Gene Proximity to Nuclear Speckles Drives Efficient mRNA Splicing

Over one hundred years ago, when Santiago Ramón y Cajal observed neurons microscopically, he saw fibrillous and spotted structures inside their nuclei.¹ Researchers later discovered that these nuclear compartments, dubbed nuclear bodies, lacked membranes but contained clusters of molecules that participated in specific functions. One such nuclear body, the speckle, contains spliceosomes that are known to be involved in mRNA splicing. Disruption of speckles leads to a range of diseases and developmental disorders, yet how speckles drive splicing remains unclear.^2,3

One idea has been that speckles are splicing factories. The splicing reaction would happen inside the speckle, and then the spliced product would leave. “What people found is that’s not what happens,” said Mitchell Guttman, a molecular biologist at the California Institute of Technology. “The reason for their highest concentration of splicing factors is very much like the same reason that, if I look for where's the highest concentration of bed sheets in your house, it's not going to be on your bed, it's going to be in your linen closet, right? The location where you store them when you're not using them. And the same was thought to be true for nuclear speckles.”

The idea that speckles might be storage sites for spliceosomes triggered Guttman to look deeper into how nuclear organization might affect the splicing process. In a recent article, he showed that when genes are preferentially positioned near speckles, mRNA splicing is significantly more efficient.⁴

“It’s huge for the RNA processing field, because now it really gives a functional significance to speckles,” said cell biologist Andrew Belmont at the University of Illinois, Urbana-Champaign who was not affiliated with the study.

Guttman and his team are interested in how nuclear organization influences different quantitative aspects of gene regulation. Toward this end, they previously developed a method to identify distinct genomic regions that were “speckle far” and “speckle close” in mouse embryonic stem (ES) cells.⁵ In their new study, they integrated a reporter gene into each of these genomic regions using CRISPR, along with CRISPR-associated protein 9 gene-editing technology.

The reporter gene was bidirectional. One transcriptional direction produced spliced RNA that was tagged with green fluorescent protein (GFP) and the other direction produced unspliced RNA labeled with a blue fluorescent protein (BFP). When the reporter was integrated into genomic regions close to speckles, the ratio of GFP to BFP was significantly higher.

Mouse ES cells are totipotent, expressing many diverse genes, but Guttman wanted to know whether genomic architecture shifted tissue-specific genes closer to speckles. To find out, he studied the Ttn gene that expresses titin, a key component of the contractile apparatus, in mouse myocytes. Guttman showed that Ttn was positioned closer to speckles in myocytes than in ES cells, and its corresponding mRNA in myocytes was more efficiently spliced than in ES cells. Further, other genes located in the genomic vicinity of Ttn in myocytes were spliced more efficiently.

From these findings, Guttman concluded that the speckle emits spliceosomes. “Think about planets orbiting around the sun,” he said. “As you get farther and farther away, you get a dramatic decrease in heat transfer.” Similarly, for the spliceosome-emitting speckles, Guttman explained, if the target mRNA is nearby, the spliceosome has a smaller area to search within the nucleus, making the mRNA easier to find.

“This idea that functional kinetic coupling of two very diverse but critically integrated processes is achieved through physical spatial organization, remodeling of DNA architecture, is something that I wouldn't have necessarily anticipated,” Guttman said, “but I think it's incredibly exciting, and more generally, in thinking about novel mechanisms of how quantitative regulation occurs in the cell.”

Next, Guttman and his team want to uncover how genome structure organizes to enable the specific and precise kinetics of mRNA splicing. “Even though all these molecules are moving randomly, stochastically in the nucleus, you get this coalescence that is driven by mutual affinity between these two components. That’s how we think about why you actually get this organization,” Guttman said.

“Already the number of papers looking at nuclear speckles has gone up in the last few years,” said Belmont, “but now, this strong functional significance will convince even more people to consider the role of nuclear speckles in whatever process they're studying related to RNA processing in the context of the nuclear organization.”