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cP formation via RNA cleavage by ribonucleases. (A,B) Cleavage reactions catalyzed by RNase A (A) and ANG (B). (C) Structure of ANG catalytic site (human wild type, PDB ID: 1B1I). Three catalytic residues are shown in pink, and the residues forming a substrate binding pocket and/or associating with Gln117 are shown in green. Orange dotted lines indicate the hydrogen bonds that particularly support Gln117’s obstructive position (Leonidas et al., 1999, 2002; Holloway et al., 2004).
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Cellular RNA molecules contain phosphate or hydroxyl ends. A 2′,3′-cyclic phosphate (cP) is one of the 3′-terminal forms of RNAs mainly generated from RNA cleavage by ribonucleases. Although transcriptome profiling using RNA-seq has become a ubiquitous tool in biological and medical research, cP-containing RNAs (cP-RNAs) form a hidden transcriptome...
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Citations
... Autocatalytic hydrolytic cleavage of RNA strands produces 2',3'-cP residues [21] and such residues can also be formed after enzymatic cleavage [22]. To avoid elaborate assay modifications, we have focused on using the 2',3'-cPcompatible PNPase. ...
Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical methods before clinical use. The current study first demonstrates the effect of heat degradation on transcript translatability and then describes a novel enzymatic approach to assess the integrity of conventional mRNA and long self-amplifying mRNA. By first hybridizing oligo-T to the poly(A) tail of intact mRNA and subsequently digesting the unhybridized RNA fragments with a 3’-5’ exoribonuclease, individual nucleotides can be selectively released from RNA fragments. The adenosine-based fraction of these nucleotides can then be converted into ATP and detected by luminescence as a sensitive indicator of mRNA byproducts. We developed a polynucleotide phosphorylase (PNPase)-based assay that offers fast and sensitive evaluation of mRNA integrity, regardless of its length, thus presenting a novel and fully scalable alternative to chromatographic-, electrophoresis-, or sequencing-based techniques.
... The cleavage of tRNA by T2 RNases has been shown to result mostly in 3ʹ tRNA-derived fragments with 5ʹ-OH ends and 5ʹ fragments with a terminal 2ʹ,3ʹ-cyclic phosphate (cP) or 3' phosphate (P), both of which are recalcitrant to the sRNA sequence library preparation used in this work. Only a low percentage of tRNA-derived fragments possess the conventional 3ʹ-OH and 5'-P ends, and therefore are likely sequenced (27,(39)(40)(41). To overcome these limitations, we selected four tRNAs, tRNA Gly , tRNA Glu , tRNA Ala , and tRNA Lys , and analyzed them using RNA gel blot analysis ( Fig. 6 and SI Appendix, Fig. S12). ...
Transgenic expression of a double-stranded RNA in plants can induce silencing of homologous mRNAs in fungal pathogens. Although such host-induced gene silencing is well-documented, the molecular mechanisms by which RNAs can move from the cytoplasm of plant cells across the plasma membrane of both the host cell and fungal cell are poorly understood. Indirect evidence suggests that this RNA transfer may occur at a very early stage of the infection process, prior to breach of the host cell wall, suggesting that silencing RNAs might be secreted onto leaf surfaces. To assess whether Arabidopsis plants possess a mechanism for secreting RNA onto leaf surfaces, we developed a protocol for isolating leaf surface RNA separately from intercellular (apoplastic) RNA. This protocol yielded abundant leaf surface RNA that displayed an RNA banding pattern distinct from apoplastic RNA, suggesting that it may be secreted directly from the leaf surface rather than exuded through stomata or hydathodes. Notably, this RNA was not associated with either extracellular vesicles or protein complexes; however, RNA species longer than 100 nucleotides could be pelleted by ultracentrifugation. Pelleting was inhibited by the divalent cation chelator EGTA, suggesting that these RNAs may form condensates on the leaf surface. These leaf surface RNAs are derived almost exclusively from Arabidopsis, but come from diverse genomic sources, including rRNA, tRNA, mRNA, intergenic RNA, microRNAs, and small interfering RNAs, with tRNAs especially enriched. We speculate that endogenous leaf surface RNA plays an important role in the assembly of distinct microbial communities on leaf surfaces.
