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Nuclear Argonaute proteins, guided to nascent target RNAs by their bound small RNAs, elicit co-transcriptional silencing through heterochromatin formation at transposon insertions and repetitive genomic loci. The molecular mechanisms involved in this process are incompletely understood. Here, we propose that the SFiNX complex, a silencing mediator downstream of nuclear Piwi-piRNA complexes inDrosophila, enables co-transcriptional silencing via the formation of molecular condensates. Condensate formation is stimulated by nucleic acid binding and requires SFiNX dimerization, mediated by the dynein light chain protein, LC8/Cutup. LC8’s function within SFiNX can be bypassed with a heterologous dimerization domain, suggesting that dimerization is a constitutive feature of SFiNX. Mutations preventing LC8-mediated SFiNX dimerization result in loss of condensate formationin vitroand inability of Piwi to initiate heterochromatin formation and silence transposonsin vivo. Formation of molecular condensates might be a general mechanism that underlies effective heterochromatin establishment at small RNA target loci in a co-transcriptional manner.

Nuclear Argonaute proteins, guided by their bound small RNAs to nascent target transcripts, mediate cotranscriptional silencing of transposons and repetitive genomic loci through heterochromatin formation. The molecular mechanisms involved in this process are incompletely understood. Here, we show that the SFiNX complex, a silencing mediator downstream from nuclear Piwi-piRNA complexes in Drosophila, facilitates cotranscriptional silencing as a homodimer. The dynein light chain protein Cut up/LC8 mediates SFiNX dimerization, and its function can be bypassed by a heterologous dimerization domain, arguing for a constitutive SFiNX dimer. Dimeric, but not monomeric SFiNX, is capable of forming molecular condensates in a nucleic acid-stimulated manner. Mutations that prevent SFiNX dimerization result in loss of condensate formation in vitro and the inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. We propose that multivalent SFiNX-nucleic acid interactions are critical for heterochromatin establishment at piRNA target loci in a cotranscriptional manner.

The export of mRNA from nucleus to cytoplasm requires the conserved and essential transcription and export (TREX) complex (THO–UAP56/DDX39B–ALYREF). TREX selectively binds mRNA maturation marks and licenses mRNA for nuclear export by loading the export factor NXF1–NXT1. How TREX integrates these marks and achieves high selectivity for mature mRNA is poorly understood. Here, we report the cryo-electron microscopy structure of the human THO–UAP56/DDX39B complex at 3.3 Å resolution. The seven-subunit THO–UAP56/DDX39B complex multimerizes into a 28-subunit tetrameric assembly, suggesting that selective recognition of mature mRNA is facilitated by the simultaneous sensing of multiple, spatially distant mRNA regions and maturation marks. Two UAP56/DDX39B RNA helicases are juxtaposed at each end of the tetramer, which would allow one bivalent ALYREF protein to bridge adjacent helicases and regulate the TREX–mRNA interaction. Our structural and biochemical results suggest a conserved model for TREX complex function that depends on multivalent interactions between proteins and mRNA., eLife digest The DNA of human and other eukaryotic cells is stored inside a compartment called the nucleus. DNA carries the genetic code and provides a blueprint for all of the cell’s proteins. However, protein production occurs outside the nucleus, in the main body of the cell. To transmit genetic information from one compartment to the other, the DNA sequences are first transcribed into another molecule called messenger RNA, or mRNA for short. Once made, mRNA exits the nucleus and enters the cell’s main body to encounter the machinery that translates its sequence into a protein. Before mRNA can exit the nucleus, it must first undergo a series of modifications, which result in the mRNA molecule being successively bound to specific proteins. Once mRNA has passed through these steps, it is recognized by the transcription-and-export complex, or TREX for short, which is comprised of several proteins. When TREX binds to mRNA, it adds on a final protein which allows the mRNA molecule to be transported out of the nucleus. However, it remained unclear how TREX selects the completed mRNA-protein complexes that are ready for export while at the same time recognizing the wide variety of mRNA molecules produced by cells. Now, Pühringer and Hohmann et al. have identified the first three-dimensional structure of the core of the human TREX complex using a technique called cryo-electron microscopy. This revealed that the seven proteins of the TREX core assemble into a large complex that has four copies of each protein. The structure suggests that TREX can bind to mRNA and its attached proteins in various ways. These different binding arrangements may help the complex select which mRNA molecules are fully modified and ready to be exported. The structure also sheds light on how mutations in this complex can lead to diseases such as Beaulieu–Boycott–Innes syndrome (BBIS). This work will help guide future research into the activity of TREX, including how its structure changes when it binds to mRNA and deposits the final transport protein. Identifying these structures will make it easier to design experiments that target specific aspects of TREX activity and provide new insights into how these complexes work.

Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 small nuclear ribonucleoprotein particle (snRNP) associates with U4/U6.U5 tri-snRNP through the U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The 3' region of the U2 snRNP is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5' splice site. The U6 ACAGAGA sequence forms a hairpin that weakly tethers the 5' splice site. The B complex proteins Prp38, Snu23 and Spp381 bind the Prp8 N-terminal domain and stabilize U6 ACAGAGA stem-pre-mRNA and Brr2-U4 small nuclear RNA interactions. These results provide important insights into the events leading to active site formation.

The removal of noncoding introns from pre-messenger RNA (pre-mRNA) is an essential step in eukaryotic gene expression and is catalyzed by a dynamic multi-megadalton ribonucleoprotein complex called the spliceosome. The spliceosome assembles on pre-mRNA substrates by the stepwise addition of small nuclear ribonucleoprotein particles and numerous protein factors. Extensive remodeling is required to form the RNA-based active site and to mediate the pre-mRNA branching and ligation reactions. In the past two years, cryo-electron microscopy (cryo-EM) structures of spliceosomes captured in different assembly and catalytic states have greatly advanced our understanding of its mechanism. This was made possible by long-standing efforts in the purification of spliceosome intermediates as well as recent developments in cryo-EM imaging and computational methodology. The resulting high-resolution densities allow for de novo model building in core regions of the complexes. In peripheral and less ordered regions, the combination of cross-linking, bioinformatics, biochemical, and genetic data is essential for accurate modeling. Here, we summarize these achievements and highlight the critical steps in obtaining near-atomic resolution structures of the spliceosome.

The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

Noncoding introns are removed from nuclear precursor messenger RNA (pre-mRNA) in a two-step phosphoryl transfer reaction by the spliceosome, a dynamic multimegadalton enzyme. Cryo-electron microscopy (cryo-EM) structures of the Saccharomyces cerevisiae spliceosome were recently determined in eight key states. Combined with the wealth of available genetic and biochemical data, these structures have revealed new insights into the mechanisms of spliceosome assembly, activation, catalysis, and disassembly. The structures show how a single RNA catalytic center forms during activation and accomplishes both steps of the splicing reaction. The structures reveal how spliceosomal helicases remodel the spliceosome for active site formation, substrate docking, reaction product undocking, and spliceosome disassembly and how they facilitate splice site proofreading. Although human spliceosomes contain additional proteins, their cryo-EM structures suggest that the underlying mechanism is conserved across all eukaryotes. In this review, we summarize the current structural understanding of pre-mRNA splicing.

Integrated structural biology recently elucidated the architecture of Mediator and its position on RNA polymerase (Pol) II. Here we summarize these achievements and list open questions on Mediator structure and mechanism.

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 A and 3.6 A, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE ‘extended winged helix’ domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE ‘E-ribbon’ domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein–protein and protein–DNA contacts. The cryo-electron microscopy structures of yeast initiation complexes containing the transcription factors TBP, TFIIA, TFIIB, TFIIE, and TFIIF and containing either closed or open promoter DNA are reported, providing mechanistic insights into DNA opening and template-strand loading. The initiation of gene transcription in eukaryotes is tightly controlled at the promoter of each gene through the actions of the pre-initiation complex (PIC), a large multi-subunit composed of general transcription factors, and RNA polymerase II (Pol II) assembles at the promoter to ensure correct loading of Pol II and opening of the duplex DNA for transcription into RNA. Two papers published in this issue report detailed cryo-electron microscopy structures of the Pol II machinery at near-atomic resolution. Eva Nogales and colleagues present structural models of the human PIC at all major steps during transcription initiation at near-atomic resolution. They provide new mechanistic insights into the processes of promoter melting and transcription bubble stabilization, as well as proposing an almost complete structural model of all of the PIC components bound to duplex DNA. Patrick Cramer and colleagues report structures of yeast initiation complexes containing all of the basal transcription factors except TFIIH, and containing either closed or open promoter DNA. They show that DNA opening can occur in the absence of TFIIH, and provide mechanistic insights into DNA opening and template-strand loading. The structures reveal the high structural conservation between yeast and human transcription initiation systems.

