Pre-mRNA that codes for inflammatory protein identified

In a discovery that upends a longstanding tenet of human biology, University of Utah School of Medicine researchers have shown that a key process in gene regulation can occur in human platelets, unique cells that are unusual because they don't have a nucleus (anucleate).

Scientists long have thought the transformation of pre-mRNA into mature mRNA--called splicing--happens only in a cell's nucleus. But using stem cells from human umbilical cord blood to engineer the precursor cell that forms platelets and platelets isolated from the blood of study subjects, the Utah researchers found that splicing also takes place in the cytoplasm of circulating platelets.

The U researchers, who report their findings in the Aug. 12 edition of Cell, also identified the pre-mRNA in blood platelets that codes for Interleukin 1â (IL-1â), a key protein in an ancient molecular system that plays major roles in inflammation, defense against infection, organ development, and disease. When blood platelets are activated through biochemical signals in response to injury, the IL-1â pre-mRNA is processed into the mature mRNA and then directs production of the critical inflammatory protein.

Finding that platelets can splice the IL-1â pre-mRNA was completely unexpected and emerged while the researchers were engaged in earlier studies of how platelets communicate with certain leukocytes (white blood cells). During that investigation they found evidence of platelets making new proteins, which led them to pursue the mechanisms that are involved, said Guy A. Zimmerman, M.D., professor of internal medicine and one of the study's co-authors.

"The idea that blood platelets could make proteins without having a nucleus had been thought heretical," said Zimmerman, who also heads the U's Program in Human Molecular Biology and Genetics at the Eccles Institute of Human Genetics. "To find that splicing takes place outside the nucleus has potential implications beyond the platelet. It suggests that, under the right circumstances, splicing can take place a long way from the usual command and control mechanisms (nucleus), and that opens new possibilities for where this key process could occur in other cells types."

Zimmerman theorizes that splicing outside the nucleus is one of a number of intricate ways the body maintains more precise control over gene expression. Many of these control mechanisms are just coming to light.

Platelets are abundant cells that circulate in human blood and have many functions. Their primary role is to form "plugs" that stop bleeding from injured blood vessels. They also release factors that promote tissue repair and mediate inflammation. But when platelets malfunction, they can cause clots that are not required to repair injury and that then contribute to life-threatening disease, such as blocking of arteries that supply the heart; when blood has too few platelets and clotting is impaired, hemorrhaging can occur.

Blood platelets are formed in bone marrow when a parent cell, a megakaryocyte, sends out arm-like extensions called proplatelets. Platelets bud off from these extensions, separating from the cell body and nucleus, and mature platelets then enter the bloodstream.

Andrew S. Weyrich, Ph.D., research associate professor of internal medicine and the study's corresponding author, devised a way to induce stem cells to turn into megakaryocytes that could be grown in the laboratory.

Platelets are among the few cells that don't have a nucleus; for this reason they were thought incapable of turning on genes and synthesizing proteins. But Weyrich and Zimmerman and have shown in recent work that blood platelets contain many messenger RNAs (mRNAs). mRNAs are the molecules that translate the genetic information from DNA and direct the synthesis of proteins.

The genetic information that mRNAs translate to produce proteins is contained in DNA. The process of transcription encodes this information into pre-mRNAs that are acted on by a complex of molecules called the spliceosome. Along with the necessary genetic information, pre-mRNAs also contain sequences of code not needed for making proteins. These unnecessary sequences are excised during splicing. When all the unnecessary sequences have been spliced out, the spliceosome reassembles the mRNA into its mature form with only the genetic information needed to produce a protein.

In all cases prior to this study, splicing had taken place in the nucleus, after which the mature mRNA leaves the nucleus to direct production of the protein in the surrounding cytoplasm.

Identifying how the pre-mRNA matures to direct production of the IL-1â inflammatory protein sheds light on the role of platelets in both defending against injury and infection and contributing to disease, according to Zimmerman. IL-1â induces inflammatory responses in a number of other cells, and in particular, certain leukocytes and the endothelial cells that line blood vessels. In response to IL-1â, endothelial cells make factors that attract inflammatory cells and contribute to clot formation. But if the production of IL-1â goes haywire, it can cause disease, such as blocking arteries by depositing leukocytes and inducing clots that stop blood flow.

The discoveries have immediate clinical relevance, according to Zimmerman.

"This gives new insight into how disease mechanisms work," he said. "If we could learn how to interrupt it, we might be able to turn down the inflammatory process."

Melvin M. Denis, who was an M.D.-Ph.D. student at the U medical school when he died in an avalanche last year, and Neal D. Tolley, of the Eccles Institute of Human Genetics, are co-first authors of the study. Other investigators on the Utah research team, directed by Weyrich and Zimmerman, also made major contributions.