... Zhang et al., 2016). Based on the modification positions, small RNAs can be classified into three groups: (1) modifications at the 5 0 or 3 0 ends of small RNAs, including 5 0 -triphosphate (5 0 -PPP), 5 0 -cap, 5 0 -hydroxyl (5 0 -OH), 3 0 -phosphate (3 0 -P), 2 0 ,3 0 -cP and 3 0 -aminoacyl (3 0aa), which are often formed by the cleavage or enzymatic processing of small RNA precursors (Crocker et al., 2022;Shigematsu et al., 2018;Shigematsu et al., 2021); (2) modifications in the ribose backbone, but not the purine/ F I G U R E 3 Dual-end adapter addition for sRNA library construction using Group II Intron RTases or by random-priming. The sRNA samples undergo common treatment to attain 3 0 -OH prior to library construction. ...
... The most common modification at the 3 0 end of small RNAs is 2 0 ,3 0 -cP, with one phosphate bridging the 2 0 -and 3 0positions of ribose (Shigematsu et al., 2018). In vertebrates, angiogenin homologs mediate the cleavage of tRNA anticodon-loops, resulting in a 5 0 -tsRNA with 2 0 ,3 0 -cP and a 3 0 -tsRNA with 5 0 -OH (Sheng & Xu, 2016;Shigematsu et al., 2018). ...
... The most common modification at the 3 0 end of small RNAs is 2 0 ,3 0 -cP, with one phosphate bridging the 2 0 -and 3 0positions of ribose (Shigematsu et al., 2018). In vertebrates, angiogenin homologs mediate the cleavage of tRNA anticodon-loops, resulting in a 5 0 -tsRNA with 2 0 ,3 0 -cP and a 3 0 -tsRNA with 5 0 -OH (Sheng & Xu, 2016;Shigematsu et al., 2018). Most 5 0 -tsRNAs function seemingly as repressors of mRNA translation Keam et al., 2014;Sheng & Xu, 2016;Shigematsu et al., 2021). ...
Small RNAs (sRNAs) with sizes ranging from 15 to 50 nucleotides (nt) are critical regulators of gene expression control. Prior studies have shown that sRNAs are involved in a broad range of biological processes, such as organ development, tumorigenesis, and epigenomic regulation; however, emerging evidence unveils a hidden layer of diversity and complexity of endogenously encoded sRNAs profile in eukaryotic organisms, including novel types of sRNAs and the previously unknown post‐transcriptional RNA modifications. This underscores the importance for accurate, unbiased detection of sRNAs in various cellular contexts. A multitude of high‐throughput methods based on next‐generation sequencing (NGS) are developed to decipher the sRNA expression and their modifications. Nonetheless, distinct from mRNA sequencing, the data from sRNA sequencing suffer frequent inconsistencies and high variations emanating from the adapter contaminations and RNA modifications, which overall skew the sRNA libraries. Here, we summarize the sRNA‐sequencing approaches, and discuss the considerations and challenges for the strategies and methods of sRNA library construction. The pros and cons of sRNA sequencing have significant implications for implementing RNA fragment footprinting approaches, including CLIP‐seq and Ribo‐seq. We envision that this review can inspire novel improvements in small RNA sequencing and RNA fragment footprinting in future.
This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA
RNA Processing > Processing of Small RNAs
Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs
... Although miRNAs have dominated current research on both ex-short noncoding RNAs (sncRNAs) and endogenous ssRNA ligands of TLRs, recent advances in our understanding of "previously hidden" sncRNAs have widened the pool of the candidate ssRNA molecules of TLR7/8 ligands. sncRNAs generally possess either a hydroxyl group (OH), a phosphate (P), or a 2′,3′-cyclic phosphate (cP) at their termini (26). To date, most sncRNA sequencing studies have relied on a standard small RNA-seq method in which 5′-and 3′-adaptors (AD) can be ligated only to the 5′-P and 3′-OH ends of RNAs; thus, current sncRNA analyses significantly underrepresent non-miRNA-sncRNAs lacking the 5′-P or 3′-OH ends (26)(27)(28). ...
