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• W2994255736 abstract "Kiyoshi, age 68, in his lab within the new Laboratory of Molecular Biology in Cambridge. Taken on October 20, 2017View Large Image Figure ViewerDownload Hi-res image Download (PPT)Kiyoshi Nagai has been discovering how proteins interact with RNA in ever-larger complexes for over three decades and, as some of his competitors have commented, he set the pace. His research culminated in visualization of the huge, dynamic spliceosome complex in the many different states it assumes while catalyzing pre-mRNA splicing. Each advance tested the limits of what could be accomplished with the available methodology. His death came at the summit of his achievements. Kiyoshi was born on 25th June 1949 in Osaka, Japan. His father was a dedicated medical doctor and physiologist whom Kiyoshi was keen to follow into a scientific career. His mother was an accomplished cook who prepared not only fine, traditional Japanese food but also Chinese, Italian, French, and German dishes. This had a lifelong effect; Kiyoshi loved cooking for his family and friends—and eating. In the lab or office, he would frequently declare, “I’m so hungry!” particularly as dinnertime approached. Kiyoshi also loved music and played the cello with friends in a chamber orchestra throughout his life. After earning high grades in school, Kiyoshi entered Osaka University to study biophysics. He stayed on, completed a master’s degree, and began his doctorate there in 1974. His PhD study was hemoglobin structures. Only a few months into his research, and just after marrying his fiancée Yoshiko, Kiyoshi came as a visiting student to the MRC Laboratory of Molecular Biology (LMB), Cambridge, UK, to work with John Kilmartin and Max Perutz on the spectroscopy of different hemoglobin conformational states. Along with John Kendrew, Max had pioneered the field of protein X-ray crystallography with their structures of hemoglobin and myoglobin. Max was Kiyoshi’s scientific hero, and the chance to work with him must have been thrilling. Kiyoshi stayed for 18 months, during which time his son Ken was born. He met founders of molecular biology such as Francis Crick, Fred Sanger, Aaron Klug, and Sidney Brenner. The atmosphere of 1970s LMB made a great impression on Kiyoshi—and he liked it! Kiyoshi returned to Japan, continued his hemoglobin work, and made important advances. He earned his doctorate and soon took an Assistant Professor position in the Physiology Department of Nara Medical College, following in his father’s footsteps. His daughter Yuko was born at this time. Meanwhile, Max Perutz was pondering how hemoglobin had evolved. The effects of some mutations were known from natural hemoglobin variants, but many of Max’s questions could only be answered by engineering novel hemoglobins. It was an ambitious project, but Max thought Kiyoshi could achieve it. He invited Kiyoshi back to the LMB, this time as his post-doc. Kiyoshi returned in 1981 and started developing a system to produce large amounts of hemoglobin in Escherichia coli. It was a high-risk venture. His colleagues from the time say he was tenacious. Even when it seemed his project had been scooped, he didn’t relent. Kiyoshi understood how important such a system was—if he could make haemoglobin, why not the multitude of other proteins found in nature? Success came 3 years later, when Kiyoshi joined the β-globin gene to a short coding sequence from the lambda phage cII gene under control of the phage promoter and inserted it into a plasmid. The cII leader could be subsequently removed by protease cleavage. It was the first system that produced enough material for biochemical or crystallographic studies and remained widely used for several years. It delivered what Max had hoped for, and another highly productive period of hemoglobin research ensued. Kiyoshi’s group worked on making a hemoglobin-based blood substitute and studying hemoglobin from animals as diverse as crocodiles and bar-headed geese. The LMB gave Kiyoshi tenure in 1987. It was the perfect position, and he remained there throughout his life. The director, Aaron Klug, pointed him toward crystallography of RNA-protein complexes. A few structures of proteins bound to DNA had been determined, but none with RNA. Whereas DNA was generally found in the iconic double helix conformation, RNA was known to adopt many complex secondary and tertiary structures—more like protein. RNA was notorious at the time for being a highly labile molecule, which would degrade at the drop of a hat. Crystallizing protein-RNA complexes was clearly a difficult, challenging venture; Kiyoshi plunged in. A small domain, the RNA recognition motif (RRM), had been identified in proteins of the recently discovered pre-mRNA splicing machinery. Kiyoshi’s group started overexpressing RRMs in E. coli. The first success was with RRM1 of U1-A protein, a component of U1 snRNP. Its structure was published in 1990 and the complex with its cognate RNA hairpin in 1994. This was followed by the structure of the U2-B″/A′ protein heterodimer in complex with U2 snRNA stem-loop IV. There were also structures of RNA-protein complexes from the signal recognition particle (SRP), which is involved in co-translational transport of proteins through or into membranes. These all gave important insights into how proteins recognize RNA. Kiyoshi also began work on the structure of Sm proteins. There are seven such proteins, sharing high sequence homology, which bind the Sm site of snRNAs. The Sm site is a U-rich region in the snRNA of U1, U2, U4, and U5 snRNP. Sm protein expression was not trivial but was achieved in collaboration with Reinhard Lührmann’s lab. Two of these protein subcomplexes were co-crystallized, and from their structures, and some cunning biochemistry, we modeled the assembly as a seven-membered ring. At this point, the group had solved so many subcomplexes from the pre-mRNA splicing machinery that Kiyoshi decided to concentrate on solving structures that would reveal its mechanism. In pre-mRNA splicing, introns are excised and exons ligated to produce a coding mRNA molecule. This is catalyzed in two steps (branching and ligation) by the spliceosome, which consists of five snRNPs (U1, U2, U4, U5, and U6) and many protein factors. Each snRNP consists of a characteristic snRNA (e.g., U1 snRNA), which