Structure of a human catalytic step I spliceosome Zhan, Xiechao; Yan, Chuangye; Zhang, Xiaofeng ...
Science (American Association for the Advancement of Science),
02/2018, Letnik:
359, Številka:
6375
Journal Article
Recenzirano
Odprti dostop
Splicing by the spliceosome involves branching and exon ligation. The branching reaction leads to the formation of the catalytic step I spliceosome (C complex). Here we report the cryo-electron ...microscopy structure of the human C complex at an average resolution of 4.1 angstroms. Compared with the
C complex, the human complex contains 11 additional proteins. The step I splicing factors CCDC49 and CCDC94 (Cwc25 and Yju2 in
, respectively) closely interact with the DEAH-family adenosine triphosphatase/helicase Prp16 and bridge the gap between Prp16 and the active-site RNA elements. These features, together with structural comparison of the human C and C* complexes, provide mechanistic insights into ribonucleoprotein remodeling and allow the proposition of a working mechanism for the C-to-C* transition.
The glucose transporter GLUT1 catalyses facilitative diffusion of glucose into erythrocytes and is responsible for glucose supply to the brain and other organs. Dysfunctional mutations may lead to ...GLUT1 deficiency syndrome, whereas overexpression of GLUT1 is a prognostic indicator for cancer. Despite decades of investigation, the structure of GLUT1 remains unknown. Here we report the crystal structure of human GLUT1 at 3.2 Å resolution. The full-length protein, which has a canonical major facilitator superfamily fold, is captured in an inward-open conformation. This structure allows accurate mapping and potential mechanistic interpretation of disease-associated mutations in GLUT1. Structure-based analysis of these mutations provides an insight into the alternating access mechanism of GLUT1 and other members of the sugar porter subfamily. Structural comparison of the uniporter GLUT1 with its bacterial homologue XylE, a proton-coupled xylose symporter, allows examination of the transport mechanisms of both passive facilitators and active transporters.
Structural basis of pre-mRNA splicing Hang, Jing; Wan, Ruixue; Yan, Chuangye ...
Science (American Association for the Advancement of Science),
09/2015, Letnik:
349, Številka:
6253
Journal Article
Recenzirano
Splicing of precursor messenger RNA is performed by the spliceosome. In the cryogenic electron microscopy structure of the yeast spliceosome, U5 small nuclear ribonucleoprotein acts as a central ...scaffold onto which U6 and U2 small nuclear RNAs (snRNAs) are intertwined to form a catalytic center next to Loop I of U5 snRNA. Magnesium ions are coordinated by conserved nucleotides in U6 snRNA. The intron lariat is held in place through base-pairing interactions with both U2 and U6 snRNAs, leaving the variable-length middle portion on the solvent-accessible surface of the catalytic center. The protein components of the spliceosome anchor both 5′ and 3′ ends of the U2 and U6 snRNAs away from the active site, direct the RNA sequences, and allow sufficient flexibility between the ends and the catalytic center. Thus, the spliceosome is in essence a protein-directed ribozyme, with the protein components essential for the delivery of critical RNA molecules into close proximity of one another at the right time for the splicing reaction.
Precursor messenger RNA (pre-mRNA) splicing is executed by the spliceosome. In the past 3 years, cryoelectron microscopy (cryo-EM) structures have been elucidated for a majority of the yeast ...spliceosomal complexes and for a few human spliceosomes. During the splicing reaction, the dynamic spliceosome has an immobile core of about 20 protein and RNA components, which are organized around a conserved splicing active site. The divalent metal ions, coordinated by U6 small nuclear RNA (snRNA), catalyze the branching reaction and exon ligation. The spliceosome also contains a mobile but compositionally stable group of about 13 proteins and a portion of U2 snRNA, which facilitate substrate delivery into the splicing active site. The spliceosomal transitions are driven by the RNA-dependent ATPase/helicases, resulting in the recruitment and dissociation of specific splicing factors that enable the reaction. In summary, the spliceosome is a protein-directed metalloribozyme.
Each cycle of pre–messenger RNA splicing, carried out by the spliceosome, comprises two sequential transesterification reactions, which result in the removal of an intron and the joining of two ...exons. Here we report an atomic structure of a catalytic step I spliceosome (known as the complex) from Saccharomyces cerevisiae, as determined by cryo–electron microscopy at an average resolution of 3.4 angstroms. In the structure, the 2'-OH of the invariant adenine nucleotide in the branch point sequence (BPS) is covalently joined to the phosphate at the 5' end of the 5' splice site (5'SS), forming an intron lariat. The freed 5' exon remains anchored to loop I of U5 small nuclear RNA (snRNA), and the 5'SS and BPS of the intron form duplexes with conserved U6 and U2 snRNA sequences, respectively. Specific placement of these RNA elements at the catalytic cavity of Prp8 is stabilized by 15 protein components, including Snu114 and the splicing factors Cwc21, Cwc22, Cwc25, and Yju2. These features, representing the conformation of the spliceosome after the first-step reaction, predict structural changes that are needed for the execution of the second-step transesterification reaction.
