When immune T cells encounter the enemy-a virus, bacteria or other foreign invader-they transform from a state of quiet readiness to fully armed fighters. This process of T cell activation, which occurs in a day or less, requires the cell to produce a host of new proteins to fill its arsenal.

The goal of IDI Senior Investigator Anjana Rao's research is to understand how this rapid alteration in the protein profile, or proteome, of T cells occurs, and how it promotes an effective immune response.

Recently, Rao postdoctoral fellow Shalini Oberdoerffer and colleagues uncovered an important new piece of the puzzle. In a paper published in the August 1 issue of Science, the researchers describe a key regulatory protein that is induced during T cell activation. The novel protein, called heterogeneous ribonucleoprotein L-like (hnRNPLL), controls the differential processing of dozens of T cell messenger RNAs, thus altering the proteins that are finally produced.

"Our results show that modulation of RNA splicing is a very strong mechanism for shifting the proteome in T cells after activation," Oberdoerffer said.

The work pinpoints hnRNPLL as a central regulator of RNA splicing in response to the environmental signals that activate T cells, Rao added.

Oberdoeffer and coworkers set out to answer a specific question about the regulation of a single cell surface protein during T cell activation. The protein, CD45, is a transmembrane signaling molecule that is abundant on both T and B immune cells. Alternative pre-messenger RNA splicing of exons 4 through 6 produces 5 different variations of CD45 in human cells. Unstimulated T cells and most B cells express longer forms of the protein, called CD45RA. Activated T cells and memory T cells express a shorter form, CD45RO. Despite 20 years of study, the functions of the CD45 isoforms, and the reasons why and how they are produced in activated T cells, was largely unknown.

To find out what regulates the CD45 RA to RO shift during T cell activation, Oberdoerffer and colleagues tested 450 different proteins, all known or potential RNA splicing factors. In collaboration with coauthor Nir Hacohen of Massachusetts General Hospital and the Broad Institute, the investigators used short hairpin RNAs to shut each factor off one at a time in T cells that were then treated to trigger the RA to RO switch. Only one of the proteins tested completely eliminated alternative splicing to the CD45RO isoform. This hit was hnRNPLL, a relatively uncharacterized protein with high homology to the known splicing factor hnRNPL.

In subsequent experiments, Oberdoerffer determined that hnRNPLL protein increased when T cells were activated, that it bound CD45 mRNA, and that it was necessary and sufficient to cause the RA to RO switch. These findings are all consistent with the idea that hnRNPLL is the factor that promotes alternative splicing of CD45 when T cells are activated, and the investigators found a similar role for the protein in determining CD45 isoform expression in B cells.

CD45 is just one of hundreds of proteins whose expression is modified during T cell activation, leading Oberdoerrfer and Rao to ask if hnRNPLL might regulate other genes as well. Using a technique to measure global splicing changes in activated T cells, they found 132 other genes that changed their splicing pattern in response to hnNPLL, including diverse players involved in cell proliferation, activation and death.

"Our data strongly suggest that hnRNPLL is a 'master regulator' of alternative splicing in activated T cells," Rao and coauthors write in the paper. "Induction of hnRNPLL during the process of T cell activation and differentiation may represent a mechanism by which the cell can rapidly shift its transcriptome to favor proliferation and inhibit cell death."

The next step, Rao says, is to understand the signaling pathways that regulate hnRNPLL. "There is going to be an entire complex of splicing factors that work together with hnRNPLL, and therefore there is also going to be a complete signaling pathway that leads into its induction," she said.

The findings could help solve the long-standing mystery of how the CD45 isoforms work, and how the splicing affects the protein's function in different cells. Using their new knowledge about hnRNPLL, the investigators can now make cells that express only the RO or RA forms of CD45, which offers them a new tool to look at the isoform function.

Other authors on the study include Rao lab member Daniel Neems, and Luis Ferreira Moita and Rui Freitas, formerly of the Broad Institute and now at the University of Lisbon, Portugal.

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