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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 Å and 3.6 Å, 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., For transcription initiation, Pol II assembles with the basal transcription factors (TF) IIB, TFIID (or its subunit TBP), TFIIE, TFIIF, and TFIIH (1-4) on double-stranded promoter DNA to form the [...]

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 A 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., Transcription initiation at eukaryotic protein-coding genes requires RNA polymerase (Pol) II and the general transcription factors TFIIB, -D, -E, -F, and -H. In the canonical view of initiation (1,2), promoter [...]

Precursor-mRNA (pre-mRNA) splicing requires the assembly, remodelling and disassembly of the multi-megadalton ribonucleoprotein complex called the spliceosome 1 . Recent studies have shed light on spliceosome assembly and remodelling for catalysis 2-6 , but the mechanism of disassembly remains unclear. Here we report cryo-electron microscopy structures of nematode and human terminal intron lariat spliceosomes along with biochemical and genetic data. Our results uncover how four disassembly factors and the conserved RNA helicase DHX15 initiate spliceosome disassembly. The disassembly factors probe large inner and outer spliceosome surfaces to detect the release of ligated mRNA. Two of these factors, TFIP11 and C19L1, and three general spliceosome subunits, SYF1, SYF2 and SDE2, then dock and activate DHX15 on the catalytic U6 snRNA to initiate disassembly. U6 therefore controls both the start 5 and end of pre-mRNA splicing. Taken together, our results explain the molecular basis of the initiation of canonical spliceosome disassembly and provide a framework to understand general spliceosomal RNA helicase control and the discard of aberrant spliceosomes., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Pre-mRNA splicing by the spliceosome requires the biogenesis and recycling of its small nuclear ribonucleoprotein (snRNP) complexes, which are consumed in each round of splicing. The human U5 snRNP is the ~1 MDa 'heart' of the spliceosome and is recycled through an unknown mechanism involving major architectural rearrangements and the dedicated chaperones CD2BP2 and TSSC4. Late steps in U5 snRNP biogenesis similarly involve these chaperones. Here we report cryo-electron microscopy structures of four human U5 snRNP-CD2BP2-TSSC4 complexes, revealing how a series of molecular events primes the U5 snRNP to generate the ~2 MDa U4/U6.U5 tri-snRNP, the largest building block of the spliceosome., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Newly made mRNAs are processed and packaged into mature ribonucleoprotein complexes (mRNPs) and are recognized by the essential transcription-export complex (TREX) for nuclear export 1,2 . However, the mechanisms of mRNP recognition and three-dimensional mRNP organization are poorly understood 3 . Here we report cryo-electron microscopy and tomography structures of reconstituted and endogenous human mRNPs bound to the 2-MDa TREX complex. We show that mRNPs are recognized through multivalent interactions between the TREX subunit ALYREF and mRNP-bound exon junction complexes. Exon junction complexes can multimerize through ALYREF, which suggests a mechanism for mRNP organization. Endogenous mRNPs form compact globules that are coated by multiple TREX complexes. These results reveal how TREX may simultaneously recognize, compact and protect mRNAs to promote their packaging for nuclear export. The organization of mRNP globules provides a framework to understand how mRNP architecture facilitates mRNA biogenesis and export., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

In eukaryotes, the expression of genetic information begins in the cell nucleus with precursor messenger RNA (pre-mRNA) transcription and processing into mature mRNA. The mRNA is subsequently recognized and packaged by proteins into an mRNA ribonucleoprotein complex (mRNP) and exported to the cytoplasm for translation. Each of the nuclear mRNA maturation steps is carried out by a dedicated molecular machine. Here, we highlight recent structural and mechanistic insights into how these machines function, including the capping enzyme, the spliceosome, the 3'-end processing machinery, and the transcription-export complex. While we increasingly understand individual steps of nuclear gene expression, many questions remain. For example, we are only beginning to reveal how mature mRNAs are recognized and packaged for nuclear export and how mRNA maturation events are coupled to transcription and to each other. Advances in the preparation of recombinant and endogenous protein-nucleic acid complexes, cryo-electron microscopy, and machine learning promise exciting insights into the mechanisms of nuclear gene expression and its spatial organization., Competing Interests: Conflict of interest None declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (© 2021 Schnabl et al.; Published by Cold Spring Harbor Laboratory Press.)

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., Competing Interests: TP, UH, LF, BP, US, JB, CP No competing interests declared, (© 2020, Pühringer et al.)

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., (Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved.)

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 factors 1 . For branching, the intron 5' splice site and the branch point sequence are selected and brought by the U1 and U2 snRNPs into the prespliceosome 1 , which is a focal point for regulation by alternative splicing factors 2 . 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 reactions 3 , however, the structural basis of the early events in spliceosome assembly remains poorly understood 4 . 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 disease 5,6 . 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 spliceosome 7,8 . Taken together, the data provide a working model to investigate the early steps of spliceosome assembly.

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.

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.

Integrated structural biology recently elucidated the architecture of Mediator and its position on RNA polymerase II. Here we summarize these achievements and list open questions on Mediator structure and mechanism., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

Gene transcription by RNA polymerase (Pol) II requires the coactivator complex Mediator. Mediator connects transcriptional regulators and Pol II, and is linked to human disease. 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 modules. The head module constitutes one-half of the essential Mediator core, and comprises the conserved subunits Med6, Med8, Med11, Med17, Med18, Med20 and Med22. Recent X-ray analysis of the S. cerevisiae head module at 4.3 Å resolution led to a partial architectural model with three submodules called neck, fixed jaw and moveable jaw. Here we determine de novo the crystal structure of the head module from the fission yeast Schizosaccharomyces pombe at 3.4 Å resolution. Structure solution was enabled by new structures of Med6 and the fixed jaw, and previous structures of the moveable jaw and part of the neck, 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.