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RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs) 1 , 2 . However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3′ overhang and a terminal loop 3 – 11 . Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER (also known as DICER1). Our analyses revealed a deeply conserved cis -acting element, termed the ‘GYM motif’ (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified ‘ruler’-like counting mechanisms from the 5′ and 3′ ends of pre-miRNA 3 – 6 . Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics. Massively parallel assays reveal a highly conserved sequence motif termed the GYM motif, which potentiates RNA interference by directing Dicer-mediated small RNA processing.

During pre-mRNA splicing, exons in the primary transcript are precisely connected to generate an mRNA. Intron lariat RNAs are formed as by-products of this process. In addition, some exonic circular RNAs (circRNAs) may also result from exon skipping as by-products. Lariat RNAs and circRNAs are both RNase R resistant RNAs. RNase R is a strong 3' to 5' exoribonuclease, which efficiently degrades linear RNAs, such as mRNAs and rRNAs; therefore, the circular parts of lariat RNAs and the circRNAs can be segregated from eukaryotic total RNAs by their RNase R resistance. Thus, RNase R resistant RNAs could provide unexplored splicing information not available from mRNAs. Analyses of these RNAs identified repeating splicing phenomena, such as re-splicing of mature mRNAs and nested splicing. Moreover, circRNA might function as microRNA sponges. There is an enormous variety of endogenous circRNAs, which are generally synthesized in cells and tissues.

The CRISPR-associated protein Cpf1 from Francisella novicida is a novel enzyme with specific, dual-endoribonuclease–endonuclease activities in precursor crRNA processing and crRNA-programmable cleavage of target DNA. Cpf1 enzyme in CRISPR immunity The bacterial immune system, CRISPR, utilizes a small RNA guide, or crRNA, to target a nucleolytic CRISPR complex to DNA with a complementary sequence. This process has been widely exploited for various types of genome engineering. Previously described CRISPR systems utilize one nuclease, such as Cas6, to generate the mature crRNA, and a second, such as Cas9, to cleave the target DNA. Two studies illustrate a different approach that involves the Cpf1 protein. Emmanuelle Charpentier and colleagues report that type V-A Cpf1 protein from Francisella novicida functions as a minimalistic CRISPR system. It is a dual-nuclease enzyme that can perform both the pre-crRNA processing and DNA cleavage activities, having distinct active domains for the two substrates. Zhiwei Huang and colleagues solve the crystal structure of monomeric Lachnospiraceae bacterium Cpf1 protein bound to crRNA, showing how binding induces conformational changes in the nuclease. CRISPR–Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA 1 . The well-studied types I, II and III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA) 2 , 3 , 4 , 5 and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA 6 , 7 , 8 , 9 , 10 , 11 , 12 . In type II systems, RNase III cleaves pre-crRNA base-paired with trans -activating crRNA (tracrRNA) in the presence of Cas9 (refs 13 , 14 ). The mature tracrRNA–crRNA duplex then guides Cas9 to cleave target DNA 15 . Here, we demonstrate a novel mechanism in CRISPR–Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5′-YTN-3′ protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5′ overhang 16 . The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR–Cas systems so far described.

The contribution of lncRNAs to tumour progression and the regulatory mechanisms driving their expression are areas of intense investigation. Here, we characterize the binding of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) to a nucleic acid structural element located in exon 12 of PNUTS (also known as PPP1R10) pre-RNA that regulates its alternative splicing. HnRNP E1 release from this structural element, following its silencing, nucleocytoplasmic translocation or in response to TGFβ, allows alternative splicing and generates a non-coding isoform of PNUTS . Functionally the lncRNA- PNUTS serves as a competitive sponge for miR-205 during epithelial–mesenchymal transition (EMT). In mesenchymal breast tumour cells and in breast tumour samples, the expression of lncRNA- PNUTS is elevated and correlates with levels of ZEB mRNAs. Thus, PNUTS is a bifunctional RNA encoding both PNUTS mRNA and lncRNA- PNUTS , each eliciting distinct biological functions. While PNUTS mRNA is ubiquitously expressed, lncRNA- PNUTS appears to be tightly regulated dependent on the status of hnRNP E1 and tumour context. Grelet et al. find that hnRNP E1 release from PNUTS pre-RNA in response to TGFβ generates a lncRNA that acts as competitive sponge for miR-205, promoting epithelial–mesenchymal transition in cancer.

Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs) 1 , 2 . Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs—unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified ‘GYM motif’ 3 . The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5′ end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5′ terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5′ pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases. The active-state structure of human DICER bound to pre-miRNA reveals the structural basis for the specificity of DICER in how it selects substrates in a sequence dependent manner, and sheds light on DICER-related diseases.

Key Points Analysis of the recent atomic-resolution cryo-electron microscopy structures of the spliceosome reveals that during splicing all spliceosomal complexes share a rigid core of 20 RNAs and proteins. The active site of the spliceosome is positioned in the catalytic cavity of pre-mRNA-splicing factor 8 (Prp8), is stabilized by surrounding proteins and remains static throughout the two steps of transesterification. The catalytic metals in the active site are coordinated by U6 small nuclear RNA and the catalytic triplex. The interconversion of the various spliceosomal complexes is driven by eight conserved, RNA-dependent ATPase/helicases. The spliceosome is a protein-directed metalloribozyme. Atomic-resolution structures have recently been obtained for the intact spliceosome at different stages of the splicing cycle. These structural data have proved that the spliceosome is a protein-directed metalloribozyme and have increased our understanding of pre-mRNA splicing mechanisms, explaining a large body of existing genetic and biochemical data. Precursor messenger RNA (pre-mRNA) splicing is an essential step in the flow of information from DNA to protein in all eukaryotes. Research over the past four decades has molecularly delineated the splicing pathway, including characterization of the detailed splicing reaction, definition of the spliceosome and identification of its components, and biochemical analysis of the various splicing complexes and their regulation. Structural information is central to mechanistic understanding of pre-mRNA splicing by the spliceosome. X-ray crystallography of the spliceosomal components and subcomplexes is complemented by electron microscopy of the intact spliceosome. In this Review, I discuss recent atomic-resolution structures of the intact spliceosome at different stages of the splicing cycle. These structures have provided considerable mechanistic insight into pre-mRNA splicing and have corroborated and explained a large body of genetic and biochemical data. Together, the structural data have proved that the spliceosome is a protein-directed metalloribozyme.

Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes.

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.

Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat–intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5′-splice site is cleaved but remains close to the catalytic Mg 2+ site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5′-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2′OH. The 5′-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson–Crick interactions between the branch helix and 5′-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAG AGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation. Cryo-EM reveals the configuration of substrate pre-mRNA within the active spliceosome and suggests how remodelling occurs prior to exon ligation. Structure of the branched splicing complex The excision of introns from RNA is not a concerted process, but is rather an ordered one involving two transesterification reactions by the spliceosome. In the first step, the 5′-splice site is cleaved and the intron end is joined to make a lariat structure. Kiyoshi Nagai and colleagues have captured the Saccharomyces cerevisiae spliceosome stalled immediately after this first transesterification (branching) reaction by cryo-electron microscopy single-particle reconstruction at an overall resolution of 3.8 Å. The configuration of the RNA within the complex suggests that remodelling occurs before the second step, exon ligation.

During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex—a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1 – 4 ). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2–BS helix), which is proofread by Prp5 at this stage through an unclear mechanism 5 . Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2–BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155 HEAT ), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155 HEAT to the bulged BS-A of the U2–BS helix triggers closure of Hsh155 HEAT , which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155 HEAT . Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing. The cryo-electron microscopy structure of a newly identified, early spliceosomal complex reveals the mechanism by which the RNA helicase Prp5 enhances the fidelity of the excision of introns from precursor mRNAs.