Cryo-EM reveals the kinetic mechanism of intron self-splicing in Tetrahymena

On March 16, 2023, the research team of Su Zhaoming of Sichuan University published the latest research results entitled “Cryo-EM reveals dynamics of Tetrahymena group I intron self-splicing” online in the journal Nature Catalysis. The research group abandoned the traditional RNA preparation method, constructed the tetrahymena intron, and extended the carriers of 5- and 3-exons of different lengths, directly performed transcriptional co-folding splicing, so that the splicing process of the entire RNA molecule was closer to the natural physiological state, and captured a variety of conformational structures related to the self-splicing process at a resolution of 2.84 –3.73 Å, and the mechanism of the self-splicing kinetic process of tetrahymenatic ribozyme was elaborated relatively completely.

Ribonucleic acid (RNA) is a unique biological macromolecule that can store genetic information and form specific tertiary structures to participate in important life processes. Ribozymes are non-coding RNA molecules that have the function of catalyzing specific biochemical reactions, similar to, but not protein-dependent, enzymes in proteins. The first Ribozyme was identified by Thomas Cech’s lab in 1982, and his team discovered that ribosomal RNA precursors from Tetrahymena thermophila contained an intron, interrupting the noncoding sequence (IVS) of the gene. It is capable of self-splicing in vitro through two transesterification reactions from the mRNA in which it resides without any protein or external energy source. For his discovery of “the catalytic properties of RNA molecules”, Thomas Cech was awarded the 1989 Nobel Prize in Chemistry. Ribozyme’s discovery shattered the conventional notion that enzymes are proteins in nature and led to the development of “The RNA world hypothesis.”

Tetrahymena group I intron is the most characteristic milestone molecule for the study of RNA catalysis, RNA folding and high-order three-dimensional structure of RNA.

In solution, macromolecular conformational states alternate dynamically to form conformational ensembles, and dynamic transitions between RNA conformational aggregates are critical for regulating complex life processes. Cryo-electron microscopy (cryo-EM) has become an important technique to explore the three-dimensional folding conformational structural dynamics of bioactive RNA.

In 2021, Zhaoming Su’s team, together with Rhiju Das and Wah Chiu of Stanford University, analyzed the 3.1 Å cryo-EM structure of the full-length tetrahymenatic ribozyme for the first time, published in the journal Nature, revealing conformational changes before and after ribozyme binding substrate. However, atomic-scale models of multiple conformational changes that occur during the self-splicing of introns in Tetrahymena remain largely unknown, limiting the understanding of the kinetics and molecular mechanisms of RNA catalytic processes.

The latest research results of Su’s team were published online in the journal Nature Catalysis on March 16, 2023. Co-transcription refolding has been shown to be critical for the structural stability of active precursor RNA. Based on this, the study abandons the traditional RNA preparation method, constructs the intron of Tetrahymena, and extends the carriers of 5- and 3-exons of different lengths, directly performs transcriptional co-folding splicing, so that the splicing process of the entire RNA molecule is closer to the natural physiological state, captures a variety of conformational structures related to the self-splicing process at a resolution of 2.84 –3.73 Å, and elaborates the mechanism of the self-splicing kinetic process of Tetrahymenae ribozyme relatively completely.

Figure 1: Tetrahymena, type 1 intron self-splicing process

First, the 5-terminal exon and intron IGS (internal guide sequence) form a P1 double helix, which is docked to the splicing active site through the intermediate state from the initial state, and J8/7, J4/5 and J5/4 (joining region, J) near the active center form long-range interaction with P1 to mediate the precise docking of the P1 double helix to the splice site, completing the first transesterification reaction. Secondly, 3 exons form a new P10 double helix through conformational changes, using the same active center, completing the second step transesterification reaction; The ligated mature exons are then released, and the 5th end of the intron is refolded into a new P1 double helix returning to the starting position, ready for the next cyclization reaction.

Early functional experiments proved that exons affect the splicing process, and in this study, the team also found for the first time that in the second step of the reaction, two exons separated by more than 400 bases can also form an unexpected pseudonode structure with the intron: 5 exons and 5 introns form P0, and 5 and 3 exons form P0. The mutation of these two parts of the structure by biochemical method found that P0 inhibited the rate of the second step splicing reaction, which is likely due to the newly formed complex three-dimensional structure hindering the release of mature exons of the linkage, thereby promoting the occurrence of reverse reaction and reducing the rate of forward reaction, which may be an important structure for maintaining physiological homeostasis in biological evolution (Figure 2). Metal ions, especially Mg2+, are critical for the stabilization and catalytic reaction of RNA structures, and the researchers further identified 33 metal ions in structures closer to their natural state, including one that had never been observed in a structure before, and verified that the second step reaction was through the classical two-metal ion reaction mechanism.

In some cases, RNA forms strong site binding but does not form any contact with the metal or its inner spherical ligand. Therefore, magnesium can often be replaced with organic polyamines such as spermidine to stabilize RNA binding. The study found that spermidine molecules are present at RNA active sites and promote RNA conformational stability.

Figure 2: Tetrahymena, class I introns and exons at both ends form a new three-dimensional structure to regulate the second step splicing reaction

This study provides necessary structural information for the catalytic mechanism of ribozys and promotes the progress of ribozyme research. At the same time, the current limited RNA molecular structure database is supplemented; It enriches the understanding of the catalytic and folding mechanism of RNA molecules. This further demonstrates the potential of cryo-EM in studying the process of conformational changes in RNA dynamics. (Source: Science Network)

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