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Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3′ splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA–mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3′ss. Cryo–electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3′ region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3′ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3′ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3′ss choices.

A tight couple makes messenger RNAs Gene expression in eukaryotes first requires transcription of DNA to an RNA copy and then splicing to form the final, processed messenger RNA (mRNA). Zhang et al. investigated how gene transcription and RNA splicing are physically coupled. Using cryo–electron microscopy, they resolved the molecular structure of a complex of the transcription enzyme RNA polymerase II with part of the splicing machinery, the U1 small nuclear ribonucleoprotein particle. The results provide important details for our understanding of coupled mRNA production. Science , this issue p. 305

Summary Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 A core resolution and 4.5–5.7 A at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome’s architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome’s catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the Bact to C transition. Structural comparisons with human Bact, C∗, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.

Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2–U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 A, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 A from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP. The cryo-EM structure of the splicing intermediate known as the C* complex from human. Recent years have seen substantial progress in understanding the structure of various intermediates of the splicing process. Two groups, led by Reinhard Luhrmann and Kiyoshi Nagai, now describe the cryo-electron microscopy structures (from human and yeast cells, respectively) of the splicing intermediate known as the C* complex. The notable feature observed in this complex, relative to the preceding catalytic intermediate (the C complex), is a remodelling that positions the branch-site adenosine and the branched intron out of the catalytic core, opening up space for the 3′ exon to dock in preparation for exon ligation.

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 splicing1. Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP52–7. 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 cancers9, contains a HEAT domain (SF3B1HEAT) with an open conformation in isolated SF3b10, but a closed conformation in spliceosomes11, 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 A and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1HEAT 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 SF3B1HEAT. 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.

The spliceosome is a highly dynamic macromolecular complex that precisely excises introns from pre-mRNA. Here we report the cryo-EM 3D structure of the human B

Summary Little is known about the spliceosome's structure before its extensive remodeling into a catalytically active complex. Here, we report a 3D cryo-EM structure of a pre-catalytic human spliceosomal B complex. The U2 snRNP-containing head domain is connected to the B complex main body via three main bridges. U4/U6.U5 tri-snRNP proteins, which are located in the main body, undergo significant rearrangements during tri-snRNP integration into the B complex. These include formation of a partially closed Prp8 conformation that creates, together with Dim1, a 5′ splice site (ss) binding pocket, displacement of Sad1, and rearrangement of Brr2 such that it contacts its U4/U6 substrate and is poised for the subsequent spliceosome activation step. The molecular organization of several B-specific proteins suggests that they are involved in negatively regulating Brr2, positioning the U6/5′ss helix, and stabilizing the B complex structure. Our results indicate significant differences between the early activation phase of human and yeast spliceosomes.

Small molecule inhibitors of pre-mRNA splicing are important tools for identifying new spliceosome assembly intermediates, allowing a finer dissection of spliceosome dynamics and function. Here, we identified a small molecule that inhibits human pre-mRNA splicing at an intermediate stage during conversion of pre-catalytic spliceosomal B complexes into activated Bact complexes. Characterization of the stalled complexes (designated B028) revealed that U4/U6 snRNP proteins are released during activation before the U6 Lsm and B-specific proteins, and before recruitment and/or stable incorporation of Prp19/CDC5L complex and other Bact complex proteins. The U2/U6 RNA network in B028 complexes differs from that of the Bact complex, consistent with the idea that the catalytic RNA core forms stepwise during the B to Bact transition and is likely stabilized by the Prp19/CDC5L complex and related proteins. Taken together, our data provide new insights into the RNP rearrangements and extensive exchange of proteins that occurs during spliceosome activation. DOI: http://dx.doi.org/10.7554/eLife.23533.001

The DEAH-box NTPase Prp43 and its cofactors Ntr1 and Ntr2 form the NTR complex and are required for disassembling intron-lariat spliceosomes (ILS) and defective earlier spliceosomes. However, the Prp43 binding site in the spliceosome and its target(s) are unknown. We show that Prp43 fused to Ntr1's G-patch motif (Prp43_Ntr1GP) is as efficient as the NTR in ILS disassembly, yielding identical dissociation products and recognizing its natural ILS target even in the absence of Ntr1’s C-terminal-domain (CTD) and Ntr2. Unlike the NTR, Prp43_Ntr1GP disassembles earlier spliceosomal complexes (A, B, Bact), indicating that Ntr2/Ntr1-CTD prevents NTR from disrupting properly assembled spliceosomes other than the ILS. The U2 snRNP-intron interaction is disrupted in all complexes by Prp43_Ntr1GP, and in the spliceosome contacts U2 proteins and the pre-mRNA, indicating that the U2 snRNP-intron interaction is Prp43’s major target. DOI: http://dx.doi.org/10.7554/eLife.15564.001

The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5′ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5′ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation., The assembly of the splicesome involves several distinct stages that require the sequential action of DExD/H-box RNA helicases. Here, the authors uncover a new intermediate, the pre-B complex, that accumulates in the presence of an inactive form of the DEAD-box protein Prp28.