The essential core of the transcription coactivator Mediator consists of two conserved multiprotein modules, the head and middle modules. Whereas the structure of the head module is known, the structure of the middle module is lacking. Here we report a 3D model of a 6-subunit Mediator middle module. The model was obtained by arranging crystal structures and homology models of parts of the module based on lysine-lysine cross-links obtained by mass spectrometric analysis. The model contains a central tetramer formed by the heterodimers Med4/Med9 and Med7/Med21. The Med7/Med21 heterodimer is flanked by subunits Med10 and Med31. The model is highly extended, suggests that the middle module is flexible and contributes to a molecular basis for detailed structure-function studies of RNA polymerase II regulation.

The crystal structure of the Mediator head module from the fission yeast Schizosaccharomyces pombe is solved at 3.4 A resolution. The Mediator complex has a central role in gene regulation, linking RNA polymerase II to its many transcriptional regulators. The structure of the budding yeast seven-subunit Mediator head module indicated three domains: the neck, fixed jaw and moveable jaw. Patrick Cramer and colleagues have determined the structure of the fission yeast head module at 3.4 A resolution. The enhanced resolution allows refinement of interpretations of the initial structure and emphasizes the flexibility of the head module. The highly conserved structure is flexible, and the localization of known mutations permits the attribution of function to some regions of the molecule. Gene transcription by RNA polymerase (Pol) II requires the coactivator complex Mediator. Mediator connects transcriptional regulators and Pol II, and is linked to human disease1,2,3,4. Mediator from the yeast Saccharomyces cerevisiae has a molecular mass of 1.4 megadaltons and comprises 25 subunits that form the head, middle, tail and kinase modules5,6,7. The head module constitutes one-half of the essential Mediator core8, and comprises the conserved9 subunits Med6, Med8, Med11, Med17, Med18, Med20 and Med22. Recent X-ray analysis of the S. cerevisiae head module at 4.3 A resolution led to a partial architectural model with three submodules called neck, fixed jaw and moveable jaw10. Here we determine de novo the crystal structure of the head module from the fission yeast Schizosaccharomyces pombe at 3.4 A resolution. Structure solution was enabled by new structures of Med6 and the fixed jaw, and previous structures of the moveable jaw11 and part of the neck12, and required deletion of Med20. The S. pombe head module resembles the head of a crocodile with eight distinct elements, of which at least four are mobile. The fixed jaw comprises tooth and nose domains, whereas the neck submodule contains a helical spine and one limb, with shoulder, arm and finger elements. The arm and the essential shoulder contact other parts of Mediator. The jaws and a central joint are implicated in interactions with Pol II and its carboxy-terminal domain, and the joint is required for transcription in vitro. The S. pombe head module structure leads to a revised model of the S. cerevisiae module, reveals a high conservation and flexibility, explains known mutations, and provides the basis for unravelling a central mechanism of gene regulation.

The spliceosome catalyses the excision of introns from pre-mRNA in two steps, branching and exon ligation, and is assembled from five small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4, U5, U6) and numerous non-snRNP factors1. For branching, the intron 5′ splice site and the branch point sequence are selected and brought by the U1 and U2 snRNPs into the prespliceosome1, which is a focal point for regulation by alternative splicing factors2. The U4/U6.U5 tri-snRNP subsequently joins the prespliceosome to form the complete pre-catalytic spliceosome. Recent studies have revealed the structural basis of the branching and exon-ligation reactions3, however, the structural basis of the early events in spliceosome assembly remains poorly understood4. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae prespliceosome at near-atomic resolution. The structure reveals an induced stabilization of the 5′ splice site in the U1 snRNP, and provides structural insights into the functions of the human alternative splicing factors LUC7-like (yeast Luc7) and TIA-1 (yeast Nam8), both of which have been linked to human disease5,6. In the prespliceosome, the U1 snRNP associates with the U2 snRNP through a stable contact with the U2 3′ domain and a transient yeast-specific contact with the U2 SF3b-containing 5′ region, leaving its tri-snRNP-binding interface fully exposed. The results suggest mechanisms for 5′ splice site transfer to the U6 ACAGAGA region within the assembled spliceosome and for its subsequent conversion to the activation-competent B-complex spliceosome7,8. Taken together, the data provide a working model to investigate the early steps of spliceosome assembly. The cryo-electron microscopy structure of the Saccharomyces cerevisiae prespliceosome provides insights into splice-site selection and early spliceosome assembly events.