Structure of an Intron Lariat Spliceosome from Saccharomyces cerevisiae.

The disassembly of the intron lariat spliceosome (ILS) marks the end of a splicing cycle. Here we report a cryoelectron microscopy structure of the ILS complex from Saccharomyces cerevisiae at an average resolution of 3.5 Å. The intron lariat remains bound in the spliceosome whereas the ligated exon is already dissociated. The step II splicing factors Prp17 and Prp18, along with Cwc21 and Cwc22 that stabilize the 5' exon binding to loop I of U5 small nuclear RNA (snRNA), have been released from the active site assembly. The DEAH family ATPase/helicase Prp43 binds Syf1 at the periphery of the spliceosome, with its RNA-binding site close to the 3' end of U6 snRNA. The C-terminal domain of Ntr1/Spp382 associates with the GTPase Snu114, and Ntr2 is anchored to Prp8 while interacting with the superhelical domain of Ntr1. These structural features suggest a plausible mechanism for the disassembly of the ILS complex.

Rearrangements within the U6 snRNA Core during the Transition between the Two Catalytic Steps of Splicing.

The RNA catalytic core of spliceosomes as visualized by cryoelectron microscopy (cryo-EM) remains unchanged at different stages of splicing. However, we demonstrate that mutations within the core of yeast U6 snRNA modulate conformational changes between the two catalytic steps. We propose that the intramolecular stem-loop (ISL) of U6 exists in two competing states, changing between a default, non-catalytic conformation and a transient, catalytic conformation. Whereas stable interactions in the catalytic triplex promote catalysis and their disruptions favor exit from the catalytic conformation, destabilization of the lower ISL stem promotes catalysis and its stabilization supports exit from the catalytic conformation. Thus, in addition to the catalytic triplex, U6-ISL acts as an important dynamic component of the catalytic center. The relative flexibility of the lower U6-ISL stem is conserved across eukaryotes. Similar features are found in U6atac and domain V of group II introns, arguing for the generality of the proposed mechanism.

Intracellular Bacterial Pathogens Trigger the Formation of U Small Nuclear RNA Bodies (U Bodies) through Metabolic Stress Induction.

Invasive bacterial pathogens induce an amino acid starvation (AAS) response in infected host cells that controls host defense in part by promoting autophagy. However, whether AAS has additional significant effects on the host response to intracellular bacteria remains poorly characterized. Here we showed that Shigella, Salmonella, and Listeria interfere with spliceosomal U snRNA maturation in the cytosol. Bacterial infection resulted in the rerouting of U snRNAs and their cytoplasmic escort, the survival motor neuron (SMN) complex, to processing bodies, thus forming U snRNA bodies (U bodies). This process likely contributes to the decline in the cytosolic levels of U snRNAs and of the SMN complex proteins SMN and DDX20 that we observed in infected cells. U body formation was triggered by membrane damage in infected cells and was associated with the induction of metabolic stresses, such as AAS or endoplasmic reticulum stress. Mechanistically, targeting of U snRNAs to U bodies was regulated by translation initiation inhibition and the ATF4/ATF3 pathway, and U bodies rapidly disappeared upon removal of the stress, suggesting that their accumulation represented an adaptive response to metabolic stress. Importantly, this process likely contributed to shape the host response to invasive bacteria because down-regulation of DDX20 expression using short hairpin RNA (shRNA) amplified ATF3- and NF-κB-dependent signaling. Together, these results identify a critical role for metabolic stress and invasive bacterial pathogens in U body formation and suggest that this process contributes to host defense.

Electron microscopy of U4/U6 snRNP reveals a Y-shaped U4 and U6 RNA containing domain protruding from the U4 core RNP.

