What Are Obelisks? Unknown Genetic Entities Hidden in the Human Microbiome

As our exploration of the microbial world continues to deepen, unexpected discoveries keep adding new layers to biology. Hidden within the microbial communities of our bodies is a class of RNA molecules that belong neither to viruses nor quite to viroids, yet are still capable of replicating through their hosts. These RNA genetic elements, known as obelisks, have likely been present in transcriptome studies for years, but because they show no recognizable sequence homology to known organisms, they were easy to overlook until recently, when large-scale computational analyses pulled them out of the data. They show no obvious phylogenetic relationship to known organisms, viruses, or viroids, do not fit within traditional classification frameworks, and yet appear with striking prevalence across both human-associated and environmental microbiomes.

A defining feature of obelisks is that they are short RNA molecules with extensive secondary structure. Their genomes are usually around one thousand nucleotides in length, often forming circular RNAs, and are predicted to fold into rod-like secondary structures. Their high degree of internal complementarity resembles that of certain viroids, such as plant viroids. However, unlike viroids, obelisks contain RNA sequences that can encode proteins, whereas viroids are entirely non-coding.

Structure of an obelisk (RNA)（圖片來源：Zheludev IN et al. (2024)，採用 CC BY-NC 4.0 授權）

Obelisk-α. Obelisks exist in both positive-sense and negative-sense forms, with the negative-sense strand potentially involved in replication（圖片來源：Zheludev IN et al. (2024)，採用 CC BY-NC 4.0 授權）

The first obelisk discovered in the study was named obelisk-α, and it was found to encode two proteins, designated Oblin-1 and Oblin-2. These proteins have no known sequence counterparts in existing databases, but structural prediction suggests that Oblin-1 adopts a stable tertiary structure with a core built from multiple α helices and a pair of β strands, implying that it is likely functional rather than a random translational byproduct. Oblin-2 appears structurally simpler, consisting of a single elongated α helix with features of a leucine zipper, suggesting that it may form dimers or trimers.

(A,B) Predicted structure of the Oblin-1 protein; (C) Predicted structure of the Oblin-2 protein; (D) Oblin-2 may form dimers or trimers（圖片來源：Zheludev IN et al. (2024)，採用 CC BY-NC 4.0 授權）

Using Oblin-1 and Oblin-2 as hallmarks, the researchers expanded their search across approximately 5.4 million publicly available sequencing datasets worldwide and ultimately compiled 29,959 distinct obelisks. This makes it clear that obelisks are not rare accidents, but rather a widespread component of Earth's ecosystems. Analyses of human microbiome datasets further showed that obelisks can be detected in about 10% of human samples, while in the oral microbiome their prevalence rises to more than half of the sampled individuals. Because the search strategy relied on Oblin proteins as markers, the obelisks recovered in this study likely represent a homologous group, while non-homologous obelisks, if they exist, were not further examined here. The study also revealed that some obelisks encode only Oblin-1 and lack Oblin-2, indicating that substantial variation exists within this class.

Genomes of 15 obelisks and the predicted structures of their Oblin-1 proteins（圖片來源：Zheludev IN et al. (2024)，採用 CC BY-NC 4.0 授權）

One of the most important findings of the study was the successful identification of a host for an obelisk in the common human oral bacterium Streptococcus sanguinis. An RNA element named obelisk-S.s was shown to stably persist inside bacterial cells and to exist entirely as RNA, with no corresponding DNA form. Its replication therefore likely depends directly on the host cell's transcriptional machinery. This suggests that obelisks may represent a class of bacterium-associated RNAs, although whether related elements also occur in eukaryotic cells remains an open question.

Obelisks also appear to have little obvious effect on their bacterial hosts. Under nutrient-replete conditions, bacteria that lost obelisk-S.s showed almost no detectable differences in growth rate, doubling time, or final culture density. This dispensable nature gives obelisks the appearance of a commensal or low-impact colonist. At least under the conditions tested so far, they do not seem to harm their bacterial hosts, nor do they appear to provide any essential contribution to core host physiology.

The replication mechanism of obelisks remains unknown. Like viroids, they may replicate by directly exploiting host RNA polymerases. In addition, some obelisks contain motifs corresponding to self-cleaving ribozymes, structures commonly associated with rolling-circle replication. This raises the possibility that at least some obelisks propagate their genetic material through a similar RNA-based mechanism.

Another intriguing observation is that obelisks leave almost no detectable trace in bacterial CRISPR systems. CRISPR acts as a bacterial immune memory, typically recording past encounters with foreign genetic invaders such as bacteriophages. Yet in large-scale analyses, there was almost no convincing evidence that obelisks had been captured as CRISPR spacers. This may indicate that their relationship with their hosts is relatively mild and fails to trigger a strong immune response, or that their mode of persistence makes them difficult for host cells to recognize as foreign threats.

Obelisks combine features that seem to belong to different biological categories. Their genomes resemble viroid-like RNA elements, yet they also carry protein-coding capacity more reminiscent of viruses, while differing from both known viroids and viruses in their replication strategies and biological behavior. Because of this unusual combination of traits, obelisks may represent a previously unrecognized form of genetic information carrier.

Author: Shui-Ye You

Reference:

Zheludev IN et al. (2024). Viroid-like colonists of human microbiomes. Cell.