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Screening Study IDs Functions for Conserved Proteins in 'Unknome' Collection


NEW YORK – Using a systematic screening strategy, a UK team has started characterizing proteins from its "unknome" database that are conserved across species but have yet-to-be-determined functions.

"It has become clear that scientific research tends to focus on well-studied proteins, leading to a concern that poorly understood genes are unjustifiably neglected," co-senior and co-corresponding author Sean Munro, a researcher with the MRC Laboratory of Molecular Biology, and his colleagues wrote in PLOS Biology on Tuesday. "To address this, we have developed a publicly available and customizable 'unknome database' that ranks proteins based on how little is known about them."

Focusing on 260 proteins that are conserved in fruit flies and humans, the team used RNA interference (RNAi) screening to systematically characterize the consequences of knocking out the "unknome" proteins in a Drosophila model organism — from functional roles in fertility or development to stress response, movement, or protein quality control.

"The main purpose of our functional studies was to show that well-conserved proteins that have no known function are doing important things that can be detected in laboratory conditions rather than being required to deal with some obscure set of environmental conditions or pathogens," Munro said in an email.

The researchers turned to CRISPR-Cas9-based gene editing to validate a subset of the functional roles suggested by the RNAi screening. In editing experiments centered on a protein encoded by the CG11103 gene, which corresponds to the TM2D2 gene in humans, for example, they identified a Notch signaling pathway role that was reflected in the wing growth changes found in fruit flies lacking the gene.

Along with results from a prior study that found endosomal roles for TM2D and related proteins in human cells, the team speculated that proteins containing the TM2 domain may have "fundamental roles in cell function rather than an exclusive role in Notch signaling."

"Taken together, this genetic validation data confirms that the RNAi screening approach, despite its known caveats, has given accurate phenotypic information for at least a substantial subset of the hits from our RNAi screens of the unknome set of genes," the authors explained.

The screen also highlighted genes suspected of contributing to fertility. There, investigators individually knocked out 198 genes with unknown functions in the germline of male or female fruit flies that were subsequently tracked over time to follow fertility features such as brood size. After validation testing with CRISPR-Cas9-based gene editing, they highlighted two conserved genes that are required for fertility in male fruit flies.

More broadly, their results point to the possibility of unearthing still other contributors to new or known biological processes by systematically digging into "neglected" portions of the proteome spelled out in the unknome collection in the future.

"The unknome database is a key aspect of our work as we hope that others will use it as a tool to select interesting, understudied proteins in their favorite model organism, perhaps with a focus on a particular organelle or class of protein," Munro said. "It will also allow people to quickly find out what is known about the relatives of their favorite protein in other organisms." 

For example, Munro's team is continuing to assess proteins identified in the current screen, particularly focusing on proteins with potential ties to the membrane trafficking-related processes that the group studies. To help such efforts along, the investigators are using machine learning methods to try to predict potential functions for proteins based on expression and evolutionary conservation clues, Munro said.

"Overall, our approach demonstrates that significant and unexplored biology is encoded in the neglected parts of proteomes," he and his colleagues wrote.