RNA splicing is one of the important processes in regulating gene expression in eukaryotes. In the 1970s, scientists first discovered the discontinuity of eukaryotic genes, indicating that after genetic information is transferred from DNA to RNA, it needs to undergo "cutting" and "splicing" of effective genetic information. This splicing of effective genetic information and the removal of "invalid" genetic information is called RNA splicing. RNA splicing is widely present in eukaryotes. With the evolution of species, the number of genes containing introns increases, and the frequency of RNA splicing also increases accordingly, making it possible for one gene to encode multiple proteins.

On May 25, 2018, Professor Shi Yigong's research group from the School of Life Sciences at Tsinghua University published a major research result in the form of a long article in the journal Science on the assembly mechanism and structure of spliceosomes. This article reports on two key conformations of fully assembled spliceosomes in brewing yeast during the pre activation stage - pre catalytic spliceosome precursor (defined as "pre-B complex") and pre catalytic spliceosome (defined as "B complex"). These two high-resolution 3D structures with overall resolutions of 3.3-4.6 angstroms and 3.9 angstroms respectively demonstrate the recognition status and dynamic changes of the 5 'splice site and branch point (BPS) of pre mRNA during the assembly process of spliceosomes, answering important questions such as the recognition mechanism of the 5' splice site and branch point of pre mRNA before spliceosome activation, how the 5 'splice site and branch point gradually enter the active site during the activation process, how spliceosomes gradually assemble and complete activation through structural recombination.

The pre-B complex, composed of U1 snRNP, U2 snRNP, and U4/U6. U5 tri snRNP, is currently considered to be the spliceosome with the largest protein composition and molecular weight. This complex has a complex structure, but the interactions between its components are not tight, making it easy for the complex to dissociate during purification. In the latest published article in Science, Shi Yigong's research team explored purification schemes multiple times and ultimately optimized a set of stable and well performing pre-B complex samples. Subsequently, single particle cryo electron microscopy technology was used to reconstruct the cryo electron microscopy structures of U1 snRNP, U2 snRNP, and U4/U6.U5 tri snRNP with partial resolutions of up to 3.3 angstroms, 3.6-4.6 angstroms, and 3.4 angstroms, and atomic models were constructed (Figure 1).

Figure 1 Three dimensional structure of pre catalytic spliceosome precursor and pre catalytic spliceosome in brewing yeast

The pre-B complex structure analyzed in this article contains five types of ribonucleoprotein (snRNP) spliceosome structures that have been analyzed worldwide. It consists of 68 proteins and 6 RNAs. In this structure, the recognition of the 5 'splice site by U1 snRNP during early splicing assembly and the interaction interface between five ribonucleoproteins were observed for the first time. At the same time, the article also reported the high-resolution three-dimensional structure of another fully assembled spliceosome, the pre catalyzed spliceosome B complex, located after the pre-B complex. Based on the structural information of the B complex, through structural comparison, it can be clearly seen that during the assembly process, the 5 'splice site of pre mRNA is initially recognized by U1 snRNP, and then transferred and paired with U6 snRNA due to conformational changes, providing a structural basis for splicing activation. In addition, the dynamic changes of branching points and the structural reorganization and conformational changes experienced by each component of the spliceosome are also clearly presented. At the end of the article, based on the structural characteristics of pre-B, the author boldly speculated on the three-dimensional structural model of the earliest incompletely assembled pre spliceosome (defined as "A complex") (as shown in Figure 2). The analysis of the structure of these two key states of spliceosomes provides the most direct and effective structural evidence for revealing the mechanism of how to identify 5 'splice sites and branching points, how to carry out structural recombination, and how to activate spliceosomes during the early assembly of spliceosomes. It will also provide a structural basis and theoretical basis for the study of alternative splicing in higher eukaryotes.

Figure 2 Prediction of three-dimensional structure of pre spliced yeast and model for splicing assembly and activation

So far, the research team of Shi Yigong has analyzed high-resolution three-dimensional structures of 9 different states of spliceosomes in yeast (as shown in Figure 3). From assembly to activation, from two-step ester transfer reaction to spliceosome dissociation, these 9 states of spliceosomes fully cover the splicing pathway, and for the first time, connect the process of spliceosome mediated RNA splicing, providing clear and comprehensive structural information for understanding the molecular mechanism of RNA splicing.

(Reprinted from ScienceNet)