We describe the electron microscopic investigation of purified U4/U6 snRNPs from human and murine cells. The U4/U6 snRNP exhibits two morphological features, a main body approximately 8 nm in diameter and a peripheral filamentous domain, 7-10 nm long. Two lines of evidence suggest that the peripheral domain may consist of RNA and to contain U6 RNA as well as the 5' portion of U4 RNA. (a) Separation of the U4/U6 snRNA interaction regions from the core domains by site-directed cleavage of the U4 snRNA with RNase H gave filament-free, globular core snRNP structures. (b) By immuno and DNA-hybridization EM, both the 5' end of U4 and the 3' end of U6 snRNA were located at the distal region of the filamentous domain, furthest from the core. These results, together with our observation that the filamentous U4/U6 domain is often Y shaped, correlate strikingly with the consensus secondary structure proposed by Brow and Guthrie (1988. Nature (Lond.), 334:213-218), where U4 and U6 snRNA are base paired in such a way that two U4/U6 helices together with a stem/loop of U4 snRNA make up a Y-shaped U4/U6 interaction domain.

Assembly of the 68- and 72-kD proteins of signal recognition particle with 7S RNA.

Signal recognition particle (SRP), the cytoplasmic ribonucleoprotein particle that mediates the targeting of proteins to the ER, consists of a 7S RNA and six different proteins. The 68- (SRP68) and 72- (SRP72) kD proteins of SRP are bound to the 7S RNA of SRP as a heterodimeric complex (SRP68/72). Here we describe the primary structure of SRP72 and the assembly of SRP68, SRP72 and 7S RNA into a ribonucleoprotein particle. The amino acid sequence deduced from the cDNA of SRP72 reveals a basic protein of 671 amino acids which shares no sequence similarity with any protein in the sequence data libraries. Assembly of SRP72 into a ribonucleoprotein particle required the presence of 7S RNA and SRP68. In contrast, SRP68 alone specifically bound to 7S RNA. SRP68 contacts the 7S RNA via its NH2-terminal half while COOH-terminal portions of SRP68 and SRP72 are in contact with each other in SRP. SRP68 thus serves as a link between 7S RNA and SRP72. As a large NH2-terminal domain of SRP72 is exposed on SRP it may be a site of contact to other molecules involved in the SRP cycle between the ribosome and the ER membrane.

Capping of mammalian U6 small nuclear RNA in vitro is directed by a conserved stem-loop and AUAUAC sequence: conversion of a noncapped RNA into a capped RNA.

The cap structure of U6 small nuclear RNA (snRNA) is gamma-monomethyl phosphate and is distinct from other known RNA cap structures (R. Singh and R. Reddy, Proc. Natl. Acad. Sci. USA 86:8280-8283, 1989). Here we show that the information for capping the U6 snRNA in vitro is within the initial 25 nucleotides of the U6 RNA. The capping determinant in mammalian U6 snRNA is a bipartite element--a phylogenetically conserved stem-loop structure and an AUAUAC sequence, or a part thereof, following this stem-loop. Wild-type capping efficiency was obtained when the AUAUAC motif immediately followed the stem-loop and when the gamma-phosphate of the initiation nucleotide was in close proximity to the capping determinant. Incorporation of a synthetic stem-loop followed by an AUAUAC sequence is sufficient to covert a noncapped heterologous transcript into a capped transcript. Transcripts with the initial 32 nucleotides of Saccharomyces cerevisiae U6 snRNA are accurately capped in HeLa cell extract, indicating that capping machinery from HeLa cells can cap U6 snRNA from an evolutionarily distant eucaryote. The U6-snRNA-specific capping is unusual in that it is RNA sequence dependent, while the capping of mRNAs and other U snRNAs is tightly coupled to transcription and is independent of the RNA sequence.

Architecture of the U5 small nuclear RNA.