More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinity-purified human B, B(act), and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, B(act), and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosome's compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing.

Exon definition is the predominant initial spliceosome assembly pathway in higher eukaryotes, but it remains much less well-characterized compared to the intron-defined assembly pathway. Addition in trans of an excess of 5′ss containing RNA to a splicing reaction converts a 37S exon-defined complex, formed on a single exon RNA substrate, into a 45S B-like spliceosomal complex with stably integrated U4/U6.U5 tri-snRNP. This 45S complex is compositonally and structurally highly similar to an intron-defined spliceosomal B complex. Stable tri-snRNP integration during B-like complex formation is accompanied by a major structural change as visualized by electron microscopy. The changes in structure and stability during transition from a 37S to 45S complex can be induced in affinity-purified cross-exon complexes by adding solely the 5′ss RNA oligonucleotide. This conformational change does not require the B-specific proteins, which are recruited during this stabilization process, or site-specific phosphorylation of hPrp31. Instead it is triggered by the interaction of U4/U6.U5 tri-snRNP components with the 5′ss sequence, most importantly between Prp8 and nucleotides at the exon–intron junction. These studies provide novel insights into the conversion of a cross-exon to cross-intron organized spliceosome and also shed light on the requirements for stable tri-snRNP integration during B complex formation.

A human spliceosomal subcomplex The spliceosome is an RNA and protein molecular machine that cuts out introns from messenger RNAs. Agafonov et al. used cryo-electron microscopy to determine the structure of the largest intermediate subcomplex on the assembly pathway for the human spliceosome (see the Perspective by Cate). The structure shows substantial differences from the equivalent yeast complex. It also reveals how the subcomplex must dock onto the rest of the spliceosome and hints at the structural changes the complex must go through to form the mature spliceosome. Science , this issue p. 1416 ; see also p. 1390

Recently we have reported about a novel stress response protein (pY or RaiA) associated with Escherichia coli ribosomes that inhibits translation at the aminoacyl-tRNA binding stage. Here we show that leucine misincorporation during in vitro poly(U) translation is inhibited by this protein much stronger than the incorporation of phenylalanine. The miscoding counteraction by RaiA is especially strong at the concentrations of magnesium ions close to those observed in vivo and diminishes at higher magnesium concentrations. The results obtained suggest that the anti-miscoding activity of RaiA could be the main function of the protein, rather than the inhibition of translation. The role of the protein in adaptation of cells to environmental stress is discussed.

We have recently isolated and characterized a novel protein associated with Escherichia coli ribosomes and named protein Y (pY). Here we show that the ribosomes from bacterial cells growing at a normal physiological temperature contain no pY, whereas a temperature downshift results in the appearance of the protein in ribosomes. The protein also appears in the ribosomes of those cells that reached the stationary phase of growth at a physiological temperature. Our experiments with cell-free translation systems demonstrate that the protein inhibits translation at the elongation stage by blocking the binding of aminoacyl-tRNA to the ribosomal A site. The function of the protein in adaptation of cells to environmental stress is discussed.

Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia , an ORF located upstream the phe operon of E. coli . This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed.

The hot tritium bombardment technique [Goldanskii, V. I., Kashirin, I. A., Shishkov, A. V., Baratova, L. A. & Grebenshchikov, N. I. (1988) J. Mol. Biol. 201, 567–574] has been applied to measure the exposure of proteins on the ribosomal surface. The technique is based on replacement of hydrogen by high energy tritium atoms in thin surface layer of macromolecules. Quantitation of tritium radioactivity of each protein has revealed that proteins S1, S4, S5, S7, S18, S20, and S21 of the small subunit, and proteins L7/L12, L9, L10, L11, L16, L17, L24, and L27 of the large subunit are well exposed on the surface of the Escherichia coli 70 S ribosome. Proteins S8, S10, S12, S16, S17, L14, L20, L29, L30, L31, L32, L33, and L34 have virtually no groups exposed on the ribosomal surface. The remaining proteins are found to be exposed to lesser degree than the well exposed ones. No additional ribosomal proteins was exposed upon dissociation of ribosomes into subunits, thus indicating the absence of proteins on intersubunit contacting surfaces.

