the Upf1-5'-3' complex sterically obstruct the binding of Upf2-3. Our work points to a major restructuring upon recruitment of these factors during NMD and provides insights into evolutionary divergence amongst species.

This erratum addresses an error concerning Figure 5 in the original published version of this article. During the typesetting process, Figure 5 was partially omitted, resulting in an incomplete figure being published.To correct this oversight and ensure the accuracy of the scholarly record, the complete Figure 5 is provided below for reference. Link to the original article: https://doi.org/10.55730/1300-008X.2866

The lifespan of most eukaryotic mRNAs is modulated by the gradual shortening of the poly(A) tail and removal of the associated poly(A)-binding protein. The human PAN2-PAN3 complex catalyzes initial deadenylation by shortening long poly(A) tails associated with PABPC1. Both PAN2-PAN3 and PABPC1 are evolutionarily conserved from fungi to humans. How the human complex has adapted to recognize and act on longer poly(A) tails characteristic of mammalian mRNAs remains unclear. Here, we report a method to obtain homo-polymeric poly(A) RNAs up to 240 nt, mimicking the synthesis length of poly(A) tails in mammals. We recapitulate human deadenylation properties in vitro, with PAN2-PAN3 showing greater activity on long poly(A)-PABPC1 ribonucleoprotein substrates. Single-particle cryo-electron microscopy (cryo-EM) analyses of PAN2-PAN3 bound to poly(A)-PABPC1 ribonucleoproteins uncover a longer substrate-binding path in the case of the human deadenylase compared to fungi. Altogether, these data provide a rationale for the co-evolution of deadenylase properties and poly(A) tail lengths.

The RNA exosome is a conserved multiprotein complex essential for 3'-to-5' RNA degradation in eukaryotic cells. In the cytoplasm, the exosome participates in messenger RNA surveillance and decay, while in the nucleus and nucleolus it performs a broader range of functions, from fully degrading cryptic RNAs generated by faulty or pervasive transcription to precisely trimming structured RNAs. An extended network of obligate cofactors and transient RNA helicase complexes has evolved to handle the large variety of substrates in each subcellular compartment. This network organizes in layers around the exosome core and regulates the irreversible 3'-to-5' degradative action in synergy with the features of the substrates. In this review, we discuss findings derived from genetic, cellular, biochemical, and structural analyses of nuclear and cytoplasmic exosome complexes, and integrate them into molecular movies that illustrate the mechanistic principles of this versatile and dynamic machine in RNA processing and RNA decay.

Transitions from outcrossing to selfing and from diploidy to polyploidy often co-occur in plants, likely because the ability to produce selfed seed increases the likelihood of newly formed polyploids to become established. An ideal system to study these transitions is Primula, where the shift from diploid, outcrossing progenitors to polyploid, selfing descendants co-occurred repeatedly and the genetic basis of the mating-system shift is known. In Primula, outcrossing is enforced in distylous, typically diploid species characterized by florally heteromorphic, self-incompatible individuals, whereas selfing is enabled in homostylous, typically polyploid species, characterized by florally homomorphic populations of self-compatible plants. Distyly is controlled by the S-locus supergene. Small loss-of-function mutations in the S-locus CYPT gene, which controls style length and female self-incompatibility, are associated with loss of heterostyly in diploid, ancestrally heterostylous Primula species. However, CYPT and the S-locus have never been investigated in interspecific shifts from distylous, diploid species to homostylous, polyploid species. By analyzing the first assembled genome of a homostylous, polyploid species (Primula grandis) in a comparative framework, we discovered two, nearly identical S-locus alleles in the same subgenome, consistent with the hypothesis that the species originated from a cross between a homostylous, diploid pollen donor and a long-styled, diploid pollen recipient. Conformant to theoretical predictions, the macroevolutionary loss of distyly coincided with considerable degeneration of CYPT, including multiple mutations and exon loss, while other S-locus genes remained largely unaffected. This study advances knowledge on the macroevolutionary dynamics of supergenes and genomes in shifts between breeding systems and ploidy levels.