... sncRNAs generally possess either a hydroxyl group (OH), a phosphate (P), or a 2′,3′-cyclic phosphate (cP) at their termini (26). To date, most sncRNA sequencing studies have relied on a standard small RNA-seq method in which 5′-and 3′-adaptors (AD) can be ligated only to the 5′-P and 3′-OH ends of RNAs; thus, current sncRNA analyses significantly underrepresent non-miRNA-sncRNAs lacking the 5′-P or 3′-OH ends (26)(27)(28). This notion is especially important when it comes to the sequencing analyses of ex-sncRNAs because many ex-sncRNAs lack 5′-P or 3′-OH (29)(30)(31). ...
Toll-like receptors (TLRs) are crucial components of the innate immune system. Endosomal TLR7 recognizes single-stranded RNAs, yet its endogenous ssRNA ligands are not fully understood. We previously showed that extracellular (ex-) 5′-half molecules of tRNA HisGUG (the 5′-tRNA HisGUG half) in extracellular vesicles (EVs) of human macrophages activate TLR7 when delivered into endosomes of recipient macrophages. Here, we fully explored immunostimulatory ex-5′-tRNA half molecules and identified the 5′-tRNA ValCAC/AAC half, the most abundant tRNA-derived RNA in macrophage EVs, as another 5′-tRNA half molecule with strong TLR7 activation capacity. Levels of the ex-5′-tRNA ValCAC/AAC half were highly up-regulated in macrophage EVs upon exposure to lipopolysaccharide and in the plasma of patients infected with Mycobacterium tuberculosis . The 5′-tRNA ValCAC/AAC half-mediated activation of TLR7 effectively eradicated bacteria infected in macrophages. Mutation analyses of the 5′-tRNA ValCAC/AAC half identified the terminal GUUU sequence as a determinant for TLR7 activation. We confirmed that GUUU is the optimal ratio of guanosine and uridine for TLR7 activation; microRNAs or other RNAs with the terminal GUUU motif can indeed stimulate TLR7, establishing the motif as a universal signature for TLR7 activation. These results advance our understanding of endogenous ssRNA ligands of TLR7 and offer insights into diverse TLR7-involved pathologies and their therapeutic strategies.
... Generally, there are three different groups at the termini of RNA, hydroxyl (-OH), phosphate (-P), and 2',3'-cyclic phosphate (cP). -OH and -P can present on both 5'-and 3'-ends while cP can only be found on 3'-end 27 . Although the fragmentation behaviors have been published in several literatures, the effects of the termini groups to RNA fragmentation were seldom studied. ...
In recent years, research and application of mass spectrometry (MS) on RNA molecules have begun to flourish. However, MS/MS fragmentation behaviors of RNA oligonucleotides (oligos) are far from being fully understood. In this study, we have investigated the effect of the terminal phosphate group on the fragmentation behaviors of RNA oligos using high-resolution mass spectrometry. Specifically, we synthesized eight RNA oligos containing a terminal phosphate group on either or both ends, or neither. Negative-ion mode collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) of those oligos revealed that the terminal phosphate group impacted the fragmentation behaviors of RNA oligos in a way that depended on the precursor charge state and the oligo length. Firstly, a terminal phosphate group trends to impart higher negative charges to precursor ions of RNA oligos. Secondly, a terminal phosphate group(s) bestows a predominant loss of phosphoric acid (-H 3 PO 4 ) or metaphosphoric acid anion (-[PO 3 ] ⁻ ) from RNA oligo precursors in their CID or HCD spectra, especially for precursors of intermediate charge states. This uninformative loss decreases the intensity of sequencing ions ( a- , a-B , b- , c- , d- , w- , x- , y- , z- ions), hindering the sequencing and characterization of RNA oligos by CID/HCD. The terminal phosphate groups can be removed using calf intestinal alkaline phosphatase (CIP), and we show that CIP treatment improved MS analysis of RNAs with a terminal phosphate group on either or both ends. Additionally, we found that the peak intensity of m/z 158.925, which we have identified as a dehydrated pyrophosphate anion, [HP 2 O 6 ] ⁻ , is markedly increased by the presence of a terminal phosphate group on RNA oligos. This study contributes to the knowledge base on which software tools could be developed for high-throughput characterization of RNA oligos using tandem mass spectrometry.
