This preliminary report in Science has just been posted by the authors on bioRxiv — but a complete manuscript has not yet appeared in a respectable scientific journal following peer review. If the presence of a micro-protein is correlated with growth (or inhibition of growth) of a specific type of tumor, correlation still does not necessarily connote causation. Many more studies are required in this infant field.
—DwN
‘Dark proteome’ survey reveals thousands of new human genes
Database confirms that overlooked tiny genes, encoding perhaps 30,000 tiny proteins, not recognized before
25 Nov 2024; 1:15 PM ET. By Elizabeth Pennisi
One of the biggest surprises to emerge when the human genome was first sequenced more than 20 years ago — was how few genes it contained, less than one-third or one-tenth the number some scientists had predicted. Fewer than 30,000 genes and the proteins they encode are enough to build and operate the human body, it seemed; recent tallies have moved this number even lower, to about 20,000. But a new systematic analysis of what some call the “dark proteome” suggests scientists have missed thousands of nontraditional genes that lurk in previously overlooked stretches of the genome and make smaller-than-average proteins.
The newly described genes and their products could upset aspects of human biology and accelerate medical discoveries. For example, one newfound gene makes a miniature protein that appears to be pivotal in a childhood cancer.
“Unless we know what the proteins in the dark proteome are, and how they contribute, our ability to [treat] diseases will be limited,” says Alan Saghatelian, a chemical biologist at the Salk Institute for Biological Studies who was not involved in the new research, described in a preprint posted last month on bioRxiv.
One of the leaders of the analysis, John Prensner, began peering into the dark proteome because so many of his searches among known genes for ones related to cancer turned up empty-handed. “I got interested in what the rest of the genome had to offer,” recalls Prensner, a pediatric neuro-oncologist now at the University of Michigan Medical School.
He and his colleagues expanded the standard definition of a gene, typically assumed to consist of a long protein-coding DNA sequence known as an open reading frame (ORF), which has signals telling the cell where to start and stop reading it. A cell transcribes the ORF sequence into messenger RNA, which travels to cellular factories called ribosomes that assemble amino acid sequences into proteins. A typical ORF is also preceded by a snippet of DNA (enhancers and promoter) that attracts the proteins needed for the gene to be read. And for most researchers, an ORF qualifies as a gene if it encodes a protein with 50-100 or more amino acids.
But biologists studying everything from yeast to snakes to humans have recently unearthed a plethora of so-called noncanonical ORFs, which lack those introductory snippets and are shorter than average. Yet they are often transcribed into RNA, and a method known as ribosomal profiling, or Ribo-Seq, has shown that many of the transcribed RNAs attach to ribosomes, where they may be translated into short amino acid chains — even proteins with less than a dozen animo acids.
Even then, many scientists dismissed the resulting mini-proteins as unimportant, expecting they were “noise” that were quickly degraded. It’s been very challenging to convince people these ORFs are worth a serious look, says Ji-Young Youn, a biochemist at the Hospital for Sick Children in Toronto.
But about 3 years ago, Prensner and colleagues demonstrated that cancer cells contained about 550 of these micro-proteins. Two years earlier, Sebastiaan van Heesch, a systems biologist at the Oncode Institute’s Princess Máxima Center for Pediatric Oncology, found similar numbers of the tiny proteins in heart tissue. “Sebastiaan and I were finding these genes that are very, very cool and we thought the world should know about them,” Prensner says.
So, they teamed up with gene annotation specialist Jonathan Mudge of GENCODE, a database of officially recognized genes, and ultimately recruited several dozen other researchers from 20 institutions across four continents to help assess how many human noncanonical ORFs exist. This “became a sort of super-consortium to bring order to a relatively new field,” says Fergal Martin, a computational biologist at the European Molecular Biology Laboratory’s European Bioinformatics Institute.
This group didn’t conduct experiments of their own, but plumbed what others had done, first combing through ribosome profiling papers. By 2022, the scientists had tracked down 7,264 noncanonical ORFs in the human genome. With the help of the Human Proteome Organization, which seeks to catalog all human proteins, and PeptideAtlas, which compiles mass spectrometry data on proteins, they set out to show that these ORFs make proteins.