is bound by multiple proteins. The snRNPs and protein factors assemble onto and disassemble from each intron of pre-mRNA during the splicing cycle, following an ordered pathway. Introns contain three highly conserved features: (1) the 5′ splice site (5′SS) at the exon1-intron boundary, (2) an invariant adenosine, the branch point, within a characteristic branch point sequence (BPS), and (3) the 3′ splice site (3′SS) at the intron-exon2 boundary. The 5′ end of U1 snRNA was known to base pair with the 5′SS on pre-mRNA early in splicing. The group could express all the protein components and make U1 snRNA by in vitro transcription. A multi-stage reconstitution protocol was developed, and the project looked feasible, if not trivial. It was a combinatorial problem. We could make variants of the RNA and all the proteins and potentially try to crystallize hundreds of combinations. The first crystals we obtained barely diffracted at all, and after that it became a war of attrition, trying different combinations of components and engineering crystal contacts into the RNA. Nearly 10 years later we had a crystal structure at 5.5 Å resolution. It showed how the N-terminal tail of U1-70k, which was predicted to be unstructured, lay as an extended peptide over the surface of the Sm ring. It extended 180 Å from the RRM domain to complete the binding site of U1-C protein, which interacted with a duplex of U1 snRNA 5′ ends. This mimicked U1 snRNP’s interaction with the 5′SS. It was the first structure of a spliceosomal snRNP in action. After some highly determined crystal engineering, a “minimal” U1 snRNP was reconstituted, which gave crystals diffracting to 3.3 Å and showed, at near-atomic detail, how U1 snRNP interacts with the 5′SS. It was also challenging to co-crystallize the Sm proteins with the Sm site RNA, since neither a stable complex nor well-diffracting crystals were readily forthcoming. Years of painstaking crystallography yielded a structure of the human U4 snRNP core. This confirmed the Sm ring model and revealed how the Sm proteins held the Sm site RNA in a tight, high-energy conformation. Kiyoshi’s group also worked on the structure of Prp8, the largest and most highly conserved spliceosomal protein. It had been shown to crosslink to the 5′SS, BPS, and 3′SS in active spliceosomes, suggesting it is part of, or close to, the active site. Our crystal structure showed domains resembling a bacterial group II intron reverse transcriptase and a type II restriction endonuclease. It has structural similarities to group II intron-encoded “maturase” protein, thus supporting a common origin of both pre-mRNA and group II intron splicing. Kiyoshi’s group and others were finding structures of splicing components but still had no overview of the entire machinery or insight into how it coordinated the many steps of pre-mRNA splicing. Recent advances in single-particle cryo-EM changed everything. Crystallography required large amounts of homogeneous, concentrated material. With the advent of the “resolution revolution,” far smaller quantities of less-pure material could be used to solve a structure. It became practical to purify components of the splicing machinery from native sources for structural studies. The Nagai group started with the yeast U4/U6.U5 tri-snRNP structure. Although the first maps were at a resolution of ∼5.8 Å, the 34 proteins and 3 RNAs constituting the huge assembly were identified, revealing the structures of three snRNPs together. A 3.7 Å resolution structure soon followed, showing how the single-stranded region of U4 snRNA was pre-bound in the active site of the Brr2 helicase. The orientation of Prp8 and U5 snRNA loop 1 revealed where the active site would form upon spliceosome activation. Kiyoshi’s group wasn’t alone in using cryo-EM to discover the secrets of splicing; Yigong Shi’s, Reinhard Lührmann’s, and Rui Zhao’s groups also competed. The last 4 years have been intense; the Nagai group solved a large number of yeast and human spliceosome structures. The entries and exits of the snRNPs and a host of transient protein factors on the pre-mRNA substrate have been revealed as an intricately choreographed dance coordinated by RNA helicases. This shows that a single catalytic pocket is rearranged to catalyze both steps of splicing and that the spliceosome, with over 100 protein components, is still a ribozyme. We can now see how U1 and U2 snRNPs assemble on pre-mRNA and are then joined by tri-snRNP. The structures then reveal how U1 snRNP is released from the nascent spliceosome by Prp28 helicase and how U4 snRNA is unwound from U6 snRNA by Brr2 helicase. These rearrangements bring the 5’SS into the active site, which comprises U2, U6 and U5 snRNAs, and two magnesium ions, all held in precise geometry by a dense network of proteins. Prp2 activity brings the branch point to the active site and branching (the first catalytic step) occurs. Prp16 helicase then remodels the spliceosome to replace the intron’s branch point with the 3′SS in the same active site, ready for exon ligation (the second catalytic step). Once splicing is complete, Prp22 helicase releases the spliced mRNA from the spliceosome and remodels the spliceosome further, prior to disassembly and recycling. During these final years Kiyoshi barely had breathing space between sending off one paper and starting work on the next, yet sometimes he still managed to find a few hours for work at the bench, which is where he was happiest. He was an effective and enthusiastic, but very messy, benchworker; his office desk was just as chaotic, yet he could (almost) always find things! He was an intuitive and lateral thinker. He smiled and laughed a lot, liked to have fun, and sometimes played practical jokes on his friends. He followed the careers of everyone who passed through his group, as well as many other colleagues, with keen interest and was always ready to support them in their aspirations. When he was diagnosed with advanced liver cancer a month before his 70th birthday, he paused briefly and spent more time with his family. He told few people about his condition and was soon back at work again. “Kiyoshi’s work made him so happy,” says Yoshiko, and although his family and many, many friends will miss him deeply, we know he was a man who did what he loved to the end." @default.
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