Making Invisible RNA Visible: Discriminative Sequencing Methods for RNA Molecules with Specific Terminal Formations

Abstract

Next generation sequencing of RNA molecules (RNA-seq) has become a common tool to characterize the expression profiles of RNAs and their regulations in normal physiological processes and diseases. Although increasingly accumulating RNA-seq data are widely available through publicly accessible sites, most of the data for short non-coding RNAs (sncRNAs) have been obtained for microRNA (miRNA) analyses by standard RNA-seq, which only capture the sncRNAs with 5′-phosphate (5′-P) and 3′-hydroxyl (3′-OH) ends. The sncRNAs with other terminal formations such as those with a 5′-hydroxyl end (5′-OH), a 3′-phosphate (3′-P) end, or a 2′,3′-cyclic phosphate end (2′,3′-cP) cannot be efficiently amplified and sequenced by standard RNA-seq. Due to the invisibility in standard RNA-seq data, these non-miRNA-sncRNAs have been a hidden component in the transcriptome. However, as the functional significances of these sncRNAs have become increasingly apparent, specific RNA-seq methods compatible with various terminal formations of sncRNAs have been developed and started shedding light on the previously unrecognized sncRNAs that lack 5′-P/3′-OH ends. In this review, we summarize the expanding world of sncRNAs with various terminal formations and the strategic approaches of specific RNA-seq methods to distinctively characterize their expression profiles.

Extracellular tRNA-derived RNAs as emerging activators of endosomal Toll-like receptors: a narrative review

“…Cellular sncRNA molecules generally possess either a hydroxyl group (OH), a monophosphate (P), or a 2',3'-cyclic phosphate (cP) at their termini (Figure 1A), and the terminal states of each sncRNA are determined by the catalytic machinery underlying the RNA cleavage that produces them (21,24). Although next-generation sequencing of RNA molecules (RNA-seq) has become a common tool to characterize RNA expression profiles, most sncRNA sequencing studies to date have relied on a standard small RNA-seq method in which 5'-and 3'-adaptors (AD) can be ligated only to 5'-P and 3'-OH ends of RNAs, respectively.…”

The essential roles of small non-coding RNAs and RNA modifications in normal and malignant hematopoiesis

“…In order to facilitate adapter ligation for RNA-seq of small and large RNAs, T4 polynucleotide kinase (T4PNK) is applied. It is also noticeable that T4PNK pretreatment of RNA should be applied according to the specific requirement (T4PNK is not recommended for research on miRNA due to the higher level of non-miRNA reads) (Shigematsu and Kirino, 2022). Based on PANDORA-seq, tsRNA and rsRNA have been observed largely expressed in the mouse brain, liver, and HeLa cells (Liu et al, 2023).…”

Challenges inherent to the sequencing and quantification of short non‐coding RNAs and how to address them

“…Specific sequencing and quantification of short non‐coding RNAs (sncRNAs). (A) Chemical structures of sncRNA termini (modified from our previous review 2 ). (B) Targeted sncRNAs in standard RNA‐seq, cP‐RNA‐seq, and phospho‐seq.…”

“…Standard RNA‐seq relies on 5′‐P/3′‐OH ends of sncRNAs for adaptor (AD) ligations, and thus, it cannot capture non‐miRNA‐sncRNAs without the 5′‐P/3′‐OH ends (Figure 1B). To address the issue of this invisibility of non‐miRNA‐sncRNAs, specific RNA‐seq methods compatible with various terminal formations of sncRNAs have been developed, 2 enabling characterization of the previously unrecognized sncRNAs. For example, cP‐RNA‐seq can specifically amplify and sequence 2′,3′‐cP‐containing sncRNAs 3,4 (Figure 1B), revealing the expression profiles of 5′‐transfer RNA (tRNA) halves and other 2′,3′‐cP‐containing sncRNAs 5 in diseases and germline development 3,6,7 .…”