Developing Small RNA Biomarkers through RNA Sequencing

Non-coding RNAs, also known as RNAs that lack the ability to encode proteins, are produced through transcription from the genome. However, unlike coding RNAs, they do not undergo translation into proteins. Instead, these non-coding RNAs carry out their specific biological functions at the RNA level. In terms of their size, we classify non-coding RNAs smaller than 50 nt as small non-coding RNAs (sncRNAs), encompassing miRNAs, piRNAs, rsRNAs, tsRNAs, and other types of small RNAs.

Refer to our article Small RNA Sequencing: Introduction, Workflow, and Applications for more information.


MicroRNAs (miRNAs) refer to a class of small molecule RNAs, typically 20-25 nucleotides long, that exist naturally within eukaryotes. They are non-coding in nature and have been extensively studied. miRNAs are highly conserved throughout evolution and play a crucial role in inhibiting gene expression at the post-transcriptional level. They achieve this by specifically binding to messenger ribonucleic acids (mRNAs) that are their target. Due to the limited complementarity between miRNAs and their targets, they often form incomplete pairings. Consequently, miRNAs can simultaneously regulate the expression of multiple target genes. Studies estimate that miRNAs have a role in regulating over 30% of the protein-coding genes present in the human genome. Dysregulation of miRNAs has been frequently linked to various diseases, including cancer, diabetes, and cardiovascular disorders.

Recommended Reading: Overview of MicroRNAs (miRNAs).


tsRNA (tRNA-derived small RNA) represents a recently discovered class of non-coding small RNAs that arise through specific cleavage of various tRNAs or tRNA precursors by distinct enzymes. These RNAs are also known by alternative names such as tRF, tiRNA, and tRNA halves. Extensive research has elucidated the multifaceted biological functions exhibited by tsRNAs, encompassing the regulation of stress response, tumorigenesis, and epigenetic processes.

The biological functions of tsRNA can be categorized as follows:

Regulation of mRNA stability

Similar to microRNAs, tsRNAs exert microRNA-like effects by targeting mRNAs, thereby influencing their stability.

Modulation of protein translation

(a) Enhancement of translation through ribosome biogenesis: Studies in mice and humans have demonstrated that tsRNALeuCAG binds to the mRNA of the ribosomal protein RPS28, promoting its translation.

(b) Inhibition of translation via ribosome binding: tsRNA interacts with components of ribosomes, such as the small subunit 16S rRNA, leading to a general repression of translation.

(c) Regulation of translation initiation: tsRNA can form intermolecular RNA G tetramers (RG4) to replace the translation initiation factor eIF4 complex, thus inhibiting translation. Moreover, the RG4 structure can bind to YB-1 and impede the assembly of the translation initiation complex.

Epigenetic regulation

tsRNA serves as an epigenetic regulator by modulating various epigenetic processes to influence gene expression. For instance, it can interact with PIWI proteins to promote H3K9 methylation.

Regulation of transposons

18-nt-3' tsRNAs in HeLa cells compete with mature tRNAs for binding to the primer binding site of reverse transcriptional transposons, thereby obstructing their reverse transcription.

tsRNAs carry diverse RNA modifications inherited from tRNAs, which contribute to the expanded repertoire of RNA structures and functions. However, these modifications hinder the preparation of RNA-seq libraries, rendering conventional RNA-seq methods incapable of detecting the presence of tsRNAs. Consequently, the study of tsRNA functions encounters obstacles in this regard.

tsRNA and its Implications as Disease Markers

Viral infection: The presence and levels of tsRNA exhibit significant alterations in tissue cells infected with hepatitis B virus (HBV) or hepatitis C virus (HCV).

Stress: tsRNAs circulating in the peripheral blood demonstrate a remarkable sensitivity to acute inflammation, aging, energy restriction, acute kidney disease, and tissue injury. Consequently, tsRNAs may serve as a valuable class of non-invasive biomarkers, offering insights into the aforementioned conditions.

Tumors: Aberrant regulation of tsRNAs has been observed across a diverse range of cancers. These dysregulated tsRNAs hold promising potential as disease-specific biomarkers, facilitating accurate disease diagnosis, guiding targeted therapeutic interventions, and providing prognostic indicators.

Methods of Exploring tsRNA-Protein/RNA Interactions

In the rapidly advancing field of molecular biology, understanding the intricate interactions between tsRNAs (tRNA-derived small RNAs) and proteins/RNAs is crucial. By studying these interactions, researchers can unravel the roles and functions of tsRNAs in various biological processes. This article explores four cutting-edge methods that have revolutionized the investigation of tsRNA-protein/RNA interactions: RIP-seq, CLIP-Seq, PAR-CLIP Seq, and CLASH Seq.

RIP-seq (RNA Immunoprecipitation Sequencing)

RIP-seq represents a widely employed technique employed by researchers to discern the RNA molecules that engage in interactions with specific proteins. This method encompasses the immunoprecipitation of protein-RNA complexes through the use of antibodies specifically targeting the protein of interest. Subsequently, the isolated RNA is subjected to high-throughput sequencing, thereby facilitating the identification of transfer RNA-derived small RNAs (tsRNAs) that are associated with the protein. Through its ability to provide valuable insights into the interplay between tsRNAs and specific proteins, RIP-seq serves to illuminate the functional roles assumed by these interactions.

Recommended Reading: RIP-Seq: Introduction, Features, Workflow, and Applications.

