A noncanonical microRNA derived from the snaR-A noncoding RNA targets a metastasis inhibitor

Stribling, Daniel; Lei, Yi; Guardia, Casey M.; Li, Lü; Fields, Christopher J.; Nowialis, Pawel; Opavský, René; Renne, Rolf; Xie, Mingyi

doi:10.1261/rna.078694.121

Cited by 15 publications

(18 citation statements)

References 72 publications

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“…S1B). As expected, in data sets obtained from DROSHA KO cells, we observed enrichment of hybrids containing Drosha-independent miRNAs including TSS-miRNAs (miR-320a and miR-484), mirtron (miR-877), and the recently described miR-snaR, a non-canonical miRNA derived from non-coding RNA snaR-A (Table S2) (48). MiR-320acontaining hybrids are far more abundant than other Drosha-independent miRNAs, making up to 74.58% of all the miRNA/mRNA hybrids in qCLASH data (Table S2).…”

Section: Comparison Of Clash Qclash and Mirna-specific Qclashsupporting

confidence: 83%

“…Because the 5′ and 3′ adapters contain degenerate nucleotides to account for PCR bias and minimize ligation preferences (44), we collapsed duplicate reads using the FASTX-Toolkit FASTQ Collapser, and the degenerate nucleotides were removed using Cutadapt (45,46). Chimeric reads (hybrids) were identified using the Hyb software with the default database supplemented with previously identified TSS-miRNAs and miR-snaR (20,47,48). With viennad-format files output from Hyb, we used a custom script to identify overlapping targets for specific miRNAs.…”

Section: Clash For Mirna Target Identification In Hct116 Cellsmentioning

confidence: 99%

“…The use of qCLASH allows genome-wide identification of such events that would otherwise be passed over with bioinformatic predictions or AGO-CLIP. Additionally, qCLASH can be used to identify novel miRNA with TOMiD (target oriented miRNA discovery), a database-naive bioinformatic method for identifying miRNA in chimeric reads (48). In TOMiD, reads that do not fully align to a single reference sequence are parsed for characteristics of miRNA hybrids.…”

Section: Alternative Cleavage By Drosha and Dicer And Modification By Exonucleases And Nucleotidylmentioning

confidence: 99%

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Sequencing of Argonaute-bound miRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a

Fields

Lü

Hiers

et al. 2021

Preprint

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MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched in the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed genes are enriched in eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone Calnexin as a direct miR-320a target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. Our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

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Section: Comparison Of Clash Qclash and Mirna-specific Qclashsupporting

confidence: 83%

Section: Clash For Mirna Target Identification In Hct116 Cellsmentioning

confidence: 99%

Section: Alternative Cleavage By Drosha and Dicer And Modification By Exonucleases And Nucleotidylmentioning

confidence: 99%

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Sequencing of Argonaute-bound miRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a

Fields

Lü

Hiers

et al. 2021

Preprint

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“…However, it is possible to identify cases in which both types of molecule (piRNA and other sncRNAs) could be bifunctional and even suggest alternative regulatory mechanisms to generate one or the other type. In fact, alternative, non-canonical pathways in the biogenesis of miRNAs have been reported [ 18 – 20 ]. Here, we evaluate that possibility for miRNAs/piRNAs from precursors that might generate such a situation.…”

