Work has potential implications for new therapies to treat cancer and neurodegenerative disorders

A team of Case Western Reserve University researchers has found a way to measure key characteristics of proteins that bind to RNA in cells—a discovery that could improve our understanding of how gene function is disturbed in cancer, neurodegenerative disorders or infections.

RNA—short for ribonucleic acid—carries genetic instructions within the body. RNA-binding proteins play an important role in the regulation of gene expression. Scientists already knew that the way these proteins function depends on their “binding kinetics,” a term that describes how frequently they latch on to a site in an RNA, and how long they stay there.

Until now, researchers could not measure the kinetics of RNA-binding proteins in cells. But the Case Western Reserve researchers answered this longstanding question in RNA biology. The findings open the door to a biochemical understanding of RNA protein interactions in cells.

By understanding the kinetics, researchers can quantitatively predict how an RNA binding protein regulates the expression of thousands of genes, which is critical for developing strategies that target RNA protein interactions for therapeutic purposes.

“The study marks a major step toward understanding how gene function is regulated and how to devise ways to correct errors in this regulation in diseases such as cancer, neurodegenerative disorders or infections,” said Eckhard Jankowsky, the study’s principal author and a professor of biochemistry at the university’s School of Medicine and director of the school’s Center for RNA Science and Therapeutics.

To measure the kinetics of RNA binding proteins, the researchers used a laser that sends out extremely short (femtosecond) pulses of ultraviolet light to cross-link the RNA-binding protein known as DAZL to its several thousand binding sites in RNAs. (DAZL, short for Deleted in Azoospermia-Like, is involved in germ cell development.) They then used high throughput sequencing to measure the change of the crosslinked RNA over time and determined the binding kinetics of DAZL at thousands of binding sites. The resulting “kinetic landscape” allowed the researchers to decode the link between DAZL binding and its effects on RNAs.

Kinetics of DAZL-RNA binding and dissociation in cells

a, Normalized sequencing reads for the 3′-UTR of a representative transcript (Thbs1) at increasing cross-linking times (left) and different protein concentrations and laser powers (right; scale, normalized coverage = 11 for all traces). Reads for conventional individual-nucleotide-resolution cross-linking and immunoprecipitation (iCLIP) are indicated below. b, Cross-linking time courses for two binding sites (sites 1 and 2). c, Association rate constants for 1× DAZL and 4.2× DAZL for all binding sites (n = 10,341). d, Transcriptome-wide distributions of dissociation rate constants (k_diss), association rate constants at high DAZL concentration (k_on^4.2× DAZL), binding probability at high DAZL concentration (P^4.2× DAZL) and maximum fractional occupancy (Φ_max) for all DAZL-binding sites. 

Source – Case Western Reserve University