... Consequently, while sncRNAs with the 5 0 -P/3 0 -OH ends, such as miRNAs, are efficiently sequenced by this method, sncRNAs with different terminal forms, such as 5 0 -OH, 3 0 -P, and 2 0 ,3 0 -cyclic phosphate (cP), cannot be efficiently captured. 31,32 This is especially important for the sequencing analyses of ex-sncRNAs, as many ex-sncRNAs lack 5 0 -P or 3 0 -OH. 33,34 To overcome this limitation, pretreatment of RNAs with T4 polynucleotide kinase (T4 PNK), which converts the RNA termini to 5 0 -P/3 0 -OH ends (thus rendering them available for 5 0 -/3 0 -AD ligation), has been used to capture whole ex-sncRNAs in human plasma samples. ...
Mycobacterium tuberculosis (Mtb) infection is among the world’s deadliest infectious diseases. Developing effective treatments and biomarkers for tuberculosis requires a deeper understanding of its pathobiology and host responses. Here, we report a comprehensive characterization of circulating short non-coding RNAs (sncRNAs) in plasma samples from Mtb-infected patients. We achieved this by pre-treating plasma RNAs with T4 polynucleotide kinase to convert all RNA ends to those compatible with sncRNA sequencing. We discovered a global and drastic upregulation of plasma sncRNAs in Mtb-infected patients, with tRNA-derived sncRNAs representing the most dramatically elevated class. Most of these tRNA-derived sncRNAs originated from a limited subset of tRNAs, specifically from three tRNA isoacceptors, and exhibited skewed patterns to 5′-derived fragments, such as 5′ halves, 5′ tRNA fragments (tRFs), and internal tRFs (i-tRFs) from the 5′ regions. Further, Mtb-infected patients displayed markedly upregulated and distinct profiles of both rRNA- and mRNA-derived sncRNAs. Some of these sncRNAs, which are abundant and specific to Mtb-infected patients, robustly activated human macrophages via Toll-like receptor 7 and induced cytokine production. This drastic accumulation of circulating, immunostimulatory sncRNAs in the plasma of Mtb-infected patients offers insights into the sncRNA-driven aspects of host immune response against infectious diseases and suggests a pool of potential therapeutic targets and biomarkers.
... Most ex-sncRNAs are uncaptured by standard RNA-seq 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 2 ,3 -cNMPs have been identified in eukaryotes, bacteria, and archaea. 2 ,3 -Cyclic phosphate termini are produced during RNA cleav a ge by man y endoribonucleases, either as intermediates or final products (Filipowicz 2016 ). In eukaryotes, they originate from trans-phosphorylation (Thompson et al. 1994 ) or RN A c yclase activity (Shigematsu et al. 2018 ), and in E. coli , they have been shown to originate from RNase I-dependent RNA degradation (Fontaine et al. 2018 ) or RN A c yclase activity (Genschik et al. 1997 ). RNA 3phosphate cyclases are found in most archaea. ...
Second messengers transfer signals from changing intra- and extracellular conditions to a cellular response. Over the last few decades, several nucleotide-based second messengers have been identified and characterized in especially bacteria and eukaryotes. Also in archaea, several nucleotide-based second messengers have been identified. This review will summarize our understanding of nucleotide-based second messengers in archaea. For some of the nucleotide-based second messengers, like cyclic di-AMP and cyclic oligoadenylates, their roles in archaea have become clear. Cyclic di-AMP plays a similar role in osmoregulation in euryarchaea as in bacteria, and cyclic oligoadenylates are important in the Type III CRISPR–Cas response to activate CRISPR ancillary proteins involved in antiviral defense. Other putative nucleotide-based second messengers, like 3′,5′- and 2′,3′-cyclic mononucleotides and adenine dinucleotides, have been identified in archaea, but their synthesis and degradation pathways, as well as their functions as secondary messengers, still remain to be demonstrated. In contrast, 3′-3′-cGAMP has not yet been identified in archaea, but the enzymes required to synthesize 3′-3′-cGAMP have been found in several euryarchaeotes. Finally, the widely distributed bacterial second messengers, cyclic diguanosine monophosphate and guanosine (penta-)/tetraphosphate, do not appear to be present in archaea.