Section: Utility and Discussionmentioning

confidence: 99%

piRNA-IPdb: a PIWI-bound piRNAs database to mining NGS sncRNA data and beyond

et al. 2021

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Background PIWI-interacting RNAs (piRNAs) are an abundant single-stranded type of small non-coding RNAs (sncRNAs), which initially were discovered in gonadal cells, with a role as defenders of genomic integrity in the germline, acting against the transposable elements. With a regular size range of 21-35 nt, piRNAs are associated with a PIWI-clade of Argonaute family proteins. The most widely accepted mechanisms of biogenesis for piRNAs involve the transcription of longer precursors of RNAs to be processed, by complexes of proteins, to functional size, preferentially accommodating uridine residues at the 5’ end and 3’ methylation to increase the stability of these molecules. piRNAs have also been detected in somatic cells, with diverse potential functions, indicating their high plasticity and pleiotropic activity. Discovered about two decades ago, piRNAs are a large and versatile type of sncRNA and that remain insufficiently identified and analyzed, through next-generation sequencing (NGS), to evaluate their processing, functions, and biogenesis in different cell types and during development. piRNAs’ distinction from other sncRNAs has led to controversial results and interpretation difficulties when using existing databases because of the heterogeneity of the criteria used in making the distinction. Description We present “piRNA-IPdb”, a database based uniquely on datasets obtaining after the defining characteristic of piRNAs: those small RNAs bound to PIWI proteins. We selected and analyzed sequences from piRBase that exclusively cover the binding to PIWI. We pooled a total of 18,821,815 sequences from RNA-seq after immunoprecipitation that included the binding to any of the mouse PIWI proteins (MILI, MIWI, or MIWI2). Conclusions In summary, we present the characteristics and potential use of piRNA-IPdb database for the analysis of bona fide piRNAs.

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“…While this protocol was developed to study KSHV, EBV, and MHV-68 miRNA targetomes, it has already been applied to nonviral cancers such as metastatic melanoma (Bullard et al, 2019;Gay et al, 2018;Kozar et al, 2021;Ungerleider et al, 2020). Moreover, in combination with innovative bioinformatic approaches, qCLASH has identified a novel human miRNA called miR-SNAR that is up-regulated in breast cancer cells and regulates migration (Stribling et al, 2021). In summary, this protocol simplifies the application of state-of-the-art ribonomics methods to identify high-confidence miRNA targetomes in disease-relevant models and tissues.…”

Section: Commentary Background Informationmentioning

confidence: 99%

Modified Cross‐Linking, Ligation, and Sequencing of Hybrids (qCLASH) to Identify MicroRNA Targets

Turner

Renne

2021

Current Protocols

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This protocol was designed to identify microRNA (miRNA) targetomes from smaller-input samples by performing a simplified workflow of the Cross-Linking and Sequencing of Hybrids (CLASH) technique developed in the Tollervey group. In this ribonomics-based technique, Cross-Linking and Immunoprecipitation (CLIP) of Argonaute (Ago) is combined with an RNA ligase reaction that yields covalently bound "hybrids" between miRNAs and their target RNAs. While this iteration of CLIP identifies "high-confidence" or "unambiguous" miRNA targets, the added ligation step is highly inefficient and therefore requires large numbers of cultured cells. To make this powerful approach applicable to smaller cell numbers, we created qCLASH, incorporating a workflow that performs all enzymatic reactions on bead-bound complexes and omits gel purification of immunoprecipitated Ago complexes associated with major loss of RNA. At a sequencing depth of 100 million reads per library, which is highly feasible with rapidly decreasing sequencing costs, qCLASH, when used with three biological replicates, results in thousands of high-confidence miRNA targets. qCLASH was first developed to identify viral miRNA targetomes of endothelial cells infected with Kaposi's sarcoma−associated herpesvirus. Since then, qCLASH has been applied to Epstein-Barr virus− and MHV68-infected cells, and more recently to metastatic melanoma and breast cancer cells. Currently, protocols are under development to apply qCLASH to human solid tumor specimens.

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A noncanonical microRNA derived from the snaR-A noncoding RNA targets a metastasis inhibitor

Cited by 15 publications

References 72 publications

Sequencing of Argonaute-bound miRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a

Sequencing of Argonaute-bound miRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a

piRNA-IPdb: a PIWI-bound piRNAs database to mining NGS sncRNA data and beyond

Modified Cross‐Linking, Ligation, and Sequencing of Hybrids (qCLASH) to Identify MicroRNA Targets

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