University of California (UC), San Diego, biochemists studying the evolutionary origins and history of RNA splicing and the human genome combined two-dimensional (2D) images of individual molecules to reconstruct a three-dimensional (3D) picture of a portion of RNA, i.e., group II introns. In so doing, they discovered a large-scale molecular movement associated with RNA catalysis that provides evidence for the origin of RNA splicing and its role in the diversity of life on Earth. Their research “(Cryo-EM Structures of a Group II Intron Reverse Splicing into DNA”) is published in Cell.

“We are trying to understand how the human genome has evolved starting from primitive ancestors. Every human gene has unwanted frames that are noncoding and must be removed before gene expression. This is the process of RNA splicing,” stated Navtej Toor, PhD, an associate professor in the department of chemistry and biochemistry, adding that 15% of human diseases are the result of defects in the RNA process.

“Group II introns are a class of retroelements that invade DNA through a copy-and-paste mechanism known as retrotransposition. Their coordinated activities occur within a complex that includes a maturase protein, which promotes splicing through an unknown mechanism. The mechanism of splice site exchange within the RNA active site during catalysis also remains unclear. We determined two cryo-EM structures at 3.6-Å resolution of a group II intron reverse splicing into DNA,” the investigators wrote.

Reverse Splicing into DNA Graphical Abstract.
Source: Cell

“These structures reveal that the branch-site domain VI helix swings 90°, enabling substrate exchange during DNA integration. The maturase assists catalysis through a transient RNA-protein contact with domain VI that positions the branch-site adenosine for lariat formation during forward splicing. These findings provide the first direct evidence of the role the maturase plays during group II intron catalysis. The domain VI dynamics closely parallel spliceosomal branch-site helix movement and provide strong evidence for a retroelement origin of the spliceosome.”

Toor explained that his team works to understand the evolutionary origins of 70% of human DNA—a portion made up of two types of genetic elements, which are both thought to have evolved from group II introns. Specifically, spliceosomal introns, which make up about 25% of the human genome, are noncoding sequences that must be removed before gene expression. The other 45% is comprised of sequences derived from what are called retroelements. These are genetic elements that insert themselves into DNA and hop around the genome to replicate themselves via an RNA intermediate.

“Studying group II introns gives us insight into the evolution of a large portion of the human genome,” noted Toor.

Working with the group II intron RNA nanomachine, Toor and Daniel Haack, PhD, a postdoctoral scholar at UC San Diego and first author of the paper, were able to isolate the group II intron complexes from a species of blue-green algae that lives at high temperature.

“Using a group II intron from a high-temperature organism facilitated structure determination due to the innate stability of the complex from this species,” said Haack. “The evolution of this type of RNA splicing likely led to the diversification of life on Earth.”

Haack further explained that he and Toor discovered that the group II intron and the spliceosome share a common dynamic mechanism of moving their catalytic components during RNA splicing.

“This is the strongest evidence to date that the spliceosome evolved from a bacterial group II intron,” he said.

Additionally, the findings reveal how group II introns are able to insert themselves into DNA through a process called retrotransposition. This copy-and-paste process has resulted in selfish retroelements proliferating in human DNA to comprise a large portion of the genome.

“Replication of these retroelements has played a large role in shaping the architecture of the modern human genome and has even been implicated in the speciation of primates,” noted Toor.

The researchers used cryo-electron microscopy (cryo-EM) to extract a molecular structure of the group II intron. They froze the RNA in a layer of thin ice and then shot electrons through this sample. According to the scientists, the electron microscope can magnify the image 39,000 times. The resulting 2D images of individual molecules were then put together to come up with a 3D view of the group II intron.

“This is like molecular archaeology,” described Haack. “Group II introns are living fossils that give us a glimpse into how complex life first evolved on Earth.”

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