Despite the evolutionary importance of supergenes, their properties in polyploids remain unexplored. Polyploid genomes are expected to undergo chromosomal rearrangements and gene losses over time, potentially affecting supergene architecture. The iconic distyly supergene (S-locus), controlling a floral heteromorphism with two self-incompatible morphs, has been well-documented in diploids, but remains unknown in polyploids. Primula , the classic model for distyly since Darwin, is ancestrally diploid and distylous, yet polyploid, homostylous species with a single, self-compatible floral morph evolved repeatedly. The intraspecific loss of distyly is associated with small loss-of-function mutations in the S-locus CYPT gene controlling style length and female self-incompatibility. Over longer timescales, relaxed selection on CYPT should generate greater accumulation of larger mutations, including exon and gene loss. By analyzing the first assembled genome of an allotetraploid, homostylous species ( Primula grandis ) in a comparative framework, we discovered two, nearly identical S-locus alleles in the same subgenome, suggesting it originated via inter-specific hybridization between a homostylous and a distylous progenitor. Conformant to predictions from theory, the macroevolutionary loss of distyly coincided with considerable degeneration of CYPT , while other S-locus genes remained largely unaffected, suggesting the shift to homostyly preceded and facilitated polyploid establishment. At the whole-genome level, we found minimal subgenome dominance - as expected, given the inferred recent origin of P. grandis - and highly reduced genetic diversity, congruently with its narrow distribution and self-compatibility. This study provides the first comparison of a supergene across ploidy levels and reproductive systems, contributing new knowledge on the previously unknown fate of supergenes in polyploids. ### Competing Interest Statement The authors have declared no competing interest.

The widespread loss and fragmentation of habitats have caused significant declines in biodiversity. Among plants, animal-pollinated species are particularly threatened because of the negative effects of these factors on pollinators. Heterostyly is a unique reproductive system defined by two or three floral morphs having a distinct position of anthers and style. The spatial separation of reproductive organs, accompanied by a self-incompatibility system, restricts self-pollination and favours pollinator-mediated pollen transfer between different morphs. In this review, we synthesize knowledge about the effects of loss in the area and connectivity of habitats, and related reduction in population size on heterostylous plants. We conducted a literature search to obtain an overview of studies investigating the short- and long-term consequences of the decreased area and connectivity of habitats as well as plant population size for heterostylous species. To quantify the relationship between plant population size and morph ratio bias, we applied a meta-analytical approach. First, the meta-analysis showed that reductions in population size can significantly disrupt the optimal morph ratio, leading to fewer compatible mates and lower reproductive output. Second, the literature review highlights the negative consequences of biased morph ratios for population viability and genetic diversity of heterostylous plants. Finally, heterostylous species may adapt to the loss of pollination by shifting their mating system to homostyly and selfing. This review demonstrates that habitat loss and fragmentation have various consequences for heterostylous plants, e.g. reduced population size, morph ratio bias, and disruption of pollination. With ongoing environmental changes, there are still important knowledge gaps that need to be addressed more systematically. These include the long-term impact of skewed morph frequencies on population viability, the selective forces driving variation in anther-stigma separation and intra-morph compatibility, the role of habitat loss and connectivity, and related reduction in pollinator abundance and diversity in the selection for homostyly.

Uniparental reproduction is advantageous when lack of mates limits outcrossing opportunities in plants. Baker’s law predicts an enrichment of uniparental reproduction in habitats colonized via long-distance dispersal, such as volcanic islands. To test it, we analyzed reproductive traits at multiple hierarchical levels and compared seed-set after selfing and crossing experiments in both island and mainland populations of Limonium lobatum, a widespread species that Baker assumed to be self-incompatible because it had been described as pollen-stigma dimorphic, i.e., characterized by floral morphs differing in pollen-surface morphology and stigma-papillae shape that are typically self-incompatible. We discovered new types and combinations of pollen and stigma traits hitherto unknown in the literature on pollen-stigma dimorphism and a lack of correspondence between such combinations and pollen compatibility. Contrary to previous reports, we conclude that Limonium lobatum comprises both self-compatible and self-incompatible plants characterized by both known and previously undescribed combinations of reproductive traits. Most importantly, plants with novel combinations are overrepresented on islands, selfed seed-set is higher in islands than the mainland, and insular plants with novel pollen-stigma trait-combinations disproportionally contribute to uniparental reproduction on islands. Our results thus support Baker’s law, connecting research on reproductive and island biology.