We have used comparative sequence analysis and deletion analysis to examine the secondary structure of the U5 small nuclear RNA (snRNA), an essential component of the pre-mRNA splicing apparatus. The secondary structure of Saccharomyces cerevisiae U5 snRNA was studied in detail, while sequences from six other fungal species were included in the phylogenetic analysis. Our results indicate that fungal U5 snRNAs, like their counterparts from other taxa, can be folded into a secondary structure characterized by a highly conserved stem-loop (stem-loop 1) that is flanked by a moderately conserved internal loop (internal loop 1). In addition, several of the fungal U5 snRNAs include a novel stem-loop structure (ca. 30 nucleotides) that is adjacent to stem-loop 1. By deletion analysis of the S. cerevisiae snRNA, we have demonstrated that the minimal U5 snRNA that can complement the lethal phenotype of a U5 gene disruption consists of (i) stem-loop 1, (ii) internal loop 1, (iii) a stem-closing internal loop 1, and (iv) the conserved Sm protein binding site. Remarkably, all essential, U5-specific primary sequence elements are encoded by a 39-nucleotide domain consisting of stem-loop 1 and internal loop 1. This domain must, therefore, contain all U5-specific sequences that are essential for splicing activity, including binding sites for U5-specific proteins.

The PRP4 (RNA4) protein of Saccharomyces cerevisiae is associated with the 5' portion of the U4 small nuclear RNA.

We have combined oligonucleotide-directed RNase H degradation and immunoprecipitation in a study of the association of the Saccharomyces cerevisiae PRP4 protein with the U4-U6 complex. We have found that three oligonucleotides were able to direct nearly to completion the RNase H-specific cleavage of the target RNA molecules as they exist in splicing extracts. Immunoprecipitation of the degradation products with PRP4 antibody showed that the 5' portion of U4 small nuclear RNA (snRNA) and the 3' portion of U6 snRNA coimmunoprecipitated with the PRP4 protein. Micrococcal nuclease protection experiments confirmed further that the 5' portion and 3' end of U4 snRNA were very resistant to nuclease digestion, whereas the 3' portion of U6 snRNA was protected to only a very small extent. We conclude that the PRP4 protein of S. cerevisiae is associated primarily with the 5' portion of U4 snRNA in the U4-U6 small nuclear ribonucleoprotein (snRNP).

The 3.8 Å structure of the U4/U6.U5 tri-snRNP: Insights into spliceosome assembly and catalysis.

Splicing of precursor messenger RNA is accomplished by a dynamic megacomplex known as the spliceosome. Assembly of a functional spliceosome requires a preassembled U4/U6.U5 tri-snRNP complex, which comprises the U5 small nuclear ribonucleoprotein (snRNP), the U4 and U6 small nuclear RNA (snRNA) duplex, and a number of protein factors. Here we report the three-dimensional structure of a Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at an overall resolution of 3.8 angstroms by single-particle electron cryomicroscopy. The local resolution for the core regions of the tri-snRNP reaches 3.0 to 3.5 angstroms, allowing construction of a refined atomic model. Our structure contains U5 snRNA, the extensively base-paired U4/U6 snRNA, and 30 proteins including Prp8 and Snu114, which amount to 8495 amino acids and 263 nucleotides with a combined molecular mass of ~1 megadalton. The catalytic nucleotide U80 from U6 snRNA exists in an inactive conformation, stabilized by its base-pairing interactions with U4 snRNA and protected by Prp3. Pre-messenger RNA is bound in the tri-snRNP through base-pairing interactions with U6 snRNA and loop I of U5 snRNA. This structure, together with that of the spliceosome, reveals the molecular choreography of the snRNAs in the activation process of the spliceosomal ribozyme.

Electron microscopy of assembly intermediates of the snRNP core: morphological similarities between the RNA-free (E.F.G) protein heteromer and the intact snRNP core.

