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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 cryo-electron microscopy structure of the yeast spliceosome in a pre-catalytic state provides insights into the molecular events leading to formation of the spliceosome active site. Visualization of a poised spliceosome Protein-coding regions of DNA can be interrupted by non-coding regions, or introns. A large multisubunit complex, the spliceosome, is used to excise introns from the messenger RNA before it is translated into protein. Formation of an active spliceosome complex on an intron requires stepwise assembly of subcomplexes, followed by their rearrangement and the loss of some factors. Kiyoshi Nagai and colleagues have solved the structure of the B complex spliceosome, poised in a pre-catalytic state. The detection of several factors that were not visualized in previous spliceosome structures provides new insights regarding the process by which the complex is activated.

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 cryo-electron microscopy structure of the Saccharomyces cerevisiae prespliceosome provides insights into splice-site selection and early spliceosome assembly events.

The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5ʹ splice site (5ʹSS) and branch point sequence, associates with the U4/U6.U5 tri-snRNP to form the fully assembled precatalytic pre–B spliceosome. Here, we report cryo–electron microscopy structures of the human pre–B complex captured before U1 snRNP dissociation at 3.3-angstrom core resolution and the human tri-snRNP at 2.9-angstrom resolution. U1 snRNP inserts the 5ʹSS–U1 snRNA helix between the two RecA domains of the Prp28 DEAD-box helicase. Adenosine 5ʹ-triphosphate–dependent closure of the Prp28 RecA domains releases the 5ʹSS to pair with the nearby U6 ACAGAGA-box sequence presented as a mobile loop. The structures suggest that formation of the 5ʹSS-ACAGAGA helix triggers remodeling of an intricate protein-RNA network to induce Brr2 helicase relocation to its loading sequence in U4 snRNA, enabling Brr2 to unwind the U4/U6 snRNA duplex to allow U6 snRNA to form the catalytic center of the spliceosome.

U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Å resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5′-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre. A 3.7 Å resolution structure for the yeast U4/U6.U5 tri-snRNP, a complex involved in splicing, allows a better appreciation of the architecture of the tri-snRNP, and offers new functional insights into the activation of the spliceosome and the assembly of the catalytic core. Yeast U4/U6.U5 tri-snRNP structure Following up on their 5.9 Å cryo-electron microscopy structure published less than a year ago, Kiyoshi Nagai and colleagues have now achieved a resolution of 3.7 Å for the yeast U4/U6.U5 tri-snRNP, a complex involved in splicing of messenger RNA. The improved resolution allows a better appreciation of the architecture of the tri-snRNP, and offers new functional insights into the activation of the spliceosome and the assembly of the catalytic core.

In mammals, the structural basis for the interaction between U1 and U2 small nuclear ribonucleoproteins (snRNPs) during the early steps of splicing is still elusive. The binding of the ubiquitin-like (UBL) domain of SF3A1 to the stem-loop 4 of U1 snRNP (U1-SL4) contributes to this interaction. Here, we determined the 3D structure of the complex between the UBL of SF3A1 and U1-SL4 RNA. Our crystallography, NMR spectroscopy, and cross-linking mass spectrometry data show that SF3A1-UBL recognizes, sequence specifically, the GCG/CGC RNA stem and the apical UUCG tetraloop of U1-SL4. In vitro and in vivo mutational analyses support the observed intermolecular contacts and demonstrate that the carboxyl-terminal arginine-glycine-glycine-arginine (RGGR) motif of SF3A1-UBL binds sequence specifically by inserting into the RNA major groove. Thus, the characterization of the SF3A1-UBL/U1-SL4 complex expands the repertoire of RNA binding domains and reveals the capacity of RGG/RG motifs to bind RNA in a sequence-specific manner.

Many recent advances have emerged in the telomere and telomerase fields. This Timeline article highlights the key advances that have expanded our views on the mechanistic underpinnings of telomeres and telomerase and their roles in ageing and disease. Three decades ago, the classic view was that telomeres protected the natural ends of linear chromosomes and that telomerase was a specific telomere-terminal transferase necessary for the replication of chromosome ends in single-celled organisms. While this concept is still correct, many diverse fields associated with telomeres and telomerase have substantially matured. These areas include the discovery of most of the key molecular components of telomerase, implications for limits to cellular replication, identification and characterization of human genetic disorders that result in premature telomere shortening, the concept that inhibiting telomerase might be a successful therapeutic strategy and roles for telomeres in regulating gene expression. We discuss progress in these areas and conclude with challenges and unanswered questions in the field.In this Timeline article, Shay and Wright provide a historical account of progress in our understanding of telomeres (the ends of linear chromosomes) and telomerase (the primary enzyme that maintains and extends telomere lengths). Their perspective covers seminal moments from the early discoveries through to our latest understanding of the roles of telomeres and telomerase in ageing, diverse human diseases and gene regulation.

