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Cited by 561MicroRNA in disease regulationGenomics and Chromatin DynamicsPlant Molecular Biology Research

An integrated expression atlas of miRNAs and their promoters in human and mouse

Open access
The FANTOM Consortium, de Rie, Derek, Abugessaisa, Imad, Alam, Tanvir, Arner, Erik, Arner, Peter, Ashoor, Haitham, Åström, Gaby +63 more·2017·Nature Biotechnology
DOI 10.1038/nbt.3947PMC PMC5767576
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Abstract

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.

AI snapshot

Written from the paper’s full text.

TL;DR

This FANTOM5 study integrates 492 short RNA libraries with matched CAGE data to build a human and mouse miRNA expression and promoter atlas. It identifies promoters for 1,357 human and 804 mouse miRNAs and shows that primary and mature miRNA expression levels are correlated, enabling extension to 1,829 human and 1,029 mouse CAGE libraries.

Key findings

  • Expression was confirmed for 98% (1,842/1,877) of human and 95% (1,124/1,186) of mouse miRBase release 21 pre-miRNAs; the FANTOM5 robust set comprised 795 human and 502 mouse pre-miRNAs meeting at least 4 of 5 miRBase high-confidence criteria.
  • Promoters were identified for 1,357 human and 804 mouse miRNAs; the pipeline predicted promoters for 539 robust, 623 permissive, and 3,951 candidate human pre-miRNAs, and manual curation confirmed the selected promoter for 512 (95%) robust human pre-miRNAs while adding 196 mostly intergenic promoters.
  • About half of robust pre-miRNAs had a significant Drosha CAGE peak (52% human, 64% mouse), and these peaks were absent at mirtrons; miRDeep2 found 6,543 human and 1,444 mouse candidate novel miRNAs, with 282 human and 34 mouse candidates meeting at least 4 of 5 high-confidence criteria.
  • Mature miRNA and pri-miRNA CAGE expression correlated (average Spearman r = 0.27 for intergenic, n = 180; r = 0.25 for intronic, n = 362), with higher correlation for highly differentially expressed miRNAs (r = 0.59, n = 159) and for polycistronic pri-miRNAs (r = 0.74, n = 1,372).

Methods

  • Deep-sequenced 492 short RNA libraries with matching CAGE data from 396 human and 47 mouse RNA samples, using Illumina TruSeq Small RNA preparation and HiSeq2000 50-base single-read sequencing; reads were mapped to hg19 or mm9 and annotated against miRBase release 21.
  • Used miRDeep2 for candidate novel miRNA discovery, negative-binomial modeling for Drosha CAGE peak significance, Gencode v19/RefSeq transcripts plus FANTOM5 CAGE peaks for promoter prediction, and manual curation for human robust pre-miRNAs; validated promoters with RAMPAGE, RACE, and RNA-seq data.
  • Analyzed expression with cpm normalization, cell type specificity index, cell ontology enrichment, Miru clustering, and MARA for transcription factor motif activity; validated selected miRNAs by qPCR.

Limitations

  • The paper states that most sRNAs currently annotated as miRNAs failed multiple high-confidence criteria and may belong to a different class of short non-coding RNAs, and that candidate miRNAs were mostly expressed at low levels, with fewer than 5% having sufficient tag counts on both arms for full high-confidence evaluation.
  • The paper notes that cell ontology P-values were not corrected for multiple testing, and that low-level miRNAs without significant enrichment or depletion may require profiling in further cell types or states.
  • Evident from the text: the extended atlas uses pri-miRNA CAGE as a proxy for mature miRNA levels, but overall mature–pri correlations were modest (average Spearman r = 0.25–0.27), and manual promoter curation was performed for the human robust set.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/nbt.3947

Cites

67 of 67 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 6 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    FANTOM5 and ENCODE CAGE tags at the Drosha peak were enriched in the nucleus.

    “FANTOM5 and ENCODE CAGE tags at the peak were enriched in the nucleus (Figure S9), consistent with processing by Drosha.”
    Quote in the paperSubstantial claim
  • Findingown data

    The ENCODE CAGE peak was found immediately downstream of the 3' end of the pre-miRNA locus for 19 out of 25 pre-miRNAs with a full-length sequence in FANTOM4 sRNA sequencing libraries.

    “The ENCODE CAGE peak was found immediately downstream of the 3′ end of the pre-miRNA locus (Figure S8) for 19 out of 25 pre-miRNAs with a full-length sequence in the FANTOM4 sRNA sequencing libraries23, confirming that the CAGE peak marks the Drosha cleavage site.”
    Quote in the paperSubstantial claim
  • Findingown data

    The identified miRNA promoters showed strong sequence conservation between species.