All four spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 contain a common structural element called the snRNP core. This core is assembled from the common snRNP proteins and the small nuclear RNA (snRNA). We have used electron microscopy to study the structure of two intermediates of the snRNP core assembly pathway: (1) the (E.F.G) protein complex, which contains only the smallest common proteins E, F and G; and (2) the subscore of U5 snRNP, in which the U5 RNA and the common proteins D1 and D2 are bound to the (E.F.G) protein complex. The general structure of the subscore was found to resemble that of the complete snRNP core, which contains the components of the subscore plus the common proteins B/B' and D3. Both the complete snRNP core and subscore particles are globular, with diameters of 7 to 8 nm. They show a characteristic accumulation of stain at the centre. However, some subscore images showed nicked outlines not seen with the complete snRNP cores. The (E.F.G) protein complex appeared as a ring, with an outer diameter of about 7 nm and a central hole 2 nm across. The molecular dimensions of the E, F and G proteins imply that the thickness of the (E.F.G) ring structure is only about 2 nm. Comparison of the (E.F.G) structure complex with the snRNP core and subcore structures implicates that a flat side of the ring-shaped (E.F.G) complex provides the assembly site(s) for the other components of the snRNP during core assembly: first for the D1 and D2 proteins (and probably the snRNA) during subscore formation, and then for the B/B' and D3 proteins in the completion of the snRNP core particle.

Direct binding of small nuclear ribonucleoprotein G to the Sm site of small nuclear RNA. Ultraviolet light cross-linking of protein G to the AAU stretch within the Sm site (AAUUUGUGG) of U1 small nuclear ribonucleoprotein reconstituted in vitro.

The major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 participate in the splicing of pre-mRNA. U1, U2, U4 and U5 RNAs share a highly conserved sequence motif PuA(U)nGPu, termed the Sm site, which is normally flanked by two hairpin loops. The Sm site provides the major binding site for the group of common proteins, B', B, D1, D2, D3, E, F and G, which are shared by the spliceosomal snRNPs. We have investigated the ability of common snRNP proteins to recognize the Sm site of snRNA by using ultraviolet light-induced RNA-protein cross-linking within U1 snRNP particles. The U1 snRNP particles, reconstituted in vitro, contained U1 snRNA labelled with 32P. Cross-linking of protein to this U1 snRNA occurred only in the presence of the single-stranded stretch of snRNA that makes up the conserved Sm site. Characterization of the cross-linked protein by one and two-dimensional gel electrophoresis indicated that snRNP protein G had become cross-linked to the U1 snRNA. This was confirmed by specific immunoprecipitation of the cross-linked RNA-protein complex with an anti-G antiserum. The cross-link was located on the U1 snRNA by fingerprint analysis with RNases T1 and A; this demonstrated that the protein G has been cross-linked to the AAU stretch within the 5'-terminal half of the Sm site (AAUUUGUGG). These results suggest that the snRNP protein G may be involved in the direct recognition of the Sm site.

Intranuclear distribution of U1 and U2 snRNAs visualized by high resolution in situ hybridization: revelation of a novel compartment containing U1 but not U2 snRNA in HeLa cells.

We have examined the intranuclear distribution of U1 and U2 small nuclear RNAs (snRNAs) in HeLa cells by electron microscope in situ hybridization using biotinylated DNA probes reacting at the surface of thin sections of Lowicryl-embedded cells. U1 and U2 snRNAs colocalized on perichromatin fibrils, clusters of interchromatin granules and coiled bodies. The perichromatin granules were just occasionally labeled. In addition, we identified a novel nuclear domain associated with the clusters of interchromatin granules which contains U1 but not U2 snRNA. This new compartment termed "interchromatin granule-associated zone" has a fibrillar texture, does not contain DNA and might be the equivalent of the A snurposomes described in germinal vesicles of amphibians (Wu et al., J. Cell Biol. 113, 465-483 (1991)). We propose that the interchromatin granule-associated zones might be sites of the final maturation of the U1-pre-snRNP particle before its transfer to interchromatin granules and its subsequent assembly in the spliceosome.

Genes encoding the 7S RNA and tRNA(Ser) are linked to one of the two rRNA operons in the genome of the extremely thermophilic archaebacterium Methanothermus fervidus.

