Ribosomal RNA fragments in small RNA-seq: biological signals or artifacts?

Small RNA sequencing (small RNA-seq) is widely used to profile short RNA species, most notably microRNAs, in cells, tissues, and biofluids such as plasma, serum, urine, saliva, and cerebrospinal fluid. In practice, however, small RNA-seq libraries consistently contain a substantial fraction of reads that do not correspond to canonical microRNAs. Among these, ribosomal RNA–derived fragments (rRNA fragments; rRFs, also referred to as rsRNAs) are frequently observed, sometimes constituting a major component of the sequenced RNA population.

The recurrent detection of rRNA fragments in small RNA-seq datasets has been documented across large extracellular RNA reference studies, including systematic profiling of plasma, urine, and saliva and comparative analyses across multiple human biofluids^1,2. These studies establish that rRNA fragments are not isolated anomalies but part of a reproducible extracellular RNA landscape.

The presence of rRNA fragments raises questions regarding their origin, interpretation, and potential biological relevance. On one hand, rRNA is the most abundant RNA species in cells, making it an obvious substrate for fragmentation during RNA turnover, extracellular exposure to RNases, and sample handling. On the other hand, growing evidence suggests that at least some rRNA fragments are generated in a non-random manner and can be selectively enriched in specific extracellular compartments. Understanding whether rRNA fragments in small RNA-seq should be interpreted primarily as technical or degradation byproducts, or instead as a regulated and potentially functional RNA species, is essential for correct data interpretation, biomarker discovery, and assay design. The current literature provides some conclusions but also leaves major gaps.

Summary of current knowledge

Multiple studies have established that extracellular RNA populations are compositionally complex and biofluid-specific. Reference atlases demonstrate that non-miRNA species (including tRNA fragments, Y RNA fragments, and rRNA-derived fragments) are reproducibly detected across cohorts, laboratories, and platforms³. This observation does not depend on a single protocol or analytical pipeline, indicating that rRNA fragments are a recurring component of small RNA-seq outputs rather than sporadic contaminants.

Importantly, the presence of rRNA fragments in extracellular RNA pools is not explained solely by intracellular RNA decay. Experimental work has shown that ribosomes and ribosomal subunits can exist in non-vesicular fractions of plasma and other biofluids. Once extracellular, these ribosomes can undergo fragmentation, generating stable populations of small rRNA fragments, including characteristic 5′ fragments of 28S rRNA⁴. This provides direct evidence that a substantial fraction of extracellular rRNA fragments can be generated after cellular release.

Despite their prevalence, functional relevance has not been demonstrated for most rRNA fragments detected in biofluid small RNA-seq. To date, there is only one clear example of biological activity: an rRNA fragment packaged into extracellular vesicles secreted by airway epithelial cells was shown to alter antibiotic sensitivity in Pseudomonas aeruginosa⁵.

Additional evidence relevant to potential biological interpretation comes from intracellular systems, where rRNA fragmentation has been shown to be non-random. A large meta-analysis across public datasets revealed reproducible rRNA fragment boundaries and enrichment of specific fragments in Argonaute immunoprecipitations⁶. However, the functional significance of this non-random biogenesis has not been demonstrated, and it remains unclear whether this finding can be applied to circulating biofluids remains uncertain.

Several challenges complicate interpretation of rRNA fragments in small RNA-seq. A major unresolved issue is carrier attribution: rRNA fragments may derive from extracellular vesicles, non-vesicular ribonucleoprotein complexes, extracellular ribosomes, or co-isolated material introduced during sample processing. Isolation strategy has a strong impact on observed RNA composition, and there is currently no consensus framework to assign rRNA fragments to specific carriers. Recent methodological perspectives in the extracellular vesicle field emphasize substantial variability in isolation, RNA extraction, sequencing, and bioinformatic workflows, underscoring the need for improved standardization to enable reliable interpretation of extracellular RNA profiles⁷.

Bioinformatic ambiguity further complicates interpretation. rRNA genes are repetitive and highly similar, leading to frequent multi-mapping between nuclear and mitochondrial rRNAs or between mature and precursor rRNA regions. As a result, reported rRNA fragment profiles can vary substantially depending on reference annotation and alignment strategy, and robustness across analytical pipelines is rarely tested.

Finally, RNA modifications add an additional layer of complexity. Approaches such as PANDORA-seq demonstrate that many rRNA-derived small RNAs are invisible to standard small RNA-seq due to chemical modifications that block ligation or reverse transcription^8-10. While this establishes that rRNA fragment diversity is underestimated, it remains unclear which of these fragments are biologically meaningful in extracellular contexts.

Conclusion

rRNA-derived fragments are a consistent feature of small RNA-seq data from biofluids, shaped by both extracellular RNA biology and technical constraints. While select rRNA fragments show evidence of non-random generation and rare biological activity, most are best interpreted as products of RNA turnover and processing unless they are reproducible, carrier-associated, and tied to a biological hypothesis. Future research combining chemistry-aware small RNA-seq with rigorous fractionation, standardized bioinformatics, and functional validation will be essential to determine when rRNA fragments represent meaningful biological signals rather than pervasive background.