5´XP sRNA-seq: efficient identification of transcripts with and without 5´ phosphorylation reveals evolutionary conserved small RNA

Kugelberg, Unn; Nätt, Daniel; Skog, Signe; Kutter, Claudia; Öst, Anita

doi:10.1080/15476286.2020.1861770

Cited by 10 publications

(10 citation statements)

References 53 publications

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“…When profiling tsRNAs using RNA-seq, the specific tsRNA termini (e.g., 3′-phosphate and 2′,3′-cyclic phosphate) and RNA modifications (e.g., m 1 A, m 3 C, m 1 G, and N2-dimethylguanosine, m 2 2 G) have created challenges to prevent efficient and complete conversion of tsRNAs into cDNA libraries during standard RNA-seq protocols, resulting in biased detection and quantitation during deep sequencing [14]. Recently improved RNA-seq methods such as panoramic RNA display overcoming RNA modification aborted sequencing (PANDORA)-seq [14] and Cap-Clip acid pyrophosphatase, PNK, and AlkB (CPA)-seq [137] aim to overcome these problems by removing key RNA modifications that block adapter ligation and RT by using consecutive enzymatic treatment; these techniques await further optimization such as combination with other strategies focusing on the 5′-termini [138].…”

Section: Evolving Tsrna Functionalitiesmentioning

confidence: 99%

Origins and evolving functionalities of tRNA-derived small RNAs

Chen

Zhang

Shi

et al. 2021

Trends in Biochemical Sciences

109

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Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are among the most ancient small RNAs in all domains of life and are generated by the cleavage of tRNAs. Emerging studies have begun to reveal the versatile roles of tsRNAs in fundamental biological processes, including gene silencing, ribosome biogenesis, retrotransposition, and epigenetic inheritance, which are rooted in tsRNA sequence conservation, RNA modifications, and protein-binding abilities. We summarize the mechanisms of tsRNA biogenesis and the impact of RNA modifications, and propose how thinking of tsRNA functionality from an evolutionary perspective urges the expansion of tsRNA research into a wider spectrum, including cross-tissue/cross-species regulation and harnessing of the 'tsRNA code' for precision medicine. On the ancientness of tsRNAs/tRNAsWith the wide application of high-throughput RNA sequencing (RNA-seq), tRNA-derived small RNAs (tsRNAs) (also called tDRs or tRNA-derived fragments, tRFs) are increasingly recognized as an emerging class of functional small noncoding RNAs (sncRNAs) in various fundamental biological and disease conditions [1,2]. tsRNAs have been identified in a wide range of species across all three domains of life, including Archaea, Bacteria, and some unicellular organisms (e.g., Protozoa), where other specialized small RNA pathways, such as miRNAs, siRNAs, and Piwi-interacting RNAs (piRNAs), are lacking [3][4][5][6][7]. These observations have positioned tsRNAs among the most ancient classes of sncRNAs for intra-and intercellular functionality and communication that may pre-date the emergence of more specialized sncRNAs. Highlights tRNA-derived small RNAs (tsRNAs) are present across all three domains of life: Archaea, Bacteria, and Eukarya.

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Section: Evolving Tsrna Functionalitiesmentioning

confidence: 99%

Origins and evolving functionalities of tRNA-derived small RNAs

Chen

Zhang

Shi

et al. 2021

Trends in Biochemical Sciences

109

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“…In practice, the most widely used sncRNA-sequencing protocol is optimized for those bearing 5′-P and 3′-OH termini, and thus, the sncRNAs with 2′,3′-cP or 3′-P and/or 5′-OH termini cannot be ligated and will not be included in the cDNA library 10 . Solutions to this problem include the use of enzymes to convert the termini, such as the use of T4PNK to convert 2′,3′-cP and 3′-P into 3′-OH and 5′-OH into 5′-P before the ligation process 109 , or combining with a template-switching strategy to add a 5′ adaptor to the cDNA after the RT instead of directly adding a 5′ adaptor to the RNA 110,116 , which can resolve most problems caused by 5′-terminal modifications.…”