That was a “big challenge,” Youn notes. The consortium scoured PeptideAtlas’s archive of mass spectrometry data for small proteins that matched ORF sequences and sorted through published experiments that cataloged protein fragments detected by the human immune system, a blossoming field called immune-peptidomics. All told, they confirmed that ~40 percent of the 7,264 noncanonical ORFs they had tallied made proteins, some 3,000 in all. (An ORF can be read multiple ways to make more than one protein.)
The newly discovered mini-proteins “help provide a more complete picture of the coding portion of the [human] genome,” says Stanford University geneticist Ami Bhatt, whose own studies have explored the dark proteome of microbes.
They also give scientists new biomedical targets for study. Prensner and van Heesch had already begun to follow up on an ORF and its mini-protein they identified early in their dark proteome studies. By using the gene editor CRISPR to introduce mutations in the ORF, they could examine its protein’s importance in cancer cells. Though small, the ORF’s product is essential for the survival of medulloblastoma tumors, a brain cancer that affects children, they reported on 18 January in Molecular Cell.
“It’s not every day that you get to open a research direction and say, ‘We might have a whole new class of drug targets for patients,’” Prensner says. This mini-protein was not in the original analysis of the human genome, yet “it appears to have a critical role in medulloblastoma,” Saghatelian agrees.
Prensner is also advising a Massachusetts-based company called ProFound Therapeutics that has partnered with the drug giant Pfizer to develop treatments for obesity based on micro-protein targets. Another coauthor on the preprint, Thomas Martinez, a protein biochemist at the University of California, Irvine, and his team are chasing down tiny proteins involved in pancreatic cancer and metabolic diseases. “I am most excited about translating microproteins into therapeutic efforts, hopefully as both biomarkers and drug targets,” Martinez says. “Once this barrier is crossed, I think interest in the field will expand dramatically.”
Although Martinez is pleased with how much of the dark proteome has been uncovered, Youn believes much more remains to be found. The work her team and others have done casts just “slivers of light,” she says, on an unseen population of mini-proteins. Her team is refining mass spectrometry techniques to detect ever smaller molecules and hopes to use them to find mini-proteins that play a role in brain development.
Where does all this leave the tally of genes in the human genome? The dark proteome has clearly boosted the total, but no one knows the true number of total functional genes.
“My gut feeling it is probably not as high as 100,000” (including the ~20,000 genes already identified and classified), Martinez says, “but 50,000 is in the realm of possibility.”
doi: 10.1126/science.zxaor8l
COMMENTS:
From: Stringer, Keith
Sent: Wednesday, November 27, 2024 12:35 PM
Dan,
This is really, really cool !
Happy Thanksgiving,
Keith
Keith Stringer, MD
Pathology Department, CCHMC, Cincinnati
From: Christine Curran
Sent: Wednesday, November 27, 2024 12:25 PM
As this short woman has always emphasized, “Small is beautiful!” Actually, what I often say is that: I’m “evolutionarily advanced to use less of the Earth’s precious resources.” But “Small is beautiful” works as the abstract. I’m sure Lucia would agree.
Hope you’re well and ready to enjoy a wonderful Thanksgiving holiday!
c-2
Professor, Northern Kentucky University
From: John Stegeman
Sent: Wednesday, November 27, 2024 4:57 AM
Dan: This breakthrough finding will trigger a lot of re-thinking.
John
Woods Hole, Massacchusetts
From: Doron Lancet
Sent: Wednesday, November 27, 2024 2:47 AM
Dear Dan,
Thanks for letting me know about this highly interesting find!
I am copying my GeneCards team, with a target to possibly doubling the number of protein-coding genes in our database!
Best, Doron
Rehovot, Israel
From: Magnus Ingelman-Sundberg
Sent: Wednesday, November 27, 2024 2:28 AM
Sounds unbelievable. Or extremely interesting.
Best
Magnus
Stockholm, Sweden