Analysis of gene structure in the extremely thermophilic archaebacterium, Methanothermus fervidus, has revealed the presence of a cluster of stable RNA-encoding genes arranged 5'-7S RNA-tRNA(Ser)-16S rRNA-tRNA(Ala)-23S rRNA-5S rRNA. The genome of M. fervidus contains two rRNA operons but only one operon has the closely linked 7S RNA-encoding gene. The sequences upstream from the two rRNA operons are identical for 206 bp but diverge at the 3' base of the tRNA(Ser) gene. The secondary structures predicted for the M. fervidus 7S, 16S rRNA, tRNA(Ala) and tRNA(Ser) have been compared with those of functionally homologous molecules from moderately thermophilic and mesophilic archaebacteria. A consensus secondary structure for archaebacterial 7S RNAs has been developed which incorporates bases and structural features also conserved in eukaryotic signal-recognition-particle RNAs and eubacterial 4.5S RNAs.

Complementation of U4 snRNA in S. cerevisiae splicing extracts for biochemical studies of snRNP assembly and function.

Pre-messenger RNA splicing is a surprisingly complex and dynamic process, the details of which remain largely unknown. One important method for studying splicing involves the replacement of endogenous splicing components with their synthetic counterparts. This enables changes in protein or nucleic acid sequence to be tested for functional effects, as well as the introduction of chemical moieties such as cross-linking groups and fluorescent dyes. To introduce the modified component, the endogenous one must be removed and a method found to reconstitute the active splicing machinery. In extracts prepared from S. cerevisiae, reconstitution has been accomplished with the small, nuclear RNAs U6, U2, and U5.We describe a comparable method to reconstitute active U4 small, nuclear RNA (snRNA) into a splicing extract. In order to remove the endogenous U4 it is necessary to target it for oligo-directed RNase H degradation while active splicing is under way, i.e., in the presence of a splicing transcript and ATP. This allows complete degradation of endogenous U4 and subsequent replacement with an exogenous version. In contrast to the procedures described for depletion of U6, U2, or U5 snRNAs, depletion of U4 requires concurrent active splicing. The ability to reconstitute U4 in yeast extract allows a variety of structural and functional studies to be carried out.

Cryo-electron microscopy of spliceosomal components.

Splicing is an essential step of gene expression in which introns are removed from pre-mRNA to generate mature mRNA that can be translated by the ribosome. This reaction is catalyzed by a large and dynamic macromolecular RNP complex called the spliceosome. The spliceosome is formed by the stepwise integration of five snRNPs composed of U1, U2, U4, U5, and U6 snRNAs and more than 150 proteins binding sequentially to pre-mRNA. To study the structure of this particularly dynamic RNP machine that undergoes many changes in composition and conformation, single-particle cryo-electron microscopy (cryo-EM) is currently the method of choice. In this review, we present the results of these cryo-EM studies along with some new perspectives on structural and functional aspects of splicing, and we outline the perspectives and limitations of the cryo-EM technique in obtaining structural information about macromolecular complexes, such as the spliceosome, involved in splicing.

Electron microscopy of small nuclear ribonucleoprotein (snRNP) particles U2 and U5: evidence for a common structure-determining principle in the major U snRNP family.

We have studied by electron microscopy the structures of native small nuclear ribonucleoprotein (snRNP) particles U2 and U5 from HeLa cells. The structure of native U2 snRNP is characterized by a main body 8 nm in diameter with one additional domain about 4 nm long and 6 nm wide. Electron micrographs show that the 20S U5 snRNP, which contains at least seven U5-specific proteins in addition to the common proteins, has an elongated structure measuring 20-23 nm in length and 11-14 nm in width. Two main structural domains can be distinguished: a small head and a large elongated body about twice the size of the head. In addition to the head, the body of the 20S U5 snRNP possesses three short protuberances. The U2 and U5 core RNP particles--that is, of the snRNPs U2 and U5 without the snRNP-specific proteins, look much simpler and smaller under the electron microscope. They both are round in shape with a diameter of approximately 8 nm. With respect to their size, appearance, and fine structure, the U2 and U5 snRNP cores not only closely resemble each other but also share these properties with the core domain of U1 snRNP. We propose that the characteristic shape of each of the major snRNP species U1, U2, U4/U6, and U5 is determined by (i) a core domain containing the proteins that are common to all members of this family, which has the same shape for each member, and (ii) peripheral structures, which for snRNPs U1, U2, and U5 arise from the specific proteins, that give each of these snRNP species its characteristic shape.

