A research team led by Professor Shi Yigong from the School of Life Sciences, Tsinghua University, published two side-by-side research articles on August 21st in Science, reporting the long-sought-after structure of a yeast spliceosome at 3.6 Å resolution determined by single particle cryo-electron microscopy (cryo-EM), and the molecular mechanism of pre-messenger RNA splicing.
Gene expression is the basic principle of all living cells. During the process known as the Central Dogma, the genomic information stored in genome DNA sequences is delivered to pre-mRNAs by transcription and finally to functional proteins by translation. In eukaryotes, pre-mRNAs are intervened by coding sequences containing exons and untranslated introns. The excision of introns and ligation of exons is named as pre-mRNA splicing, and executed by spliceosome. This macromolecular machinery consists of five small nuclear ribonucleoprotein particles (snRNPs), nineteen complex Shi Yigong (second from right) with his team (NTC), NTC related (NTR), and a number of associated enzymes and cofactors. In total, more than 100 proteins and at least 5 RNAs were identified to be the core and auxiliary components of spliceosome. The involved proteins and RNAs assemble into and dissociate from spliceosome in a strict order during splicing, endowing the extreme dynamics and flexibility of the spliceosome. These features guarantee the accomplishment of the complex splicing reaction, but at the same time tangle the structural investigations of spliceosome.
Besides the basic biological importance of spliceosome, numerous diseases are related to the dysfunction of spliceosomal regulation or splicing errors. Almost 35 percent of genetic disorders result from incorrect splicing, exemplified by an unusual expression of alternative splicing that leads to frontotemporal dementia driven by tau mis-splicing. The mutation of key spliceosomal proteins like Brr2 or Prp8 can lead to Autosomal Dominant retinitis pigmentosa. Some cancers are also associated with abnormal splicing.
Professor Shi has long been intrigued by the mechanism of splicing and his group has been working on the spliceosome since 2009. In 2014, Shi’s group reported in NATURE on the first crystal structure of the Lsm complex, which is a key component of the spliceosome. Driven by his ambition and not stopped by the immediate success, Professor Shi’s group continued their research on the intact spliceosome. This year, they have reached a major milestone in obtaining an endogenous intact yeast spliceosome with high purity. This was made possible by the substantial improvement in the tandemaffinity purification. Using the most advanced cryo-EM reconstitution techniques, the spectacular 3-D atomic model was resolved, which consists of 10,574 amino acids from 37 proteins and 4 RNA molecules. The combined molecular mass was approximately 1.3 mega-Daltons. Based on the structural analysis together with existing knowledge, the group provided the first structural insight into the molecular mechanism for pre-mRNA splicing: the pliceosome is in essence a proteindirected ibozyme, with the protein omponents essential for the delivery of critical RNA molecules into the close proximity of one another at the right time for the splicing reaction.
Since the discovery of “split genes” in 1977, scientists are constantly exploring the molecular mechanism of pre-mRNA splicing. In 1983, Steitz, J.A. group isolated five U snRNPs; in the same year, Sharp, P.A. and Keller, W group set up in vitro splicing assay independently. Until now, decades of genetic and biochemical experiments have identified almost all proteins in spliceosome and uncovered some functions. Yet its structure has remained a mystery for a long time. The work primarily performed by Dr. Chuangye Yan, Jing Hang, and Ruixue Wan under Professor Shi Yigong’s supervision, settled this Holy Grail question and established the structural basis for related areas.
This work has been supported by funds from the Ministry of Science and Technology and the National Natural Science Foundation of China. The EM data was acquired on the Tsinghua Cryo-EM Facility and processed on the Explorer 100 cluster system of the Tsinghua National Laboratory for Information Science and Technology.
[update: 2015-10]