The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing 1 . Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP5 2 – 7 . Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem–loop (BSL) 8 , but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers 9 , contains a HEAT domain (SF3B1 HEAT ) with an open conformation in isolated SF3b 10 , but a closed conformation in spliceosomes 11 , which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure of the human 17S U2 snRNP at a core resolution of 4.1 Å and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1 HEAT interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1 HEAT . Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2–branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site. The cryo-EM structure of human U2 small nuclear ribonucleoprotein (snRNP) offers insights into what rearrangements are required for this snRNP to be stably incorporated into the spliceosome, and the role that the DEAD-box ATPase PRP5 may have in these rearrangements.

Ki-67 protein has been widely used as a proliferation marker for human tumor cells for decades. In recent studies, multiple molecular functions of this large protein have become better understood. Ki-67 has roles in both interphase and mitotic cells, and its cellular distribution dramatically changes during cell cycle progression. These localizations correlate with distinct functions. For example, during interphase, Ki-67 is required for normal cellular distribution of heterochromatin antigens and for the nucleolar association of heterochromatin. During mitosis, Ki-67 is essential for formation of the perichromosomal layer (PCL), a ribonucleoprotein sheath coating the condensed chromosomes. In this structure, Ki-67 acts to prevent aggregation of mitotic chromosomes. Here, we present an overview of functional roles of Ki-67 across the cell cycle and also describe recent experiments that clarify its role in regulating cell cycle progression in human cells.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the most abundant and ubiquitously expressed member of the heterogeneous nuclear ribonucleoproteins family (hnRNPs). hnRNP A1 is an RNA-binding protein associated with complexes active in diverse biological processes such as RNA splicing, transactivation of gene expression, and modulation of protein translation. It is overexpressed in several cancers, where it actively promotes the expression and translation of several key proteins and regulators associated with tumorigenesis and cancer progression. Interesting recent studies have focused on the RNA-binding property of hnRNP A1 and revealed previously under-explored functions of hnRNP A1 in the processing of miRNAs, and loading non-coding RNAs into exosomes. Here, we will report the recent advancements in our knowledge of the role of hnRNP A1 in the biological processes underlying cancer proliferation and growth, with a particular focus on metabolic reprogramming.

Telomerase adds telomeric repeats at chromosome ends to compensate for the telomere loss that is caused by incomplete genome end replication 1 . In humans, telomerase is upregulated during embryogenesis and in cancers, and mutations that compromise the function of telomerase result in disease 2 . A previous structure of human telomerase at a resolution of 8 Å revealed a vertebrate-specific composition and architecture 3 , comprising a catalytic core that is flexibly tethered to an H and ACA (hereafter, H/ACA) box ribonucleoprotein (RNP) lobe by telomerase RNA. High-resolution structural information is necessary to develop treatments that can effectively modulate telomerase activity as a therapeutic approach against cancers and disease. Here we used cryo-electron microscopy to determine the structure of human telomerase holoenzyme bound to telomeric DNA at sub-4 Å resolution, which reveals crucial DNA- and RNA-binding interfaces in the active site of telomerase as well as the locations of mutations that alter telomerase activity. We identified a histone H2A–H2B dimer within the holoenzyme that was bound to an essential telomerase RNA motif, which suggests a role for histones in the folding and function of telomerase RNA. Furthermore, this structure of a eukaryotic H/ACA RNP reveals the molecular recognition of conserved RNA and protein motifs, as well as interactions that are crucial for understanding the molecular pathology of many mutations that cause disease. Our findings provide the structural details of the assembly and active site of human telomerase, which paves the way for the development of therapeutic agents that target this enzyme. A high-resolution structure of human telomerase bound to telomeric DNA reveals details of telomerase assembly and its active site, and sheds light on how mutations alter telomerase function.