    “Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species.”
    Quote in the paperNot a substantial claim
  • Findingown data

    CAGE peaks were absent at the 3' end of pre-miRNA loci encoding mirtrons.

    “CAGE peaks were absent at the 3′ end of pre-miRNA loci encoding mirtrons (Figure S10, human; Figure S11, mouse), which are excised by the spliceosomal machinery instead of by Drosha24.”
    Quote in the paperSubstantial claim
  • Findingown data

    FANTOM5 created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA libraries with matching CAGE data from 396 human and 47 mouse RNA samples.

    “we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples.”
    Quote in the paperSubstantial claim
  • May combine two claims. 40
  • Classified as probably not a substantial claim. 10

  • May combine two claims.

    Findingown data

    Applying miRBase high-confidence criteria to FANTOM5 sRNA data, 571 human pre-miRNAs satisfied all five criteria, 224 met four, and 1076 violated two or more.

    “Applying these criteria to the FANTOM5 sRNA data, we found that 571 human pre-miRNAs satisfied all five high-confidence criteria, 224 met four of them, and 1076 violated two or more criteria (Figure 1a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression was confirmed for 98% (1842/1877) of human and 95% (1124/1186) of mouse pre-miRNAs annotated in miRBase release 21.

    “Across the sRNA libraries, expression was confirmed for 98% (1842/1877) of human and 95% (1124/1186) of mouse pre-miRNAs annotated in release 21 of the miRBase database12.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The robust set encompasses 735 human and 438 mouse mature miRNAs, and covers more than 90% of high-confidence pre-miRNAs in miRBase, 90% of miRNAs well-characterized in the scientific literature, and 91% human and 88% mouse of pre-miRNAs in MirGeneDB.

    “The robust set encompasses 735 human and 438 mouse mature miRNAs, and covers more than 90% of the high-confidence pre-miRNAs in miRBase (Figures S2 and S3), 90% of miRNAs well-characterized in the scientific literature (Figure S4), as well as 91% (human) and 88% (mouse) of pre-miRNAs included in the manually curated MirGeneDB database20 (Figure S5).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Findingown data

    The 795 human and 502 mouse pre-miRNAs satisfying at least 4 of 5 high-confidence criteria were defined as the FANTOM5 robust set, and the remaining 1076 human and 684 mouse pre-miRNAs as the permissive set.

    “The 795 human and 502 mouse (Figure S1) pre-miRNAs satisfying at least 4 out of the 5 high-confidence criteria were defined as the FANTOM5 robust set, and the remaining 1076 human and 684 mouse pre-miRNAs as the permissive set (Table S4 and S5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A CAGE peak was observed immediately downstream of the 3' end of human pre-miRNA loci in ENCODE CAGE data, and a slightly wider CAGE peak starting 1 nucleotide downstream was observed in FANTOM5 CAGE data.

    “We similarly observed a CAGE peak immediately downstream of the 3′ end of human pre-miRNA loci in the ENCODE CAGE data22, and a slightly wider CAGE peak starting 1 nucleotide downstream in the FANTOM5 CAGE data17,18 (Figure 1b, c, human; Figure S6, mouse);”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    miRDeep2 identified 7,461 human and 2,034 mouse predicted pre-miRNAs, including 918 human and 590 mouse known pre-miRNAs.

    “Candidate novel miRNAs were identified using miRDeep2 (ref. 26), resulting in 7,461 (human) and 2,034 (mouse) predicted pre-miRNAs, including 918 (human) and 590 (mouse) known pre-miRNAs.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Friedländer, M.R., Mackowiak, S.D., Li, N., Chen, W. & Rajewsky, N. miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Res. 40, 37–52 (2012). source

  • May combine two claims.

    Findingown data

    mir-128-1 was expressed in most samples, while its paralog mir-128-2 was highly enriched in brain.

    “For example, we found that mir-128-1 was expressed in most samples, while its paralog mir-128-2 was highly enriched in brain (Figure 3e).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The percentage of human pre-miRNAs with a Drosha CAGE peak decreased from 56% for those satisfying all five miRBase high-confidence criteria to 37% if one criterion was violated, while only 7% of miRNAs in the permissive set had a Drosha CAGE peak.

    “This percentage decreased from 56% for human pre-miRNAs satisfying all five of the miRBase high-confidence criteria to 37% if one of the criteria was violated, while only 7% of miRNAs in the permissive set had a Drosha CAGE peak (Figure 1a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The 282 human and 34 mouse candidate miRNAs meeting at least 4 of 5 high-confidence criteria formed the robust candidate set, while the permissive candidate set consisted of the remaining candidate miRNAs.