CLIP-Seq (Cross-Linking Immunoprecipitation Sequencing)

CLIP-Seq constitutes a potent methodology that not only enables the identification of tsRNAs engaging with proteins but also offers information regarding the precise binding sites involved. This process entails the cross-linking of RNA-protein complexes utilizing ultraviolet (UV) light, which is subsequently followed by immunoprecipitation and high-throughput sequencing. By capturing the precise sites of interaction, CLIP-Seq assists researchers in deciphering the molecular mechanisms underlying tsRNA-protein interactions.


PAR-CLIP Seq (Photoactivatable Ribonucleoside-Enhanced Cross-Linking Immunoprecipitation Sequencing) builds upon the fundamental principles of CLIP-Seq by integrating photoactivatable ribonucleoside analogs (PARs) to heighten the efficiency of cross-linking. This enhanced methodology enables more accurate identification of tsRNA binding sites, enhancing the ability to detect weak or transient interactions. By employing PAR-CLIP Seq, researchers can unveil more intricate details pertaining to tsRNA-protein interactions and discern their consequential functional ramifications.


CLASH Seq (Cross-Linking, Ligation, and Sequencing of Hybrids) is a technique that investigates both RNA-RNA and RNA-protein interactions. It involves cross-linking RNA molecules with their interacting partners, followed by ligation and sequencing of the resulting RNA-RNA chimeras. CLASH Seq enables the identification of tsRNAs that interact with other RNA molecules, such as mRNAs or lncRNAs, shedding light on the complex regulatory networks involving tsRNAs.


PIWI-interacting RNA (piRNA), a distinct class of small RNAs ranging from 24 to 31 nucleotides in length, holds considerable promise as a biomarker and innovative therapeutic target for the early detection and prognostic evaluation of malignant tumors. By associating with PIWI proteins, piRNAs exert their influence on tumor epigenetics, modulating the levels of post-transcriptional mRNAs and protein stability, and actively participating in various malignant processes, including proliferation, invasion, and metastasis. Furthermore, piRNAs have garnered more attention in scientific literature compared to tsRNAs due to their well-established roles in regulating transposon silencing and reproductive development.

PiRNAs employ several pathways to execute their functions, each of which has distinct mechanisms. First, the histone modification pathway involves piRNA-induced histone modification, leading to chromatin condensation and subsequent inhibition of transposon translocation. Second, the DNA methylation pathway entails piRNA-mediated H3K9 methylation and H3K4 demethylation, resulting in the recruitment of DNA methyltransferases. This recruitment leads to DNA methylation in LINE-1 DNA, which effectively suppresses LINE-1 transcription. The third pathway, known as the ping-pong loop pathway, involves a secondary amplification of piRNAs that coincides with the substantial depletion of transposon mRNA. This depletion ultimately represses transposition. Finally, the DNA elimination pathway plays a crucial role in specific organisms. In Tetrahymena, the scnRNA and Twi1p complexes originating from the maternal micronucleus facilitate the deletion of the entire IESs region in the merging macronucleus. Similarly, in C. acuminata, template RNAs produced in the maternal macronucleus enter the merging macronucleus, directing the deletion of IESs and reordering of disorganized MDSs. Both mechanisms effectively delete transposon gene sequences, resulting in the repression of transposition.

rsRNA (rRNA-derived small RNAs)

Can ribosomal RNAs (rRNAs), which play a crucial role in cellular housekeeping, generate small RNAs akin to tsRNAs? In 2017, a research team made a significant discovery of small non-coding RNAs (sncRNAs) derived from 28S rRNAs. Remarkably, these sncRNAs, referred to as rsRNA-28S (rRNA-derived small RNAs-28S), exhibited striking similarities to known tsRNAs in terms of their sequence characteristics. Intriguingly, a previous study involving RNA-seq analysis of small RNAs in mouse sperm had dismissed the rsRNA-28S sequence as inconsequential, thereby disregarding its potential significance. However, when several human sperm samples were meticulously screened, notable alterations in rsRNA-28S expression were observed specifically in leukocytoclastic sperm derived from individuals with inflammatory disorders of the reproductive tract. This investigation sheds light on the existence of an rsRNA variant closely resembling known tsRNAs and underscores the limitations of current genome mapping methodologies employed in small RNA sequencing analysis. Notably, rsRNA, abundantly expressed in mature spermatozoa and susceptible to environmental influences, possesses predictive capabilities on par with those of tsRNAs.

CD Genomics Small RNA Sequencing Solution

Traditional small RNA sequencing library conduction technology primarily focuses on the detection of miRNAs, piRNAs, and similar small RNAs. However, it falls short when it comes to identifying small RNAs that contain unique modifications, such as tsRNAs and rsRNAs, which undergo extensive modifications. In order to address this limitation, we have developed our small RNA sequencing platform, which allows for the comprehensive detection of small RNA profiles, including tsRNA and rsRNA.

Our small RNA sequencing platform employs enzymatic processing to remove modifications that hinder sequencing. By treating small RNAs within the 15-50 nt, we are able to eliminate modifications that interfere with accurate sequencing. This enzymatic processing ensures that we obtain a complete representation of small RNA maps, including tsRNA/rsRNA.

To further enhance our analysis, we utilize the specialized annotation platform for raw signal analysis. This powerful tool enables us to comprehensively analyze the information pertaining to various types of small non-coding RNAs (sncRNAs) present in the samples. It facilitates the identification and annotation of miRNAs, piRNAs, and most importantly, tsRNAs and rsRNAs. This comprehensive analysis lays a solid foundation for investigating the cellular and molecular functions of these lesser-known sncRNAs, opening up avenues for the exploration of previously unknown biological processes.

Small RNA Sequencing Project – CD GenomicsSmall RNA Sequencing Project – CD Genomics

* For Research Use Only. Not for use in diagnostic procedures.

Research Areas
Copyright © CD Genomics. All rights reserved.