... The ligation-based protocols require 5 phosphate and 3 hydroxyl groups for adaptor ligation and are, therefore, most efficient at sequencing miRNAs which naturally have these ends [54]. However, sRNAs such as tRFs and rsRNAs which are generated by endonuclease cleavage (e.g., cleavage of tRNAs by RNase A and T2 family endonucleases) have a 2 , 3 cyclic phosphate or a 3 phosphate, which can hinder the capture of these sRNAs in sequencing libraries [55]. Moreover, as tRNAs are highly modified RNA molecules, many reverse transcriptase (RT) enzymes cannot read through the hard-stop modifications on tRNAs and fall off prematurely, synthesizing only partial cDNAs [56]. ...
There is mounting evidence that ancestral life experiences and environment can influence phenotypes in descendants. The parental environment regulates offspring phenotypes potentially via modulating epigenetic marks in the gametes. Here, we review examples of across-generational inheritance of paternal environmental effects and the current understanding of the role of small RNAs in such inheritance. We discuss recent advances in revealing the small RNA payload of sperm and how environmental conditions modulate sperm small RNAs. Further, we discuss the potential mechanism of inheritance of paternal environmental effects by focusing on sperm small RNA-mediated regulation of early embryonic gene expression and its role in influencing offspring phenotypes.
... As compared with miRNAs, many of these sncRNAs including tsRNAs and rsRNAs are highly modified since their precursors (e.g., tRNAs and rRNAs) bear various RNA modifications such as RNA methylations (e.g., m 1 G, m 1 A, and m 3 C) (21,22,(26)(27)(28). In addition, terminal modifications (e.g., 3′-phosphate and 2′,3′-cyclic phosphate) also commonly occur during tsRNA and rsRNA biogenesis (29)(30)(31). These modifications can interfere with the reverse transcription and adaptor ligation processes in the widely used complementary DNA (cDNA) library construction protocol for standard RNA sequencing (RNA-Seq) analysis (22,29,32,33), which prevents the detection of highly modified sncRNAs by the traditional RNA-Seq method (21). ...
Small non-coding RNAs (sncRNAs) play diverse roles in numerous biological processes. While the widely used RNA sequencing (RNA-seq) method has advanced sncRNA discovery, RNA modifications can interfere with the cDNA library construction process, preventing the discovery of highly modified sncRNAs including transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) that may have important functions in disease development. To address this technical obstacle, we recently developed a novel PANDORA-seq method to overcome RNA modification-elicited sequence interferences. To identify novel sncRNAs associated with atherosclerosis development, LDL Receptor-deficient (LDLR-/-) mice were fed a low-cholesterol diet (LCD) or high-cholesterol diet (HCD) for 9 weeks. Total RNAs isolated from the intima were subjected to PANDORA-seq and traditional RNA-seq. By overcoming RNA modification-elicited limitations, PANDORA-seq unveiled a rsRNA/tsRNA-enriched sncRNA landscape in the atherosclerotic intima of LDLR-/- mice which was strikingly different from that detected by traditional RNA-seq. While microRNAs (miRNAs) were the dominant sncRNAs detected by traditional RNA-seq (55.9% of total sncRNAs), PANDORA-seq substantially increased the reads of rsRNAs and tsRNAs which account for 77.4% and 5% of total sncRNAs, respectively. PANDORA-seq also detected 1,383 differentially expressed sncRNAs induced by HCD feeding, including 1,160 rsRNAs and 195 tsRNAs. One of HCD-induced intimal tsRNAs, tsRNA-Arg-CCG may contribute to atherosclerosis development by regulating the pro-atherogenic gene expression in endothelial cells. Overall, PANDORA-seq revealed a hidden rsRNA and tsRNA population associated with atherosclerosis development. These understudied tsRNAs and rsRNAs, which are much more abundant than miRNAs in the atherosclerotic intima of LDLR-/- mice, warrant further investigations.
