For more than 50 years, ribosomal DNA (rDNA) spacer promoters have remained something of a genomic mystery. First characterized through pioneering work by the late Ron Reeder and others, these regions sit upstream of ribosomal RNA genes, yet their precise function—and the reason they are occupied by the mammalian insulator protein CTCF—has remained unclear. Now, new work from Steve and Jorja Henikoff in the Basic Sciences Division published in Science Advances uncovers an unexpected role for these spacer regions as sources of highly expressed microRNA precursors linked to cancer biology.
MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression by targeting messenger RNAs for degradation or translational repression. Although many miRNAs have been implicated in cancer, researchers are still uncovering where some of these transcripts are encoded in the genome and how they are regulated. The project began when the researchers noticed that one of the strongest candidate cis-regulatory elements across multiple tumor and normal tissue datasets had been annotated not as a conventional regulatory sequence, but as a microRNA gene.
“We were intrigued by this microRNA outlier highly expressed in both tumor and normal tissue,” explained Dr. Steve Henikoff. Mining their own previously published transcriptional and chromatin profiling datasets revealed that roughly a dozen annotated microRNAs mapped not to conventional genomic loci, but instead to ribosomal gene arrays located on the short arms of the five human acrocentric chromosomes, i.e., chromosomes with extremely off-center centromeres, each with a long arm and a short arm.
Among the most striking findings were two microRNAs—miR-1275 and miR-6724—embedded within the spacer promoter itself. Further analysis of nascent transcription datasets uncovered a single ~50 nucleotide precursor transcript spanning the annotated boundaries of both miRNAs. The transcript appeared to be exported so rapidly from the nucleus that it was nearly absent from pulse-chase datasets, becoming detectable primarily as mature processed microRNAs.
“What is remarkable,” said Dr. Henikoff, “is that both miR-1275 and miR-6724 have been identified as the most highly upregulated microRNAs in a variety of cancers.” miR-1275 alone has been associated with thousands of cancer-related studies across multiple tumor types. The authors propose that this enrichment may reflect hypertranscription, a widespread increase in global transcriptional activity that is considered a hallmark of many cancers.
Beyond the implications for cancer-associated microRNAs, the study also revisits the biology of the spacer promoter itself. The team found that the human spacer promoter forms a compact ~400-base pair CTCF-insulated loop that co-localizes with promoter pre-initiation complexes and RNA Polymerase pause-release factors including Mediator, DSIF, and NELF. These observations support a model in which the spacer promoter functions as an independently regulated transcriptional unit rather than simply serving as a passive upstream element for ribosomal RNA transcription.
Their work also highlights how important biology can remain hidden in plain sight. Although the nascent transcription datasets used in the study—including PRO-seq and NET-seq datasets—have been publicly available for years, the relevant signals were largely overlooked because ribosomal DNA regions were excluded from standard human genome assemblies such as hg19 and hg38, which underpin the vast majority of publicly available analyses. “Jorja and I attribute this oversight to the widespread reliance on standard annotations and computational pipelines for data analysis, rather than just looking at the data,” Dr. Henikoff said. In this case, simple visualization of alignment tracks and examination of outlier signals proved sufficient to uncover an unexpected transcriptional landscape within rDNA repeats.
Dr. Henikoff connected this idea to a century-old piece of scientific advice from geneticist William Bateson: “Treasure your exceptions.” The message, he noted, feels particularly relevant in an era increasingly shaped by automated computational workflows and machine-learning approaches designed to identify common patterns—potentially at the expense of the biological outliers that can lead to fundamental discoveries.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Member Dr. Steven Henikoff contributed to this research.
The spotlighted research was funded by the Howard Hughes Medical Institute.
Henikoff S, Henikoff JG. 2026. Superabundant microRNAs are transcribed from human rDNA spacer promoters insulated by CTCF. Science Advances. DOI: 10.1126/sciadv.aec1451.
Science Spotlight writer Jenny Waters is a postdoctoral research fellow in the Hsieh lab at Fred Hutch. She studies how mRNA translation coordinates bladder cancer transformation and metastasis by post-transcriptionally regulating expression of oncogenic proteins. Outside of the lab, Jenny enjoys spending time with her dogs, convincing her husband to join her on trail runs, and pretending every steep hill is just a "gentle incline."