Genetic diversity is heterogeneously distributed among populations of the same species, due to the joint effects of multiple demographic processes, including range contractions and expansions, and mating systems shifts. Here, we ask how both processes shape genomic diversity in space and time in the classical Primula vulgaris model. This perennial herb originated in the Caucasus region and was hypothesized to have expanded westward following glacial retreat in the Quaternary. Moreover, this species is a long-standing model for mating system transitions, exemplified by shifts from heterostyly to homostyly. Leveraging a high-quality reference genome of the closely related Primula veris and whole-genome resequencing data from both heterostylous and homostylous individuals from populations encompassing a wide distribution of P. vulgaris, we reconstructed the demographic history of P. vulgaris. Results are compatible with the previously proposed hypothesis of range expansion from the Caucasus region approximately 79,000 years ago and suggest later shifts to homostyly following rather than preceding postglacial colonization of England. Furthermore, in accordance with population genetic theoretical predictions, both processes are associated with reduced genetic diversity, increased linkage disequilibrium, and reduced efficacy of purifying selection. A novel result concerns the contrasting effects of range expansion versus shift to homostyly on transposable elements, for the former, process is associated with changes in transposable element genomic content, while the latter is not. Jointly, our results elucidate how the interactions among range expansion, transitions to selfing, and Quaternary climatic oscillations shape plant evolution.

3' end processing of most eukaryotic pre-mRNAs is a crucial co-transcriptional process that generally involves the cleavage and polyadenylation of the precursor transcripts. Within the human 3’ end processing machinery, the 4-subunit mammalian polyadenylation specificity factor (mPSF) recognizes the polyadenylation signal (PAS) in the pre-mRNA and recruits the polyA polymerase α (PAPOA) to it. To shed light on the molecular mechanisms of PAPOA recruitment to mPSF, we used a combination of cryogenic-electron microscopy (cryo-EM) single-particle analysis, computational structure prediction andin vitrobiochemistry to reveal an intricate interaction network. A short linear motif in the mPSF subunit FIP1 interacts with the structured core of human PAPOA, with a binding mode that is evolutionary conserved from yeast to human. In higher eukaryotes, however, PAPOA contains a conserved C-terminal motif that can interact intramolecularly with the same residues of the PAPOA structured core used to bind FIP1. Interestingly, using biochemical assay and cryo-EM structural analysis, we found that the PAPOA C-terminal motif can also directly interact with mPSF at the subunit CPSF160. These results show that PAPOA recruitment to mPSF is mediated by two distinct intermolecular connections and further suggest the presence of mutually exclusive interactions in the regulation of 3' end processing.

The interplay between translation and mRNA decay is widespread in human cells1-3. In quality-control pathways, exonucleolytic degradation of mRNA associated with translating ribosomes is mediated largely by the cytoplasmic exosome4-9, which includes the exoribonuclease complex EXO10 and the helicase complex SKI238 (refs. 10-16). The helicase can extract mRNA from the ribosome and is expected to transfer it to the exoribonuclease core through a bridging factor, HBS1L3 (also known as SKI7), but the mechanisms of this molecular handover remain unclear7,17,18. Here we reveal how human EXO10 is recruited by HBS1L3 (SKI7) to an active ribosome-bound SKI238 complex. We show that rather than a sequential handover, a direct physical coupling mechanism takes place, which culminates in the formation of a cytoplasmic exosome-ribosome supercomplex. Capturing the structure during active decay reveals a continuous path in which an RNA substrate threads from the 80S ribosome through the SKI2 helicase into the exoribonuclease active site of the cytoplasmic exosome complex. The SKI3 subunit of the complex directly binds to HBS1L3 (SKI7) and also engages a surface of the 40S subunit, establishing a recognition platform in collided disomes. Exosome and ribosome thus work together as a single structural and functional unit in co-translational mRNA decay, coordinating their activities in a transient supercomplex.

Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. Here, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by different conformational states connected to the RNA-binding status of UPF1. Our findings rationalize a key event in metazoan NMD and advance our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.

Abstract Distyly, a floral dimorphism that promotes outcrossing, is controlled by a hemizygous genomic region known as the S‐locus. Disruptions of genes within the S‐locus are responsible for the loss of distyly and the emergence of homostyly, a floral monomorphism that favors selfing. Using whole‐genome resequencing data of distylous and homostylous individuals from populations of Primula vulgaris and leveraging high‐quality reference genomes of Primula we tested, for the first time, predictions about the evolutionary consequences of transitions to selfing on S‐genes. Our results reveal a previously undetected structural rearrangement in CYPᵀ associated with the shift to homostyly and confirm previously reported, homostyle‐specific, loss‐of‐function mutations in the exons of the S‐gene CYPᵀ. We also discovered that the promoter and intronic regions of CYPᵀ in distylous and homostylous individuals are conserved, suggesting that down‐regulation of CYPᵀ via mutations in its promoter and intronic regions is not a cause of the shift to homostyly. Furthermore, we found that hemizygosity is associated with reduced genetic diversity in S‐genes compared with their paralogs outside the S‐locus. Additionally, the shift to homostyly lowers genetic diversity in both the S‐genes and their paralogs, as expected in primarily selfing plants. Finally, we tested, for the first time, long‐standing theoretical models of changes in S‐locus genotypes during early stages of the transition to homostyly, supporting the assumption that two copies of the S‐locus might reduce homostyle fitness.

Eukaryotes have evolved quality‐control mechanisms that allow cells to remove defective messenger RNAs (mRNAs). Aberrant mRNA with premature stop codons (PTCs) are recognized and degraded by a process known as nonsense‐mediated mRNA decay (NMD). The NMD pathway is conserved from lower to higher eukaryotes. In humans, the recognition of a PTC‐containing mRNA depends on both splicing and translation.In the nucleus, a complex of proteins known as the exon junction complex (EJC) is deposited on mRNA upon splicing and stays stably bound until the nucleic acid is exported to the cytoplasm. Here, the crosstalk between a ribosome stalled at a PTC and a downstream EJC elicits a cascade of interactions that ultimately lead to the recruitment of ribonucleases and mRNA degradation. The talk will focus on our current understanding of the molecular mechanisms of NMD as elucidated by structural biology.

Human cleavage and polyadenylation specificity factor (CPSF)73 (also known as CPSF3) is the endoribonuclease that catalyzes the cleavage reaction for the 3'-end processing of pre-mRNAs. The active site of CPSF73 is located at the interface between a metallo-β-lactamase domain and a β-CASP domain. Two metal ions are coordinated by conserved residues, five His and two Asp, in the active site, and they are critical for the nuclease reaction. The metal ions have long been thought to be zinc ions, but their exact identity has not been examined. Here we present evidence from inductively coupled plasma mass spectrometry and X-ray diffraction analyses that a mixture of metal ions, including Fe, Zn, and Mn, is present in the active site of CPSF73. The abundance of the various metal ions is different in samples prepared from different expression hosts. Zinc is present at less than 20% abundance in a sample expressed in insect cells, but the sample is active in cleaving a pre-mRNA substrate in a reconstituted canonical 3'-end processing machinery. Zinc is present at 75% abundance in a sample expressed in human cells, which has comparable endonuclease activity. We also observe a mixture of metal ions in the active site of the CPSF73 homolog INTS11, the endonuclease for Integrator. Taken together, our results provide further insights into the role of metal ions in the activity of CPSF73 and INTS11 for RNA 3'-end processing.