Protein complexes containing Prp19 play a central role during catalytic activation of the spliceosome, and Prp19 and its related proteins are major components of the spliceosome's catalytic core RNP. To learn more about the spatial organization of the human Prp19 (hPrp19)/CDC5L complex, which is comprised of hPrp19, CDC5L, PRL1, AD002, SPF27, CTNNBL1, and HSP73, we purified native hPrp19/CDC5L complexes from HeLa cells stably expressing FLAG-tagged AD002 or SPF27. Stoichiometric analyses indicated that, like Saccharomyces cerevisiae NTC (nineteen complex), the human Prp19/CDC5L complex contains four copies of hPrp19. Salt treatment identified a stable core comprised of CDC5L, hPrp19, PRL1, and SPF27. Protein-protein interaction studies revealed that SPF27 directly interacts with each component of the hPrp19/CDC5L complex core and also elucidated several additional, previously unknown interactions between hPrp19/CDC5L complex components. Limited proteolysis of the hPrp19/CDC5L complex revealed a protease-resistant complex comprised of SPF27, the C terminus of CDC5L, and the N termini of PRL1 and hPrp19. Under the electron microscope, purified hPrp19/CDC5L complexes exhibit an elongated, asymmetric shape with a maximum dimension of approximately 20 nm. Our findings not only elucidate the molecular organization of the hPrp19/CDC5L complex but also provide insights into potential protein-protein interactions at the core of the catalytically active spliceosome.

HIS-24 linker histone and SIR-2.1 deacetylase are involved in chromatin silencing in Caenorhabditis elegans. Depletion of SIR-2.1 results in cytoplasmic retention of HIS-24 in oocytes. However, the molecular working mechanisms of HIS-24 and SIR-2.1 are unclear. We show here a synergistic function of SIR-2.1 and HIS-24 that are together essential for maintenance of the H3K27me3 mark in the germ line of C. elegans. We demonstrate the synthetic effects of the two factors on brood size, embryogenesis, and fertility. SIR-2.1 and HIS-24 associate with the subtelomeric regions but apparently do not interact directly. We report that SIR-2.1 deacetylates H3K9 at subtelomeric regions and suggest that deacetylation of H3K9 is a prerequisite for H3K27 methylation. In turn, we found that HIS-24 specifically interacts with the histone H3 K27 region, when unmodified or in the trimethylated state. Overall, our data indicate that SIR-2.1 and HIS-24 contribute to the propagation of a specialized chromatin state at the subtelomeric regions and elsewhere in the genome.

Deactivation of release factor 1 by polyclonal antibodies in an in vitro translation system, which was used to express the esterase gene, led to the reversible elimination of naturally occurring termination. This technique allowed the antibiotic puromycin to be used as an acceptor substrate for the peptidyl residue in the peptidyl-transferase reaction. This resulted in more than 80 % yield of protein with C-terminally incorporated puromycin. pCpPuromycin that was either conjugated with the Cy3 fluorophor or biotin by N4 alkylation of cytosine, also acted as an acceptor substrate for the peptidyl-transferase reaction and was incorporated into the protein C terminus. The resulting conjugates possessed Cy3-specific fluorescence and affinity to streptavidin-coated surfaces, respectively. This left the enzymatic activity of the reporter protein unaffected. It was also shown that extension of puromycin on its 5'-hydroxyl end by up to ten deoxyoligonucleotides also allowed conjugation with the C terminus of in vitro translated protein when RF1-dependent termination was suppressed. However, the conjugation yield decreased upon addition of more than six nucleotides.

An mRNA encoding the esterase from Alicyclobacillus acidocaldarius with catalytically essential serine codon (ACG) replaced by an amber (UAG) codon was used to study the suppression in in vitro translation system. Suppression of UAG by tRNASer(CUA) was monitored by determination of the full-length and active esterase. It was shown that commonly used increase of suppressor tRNA concentration inhibits protein production and therefore limits suppression. In situ deactivation of release factor by specific antibodies leads to efficient suppression already at low suppressor tRNA concentration and allows an in vitro synthesis of fully active enzyme in high yield undistinguishable from wild-type protein.

Esterase from thermophilic bacteria Alicyclobacillus acidocaldarius can be produced up to 200μg/ml by coupled in vitro transcription/translation system derived from Escherichia coli. The synthesized thermostable enzyme can be determined by photometrical and fluorescent assays at least up to 10−8M concentration or by activity staining in the polyacrylamide gels. Enhanced green fluorescence protein-esterase fusion protein was bound to a matrix with immobilized esterase inhibitor and purified by affinity chromatography. Thus, the esterase is suited as a reporter enzyme to monitor the expression of polypeptides coupled to its N-terminus and simultaneously, as a cleavable tag for polypeptide purification.