Functionality of RNA-binding Protein RBFOX2 Revealed

A huge number of immature blood cells, also known as bone marrow stem cells, proliferate and differentiate, or mature, into normal white blood cells in healthy people. They grow rapidly in AML, however, and fail to develop into adult blood cells. Scientists have spent years looking into aspects that could explain why this is happening and possibly uncover new therapy targets. Recent focus has shifted to epigenetics, or alterations in gene expression that are not caused by mutations in the DNA sequence itself. Methylation is the most prevalent epigenetic change, and it includes the attachment of tiny chemical compounds called methyl groups to either DNA or RNA.

Adding or removing a methyl group from RNA is analogous to putting or deleting a tag onto RNA that controls gene expression via “reader” molecules that interact with the tagged RNA. Methylation of RNA in bone marrow stem cells, for example, keeps the cells proliferating, whereas removal of methyl groups pushes them toward maturation into a different type of blood cell.

The role of m6A methylation in chromatin regulation

One of the most essential parts of RNA modification is the addition of a methyl group to adenosine at the N6 position utilizing a kind of protein known as methyl transferases to generate N6-methyladenosine (m6A).

Chuan He, PhD, John T. Wilson Distinguished Service Professor of Chemistry at UChicago, discovered that m6A methylation plays an important role in post-transcriptional control of messenger RNA in the cytoplasm via writers, readers, and erasers.

Subsequent studies published in Science in 2020 and 2022 revealed that methylation can also occur on non-coding RNAs in chromatin, which is a condensed form of genetic information packed around histone proteins.

Chromatin alteration has the potential to affect hundreds of genes at once by regulating gene transcription on a global scale. He previously identified a methyltransferase complex (MTC) containing METTL3 and METTL14 that assists in the methylation of m6A on RNAs. To control early development, this methylation process can be reversed by FTO (an eraser) or detected by YTHDC1 (a reader). As a result, m6A on chromatin RNA functions as a flag for recruiting modifiers or decay machinery to change local or global chromatin states.

Scientists have discovered that m6A aids in the regulation of carRNA, which alters chromatin states in mammals and plants. However, how the RNA methyl transferase complex (MTC), which writes the m6A tag, arrives at and picks the proper position is not well understood. Furthermore, researchers are still looking for particular chromatin factors that recognize m6A-modified RNAs and interact with the recruitment of MTC and downstream modifiers.

RBFOX2 in chromatin regulation

“Now we ask the question of what are the additional reader proteins other than YTHDC1 that read/ recognize m6A-tagged carRNA and how the methyl transferase complex is brought to the desired sites on the chromatin to achieve site-specific regulation,” said He. “In the current study, we identified a protein called RNA-binding FOX1 homologue 2 (RBFOX2), a previously known splicing factor, but in this case it preferentially binds to carRNA with m6A and recruits other proteins to regulate the chromatin state.”

RBFOX2 is a well-known splicing factor that chops and connects functional RNA segments; nevertheless, its novel role is unrelated to splicing. Although most RBFOX2 binds to splicing sites, a considerable portion binds to promoter RNA.

Because of its intimate interaction with the RBM15 protein, RBFOX2 can recruit the m6A reader YTHDC1 as well as another protein, Polycomb repressive complex 2 (PRC2). The binding of YTHDC1 to PRC2 resulted in chromatin regulation through transcriptional inactivation.

“These findings highlight the role of RBFOX2 in the regulation of chromatin at m6A-modified carRNA sites through an RBM15–YTHDC1–PRC2 pathways,” said He.

Investigating the role of RBFOX2 in leukemia

To comprehend how RBFOX2 alters cell function, He and colleagues cultured malignant hematopoietic stem cells with RBFOX2 deactivated on petri dishes and discovered that the cells’ proliferative capacity was significantly reduced, indicating that RBFOX2 expression may be higher in leukemia cells.

“As we predicted, our collaborators’ investigation from a large cohort of severe acute myeloid leukemia (AML) patient samples revealed an overexpression of RBFOX2, which is highly correlated with poor prognosis,” said He.

Hematopoietic stem cells should differentiate, however due to excessive RBFOX2 expression, they are locked in a proliferative state, which He identified as problematic.

He also stated that, to his knowledge, this is the first research demonstrating RBFOX2 overexpression in severe AML patient samples. The researchers speculated that this could be used as a diagnostic and prognostic marker.

This work points the way toward developing innovative medicines that target RBFOX2 for leukemia patients, as well as encouraging the discovery of new epigenetic markers that can be utilized to better understand various types of cancer.

Epitranscriptomics pioneer

For more than a decade, the He group has been investigating the physiological importance of RNA changes during major cellular developmental events such as proliferation and differentiation.

His research aided in the development of “epitranscriptomics,” an intriguing new discipline of biology that investigates the epitranscriptome, which refers to the collective set of changed, or tagged, RNA.

In 2023, he received the prestigious Wolf Prize in Chemistry for his groundbreaking discovery of reversible RNA methylation and its significance in gene regulation. He was just awarded the 2023 Tetrahedron Prize for Creativity in Bioorganic and Medicinal Chemistry, as well as being nominated for the 2023 Falling Walls Science Breakthrough of the Year in life science.

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