Section: Improved Methods Lead To An Updated Landscape Of Sncrnasmentioning

confidence: 99%

Exploring the expanding universe of small RNAs

2022

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mall noncoding RNAs (sncRNAs) are universally distributed in all kingdoms of life-from bacteria and archaea to various eukaryotic organisms 1-3 -and have not ceased to surprise us throughout the last two decades regarding their compositional and functional diversity. Although the definition of 'small' is relatively empirical and subjective in different contexts, in this paper we mainly discuss sncRNAs of 15-50 nucleotides (nt) in length, including the relatively well-characterized small interfering RNAs (siRNAs, 20-27 nt), microRNAs (miRNAs, 21-23 nt) and Piwi-interacting RNAs (piRNAs, 21-35 nt) [4][5][6] , but with more focus on more recently discovered non-canonical sncRNAs (15-50 nt) that are derived from longer structured RNAs 7 such as transfer RNA (tRNA) 8,9 , ribosomal RNA (rRNA) 10,11 , Y RNA (yRNA) 11,12 , small nuclear RNA (snRNA) 13,14 , small nucleolar RNAs (snoRNA) 15,16 , vault RNA (vtRNA) 17,18 and even messenger RNA (mRNA) 19,20 . Studies on non-canonical sncRNAs have recently gained momentum, exemplified by the new focus on tRNA-derived small RNA (tsRNA) 8 , and are expected to expand to other categories with their systematic discovery. To facilitate communication and reduce confusion, we propose a unified naming system for these non-canonical sncRNAs (Box 1) when describing discoveries from different laboratories (usually using different names).Like many noncoding RNAs in history, the emerging non-canonical sncRNAs were initially considered as merely random degradation products of RNA turnover/metabolism and thus neglected, yet increasing evidence has begun to put them in the spotlight as regulatory sncRNAs 8,21 . This is partly due to the revelation that they are regulated by both genetic and environmental factors 18,[22][23][24][25][26][27] and that many of them are functional and associated with multiple diseases-including those linked to cancer [28][29][30] , immunity 12,31 , viral infection 32,33 , neurological disorders 34,35 , stem cells 26,[36][37][38][39] , retrotransposon control 40,41 and epigenetic inheritance 24,25,[42][43][44][45] as well as because in many cases, the exertion of their function depends on mechanisms that are distinct from those of well-studied siRNAs, miRNAs or piRNAs. Moreover, it was recently recognized that many non-canonical sncRNAs harbour various RNA modifications, some of which can prevent the detection of sncRNAs by traditional RNA sequencing (RNA-seq) 10,14,46,47 . This has promoted a recent wave of method improvements, leading to their comprehensive discovery and identification, which have in turn ignited interest in research centred on sncRNA modifications 48 . Here we briefly outline the biogenesis and functional principles of non-canonical sncRNAs and discuss recent methodological developments in promoting sncRNA discovery and accurate expression analyses as well as new techniques for direct multiplexed mapping of RNA modifications, which is necessary for decoding the full function of sncRNAs.

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“…For profiling of these missing small RNA classes, there are protocols such as ScriptMinerTM (Cambio Ltd.) utilizing enzymes, which degrade caps and polyphosphate groups on the 5 -end and make these molecules analyzable [23,47]. Another approach to capture small RNAs regardless of their 5 -end modifications is to add the 5 -adaptor after RT and target the cDNA molecules [48,49]. Lastly, the recentlyintroduced phospho-RNA-seq method utilizes T4-polynucleotide kinase, which is used for phosphorylation and dephosphorylation in order to capture small RNAs regardless of their end modifications [50].…”

Section: Two-adaptor Ligation-based Methodsmentioning

confidence: 99%

“…miRNAs are enriched in the majority of ligation-based protocols, whereas other small RNAs along with miRNAs are captured by polyadenylation methods. As polyadenylation suffers from a low mapping rate and inability to distinguish isomiRs, protocol adjustments (specialized enzymes, ligation of cDNA) might be applied in the ligation-based protocols to capture also other small RNA classes [49,80].…”

Section: Considerations and Future Prospectsmentioning

confidence: 99%

Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

2021

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MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

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5´XP sRNA-seq: efficient identification of transcripts with and without 5´ phosphorylation reveals evolutionary conserved small RNA

Cited by 10 publications

References 53 publications

Origins and evolving functionalities of tRNA-derived small RNAs

Origins and evolving functionalities of tRNA-derived small RNAs

Exploring the expanding universe of small RNAs

Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

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