Proton-coupled monocarboxylate transporters MCT1-4 catalyze the transmembrane movement of metabolically essential monocarboxylates and have been targeted for cancer treatment because of their ...enhanced expression in various tumors. Here, we report five cryo-EM structures, at resolutions of 3.0–3.3 Å, of human MCT1 bound to lactate or inhibitors in the presence of Basigin-2, a single transmembrane segment (TM)-containing chaperon. MCT1 exhibits similar outward-open conformations when complexed with lactate or the inhibitors BAY-8002 and AZD3965. In the presence of the inhibitor 7ACC2 or with the neutralization of the proton-coupling residue Asp309 by Asn, similar inward-open structures were captured. Complemented by structural-guided biochemical analyses, our studies reveal the substrate binding and transport mechanism of MCTs, elucidate the mode of action of three anti-cancer drug candidates, and identify the determinants for subtype-specific sensitivities to AZD3965 by MCT1 and MCT4. These findings lay out an important framework for structure-guided drug discovery targeting MCTs.
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•A data processing strategy for cryo-analysis of small size membrane proteins•Structures of MCT1/Basigin-2 in distinct states at 3.0–3.3 Å resolutions•Substrate recognition and proton-coupled transport mechanism were elucidated•The mode of action of three anti-cancer drug candidates was unveiled
Structures of MCT1, a small highly flexible SLC-family transporter, in inward-open and outward open conformations, provide a window on how anti-cancer leads influence transport.
Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ...ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 Å, respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3'-splice site is recognized by the junction between the 5'-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.
The voltage-gated calcium channel Ca(v)1.1 is engaged in the excitation-contraction coupling of skeletal muscles. The Ca(v)1.1 complex consists of the pore-forming subunit α1 and auxiliary subunits ...α2δ, β, and γ. We report the structure of the rabbit Ca(v)1.1 complex determined by single-particle cryo-electron microscopy. The four homologous repeats of the α1 subunit are arranged clockwise in the extracellular view. The γ subunit, whose structure resembles claudins, interacts with the voltage-sensing domain of repeat IV (VSD(IV)), whereas the cytosolic β subunit is located adjacent to VSD(II) of α1. The α2 subunit interacts with the extracellular loops of repeats I to III through its VWA and Cache1 domains. The structure reveals the architecture of a prototypical eukaryotic Ca(v) channel and provides a framework for understanding the function and disease mechanisms of Ca(v) and Na(v) channels.
Each cycle of precursor messenger RNA (pre-mRNA) splicing comprises two sequential reactions, first freeing the 5′ exon and generating an intron lariat–3′ exon and then ligating the two exons and ...releasing the intron lariat. The second reaction is executed by the step II catalytically activated spliceosome (known as the C* complex). Here, we present the cryo–electron microscopy structure of a C* complex from Saccharomyces cerevisiae at an average resolution of 4.0 angstroms. Compared with the preceding spliceosomal complex (C complex), the lariat junction has been translocated by 15 to 20 angstroms to vacate space for the incoming 3′-exon sequences. The step I splicing factors Cwc25 and Yju2 have been dissociated from the active site. Two catalytic motifs from Prp8 (the 1585 loop and the β finger of the ribonuclease H–like domain), along with the step II splicing factors Prp17 and Prp18 and other surrounding proteins, are poised to assist the second transesterification. These structural features, together with those reported for other spliceosomal complexes, yield a near-complete mechanistic picture on the splicing cycle.
Pre–messenger RNA (pre-mRNA) splicing is carried out by the spliceosome, which undergoes an intricate assembly and activation process. Here, we report an atomic structure of an activated spliceosome ...(known as the Bact complex) from Saccharomyces cerevisiae, determined by cryo–electron microscopy at an average resolution of 3.52 angstroms. The final refined model contains U2 and U5 small nuclear ribonucleoprotein particles (snRNPs), U6 small nuclear RNA (snRNA), nineteen complex (NTC), NTC-related (NTR) protein, and a 71-nucleotide pre-mRNA molecule, which amount to 13,505 amino acids from 38 proteins and a combined molecular mass of about 1.6 megadaltons.The 5' exon is anchored by loop I of U5 snRNA, whereas the 5' splice site (5'SS) and the branch-point sequence (BPS) of the intron are specifically recognized by U6 and U2 snRNA, respectively. Except for coordination of the catalytic metal ions, the RNA elements at the catalytic cavity of Prp8 are mostly primed for catalysis. The catalytic latency is maintained by the SF3b complex, which encircles the BPS, and the splicing factors Cwc24 and Prpll, which shield the 5' exon-5'SS junction. This structure, together with those determined earlier, outlines a molecular framework for the pre-mRNA splicing reaction.
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