Electron microscopy of U1 small nuclear ribonucleoprotein particles: shape of the particle and position of the 5' RNA terminus.

Small nuclear ribonucleoprotein particles U1 (U1 snRNPs) from HeLa cells were purified to homogeneity and examined in the electron microscope by negative staining with the double carbon film technique. A survey of several hundred views of the U1 snRNP particles revealed a main body about 8 nm in diameter with two characteristic protuberances 4-7 nm long and 3-4 nm wide. Anti-m3G antibodies were then used to define the site of the m3G base in the 5' cap of the U1 snRNA, close to the site where base-pairing occurs with the 5' splice sequence of pre-mRNA. The m3G base is located on the body of the U1 snRNP, close to one of the protuberances and approximately opposite the other.

Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing.

SV40 early pre-mRNA is alternatively spliced to produce large T and small t mRNAs by use of different 5'-splice sites and a shared 3'-splice site. The large T splicing pathway uses multiple lariat branch sites, whereas small t splicing, constrained by its small intron size, can use only one. We exploited this situation to test the hypothesis that RNA-RNA base pairing between U2 snRNA and the branch site sequence is important in mammalian pre-mRNA splicing by constructing and analyzing several mutations in the small t pre-mRNA branch site (UUCUAAU). All of the mutations resulted in substantial decreases in small t splicing relative to large T. To test whether these effects resulted from decreased base pairing with U2 snRNA, compensatory mutations were introduced at the appropriate positions (nucleotides 34-36) in a cloned human U2 gene. All branch site mutations tested (four separate single base substitutions and two triple mutations) were suppressed (i.e., small t splicing was increased) by the appropriate U2 mutations. These results establish that recognition of the poorly conserved mammalian pre-mRNA branch site sequence by U2 snRNP can involve base-pairing.

Alu RNA secondary structure consists of two independent 7 SL RNA-like folding units.

The amplification of genomic Alu elements by retroposition, i.e. by reintegration of reverse-transcribed RNA, suggests that Alu RNA plays an important role in this process. We report enzymatic studies of the secondary structure of Alu RNAs transcribed in vitro from two recently retroposed Alu elements. These experiments show that the dimeric organization of an Alu sequence is reflected in its RNA folding. Alu subunits fold independently, conserving secondary structure motifs of their progenitor 7 SL RNA molecule. Energy minimization analysis indicates that this folding pattern is also characteristic of different Alu and Alu-like sequences and has been conserved since primate divergence. By analogy to 7 SL RNA, the Alu RNA folding may be important for specific interactions with proteins. This could indicate a physiological function for Alu transcripts. However, this can be also seen as a structural adaptation leading to efficient retroposition of these sequence elements.

RNA polymerase II transcription and the functional organization of the mammalian cell nucleus.

The study of RNA pol II-mediated transcription regulation has been dominated by molecular biological approaches. Although these methods continue to provide important insights, other approaches are required to insure against an oversimplified view of gene expression. Improvements in EM methods and the development of the confocal light microscope have provided alternative and complementary means of investigating gene regulation. Information on the "context" in which cis- and trans-acting factors operate can be achieved with these techniques. As a result, the spatial compartmentalization of nuclear processes involved in transcriptional and post-transcriptional processing has received considerable attention.