    “The 282 human and 34 mouse candidate miRNAs meeting at least 4 of the 5 high-confidence criteria formed the robust candidate set, while the permissive candidate set consisted of the remaining candidate miRNAs (Table S4 and Figure S13, human; Table S5 and Figure S14, mouse).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Examples of enriched expression not reported previously included miR-488-5p in neural cells, miR-506-3p in light melanocytes, and miR-205-5p in epithelial cells.

    “Examples of enriched expression not reported previously included miR-488-5p in neural cells, miR-506-3p in light melanocytes, and miR-205-5p in epithelial cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    miRNA expression levels varied greatly and were highly skewed, with on average five miRNAs contributing half of the total miRNA expression in a given library.

    “The expression levels of miRNAs varied greatly and were highly skewed, with on average five miRNAs contributing half of the total miRNA expression in a given library (Figure 2b, human; Figure S17, mouse), whereas most known and candidate miRNAs were expressed at low levels (Figure 2c, human; Figure S18, mouse).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CAGE data allowed measurement of the individual contribution of each paralog to the expression of miRNAs encoded multiple times in the human genome, providing evidence for differential regulation of paralogs in different cell types and tissues.

    “Lastly, the CAGE data allowed us to measure the individual contribution of each paralog to the expression of miRNAs encoded multiple times in the human genome, providing evidence for differential regulation of paralogs in different cell types and tissues.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Manual curation by two independent annotators confirmed the selected promoter for 512 (95%) robust pre-miRNAs; the computationally selected promoter was corrected for 26 pre-miRNAs and dropped for 1 pre-miRNA.

    “Manual curation by two independent annotators confirmed the selected promoter for 512 (95%) robust pre-miRNAs; the computationally selected promoter was corrected for 26 pre-miRNAs and dropped for 1 pre-miRNA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The robust candidate set showed concordance (127/282 or 45%) with 3,524 putative miRNAs identified recently, whereas the permissive candidate set yielded a smaller overlap (352/6,261 or 6%).

    “The robust candidate set showed good concordance (127/282 or 45%) with the 3,524 putative miRNAs identified recently in a study of tissue- and primate-specific miRNAs27, whereas the permissive candidate set yielded a smaller overlap (352/6,261 or 6%).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    Previously described highly cell-type-specific miRNAs included miR-122-5p, miR-142-5p, and miR-302a-5p, which were enriched in hepatocytes, leukocytes, and pluripotent stem cells, respectively.

    “Previously described highly cell-type-specific miRNAs included miR-122-5p, miR-142-5p, and miR-302a-5p, which were enriched in hepatocytes, leukocytes, and pluripotent stem cells, respectively (Figure 2d).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The distance between the transcription start site of the pri-miRNA and the mature miRNA locus was strongly conserved between human and mouse for intronic miRNAs (Spearman correlation = 0.90; n = 78; P = 2 × 10−29 two-sided) and for intergenic miRNAs (Spearman correlation = 0.86; n = 27; P = 1 × 10−8 two-sided).

    “The distance between the transcription start site of the pri-miRNA and the mature miRNA locus was strongly conserved between human and mouse both for intronic miRNAs (Spearman correlation = 0.90; n = 78; Student t = 18.27; P = 2 × 10−29 two-sided) and for intergenic miRNAs (Spearman correlation = 0.86; n = 27; Student t = 8.33; P = 1 × 10−8 two-sided) (Figures 3b and S25).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Of miRNAs in the robust set, 54% had enriched expression in their statistically most significant cell ontology cluster, 27% were broadly expressed with depleted expression in their statistically most significant cell ontology cluster, and 19% were expressed at low levels without statistically significant enrichment or depletion in any cell ontology cluster.

    “Of miRNAs in the robust set, 54% had enriched expression in their statistically most significant cell ontology cluster, whereas 27% were broadly expressed, with depleted expression in their statistically most significant cell ontology cluster. The remaining 19% were expressed at low levels without statistically significant enrichment or depletion in any cell ontology cluster;”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Across the human robust set, an associated ENCODE RAMPAGE 5' end was found within 300 base pairs of more than 75% of the FANTOM5 curated promoters both for intergenic and intronic miRNAs, outperforming the miRGen, Chang et al., miRStart, and TSmiR collections.

    “Across the human robust set, an associated ENCODE RAMPAGE (RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression32) 5′ end was found within 300 base pairs of more than 75% of the FANTOM5 curated promoters both for intergenic and intronic miRNAs, outperforming the miRGen33, Chang et al.14, miRStart34, and TSmiR35 collections of miRNA promoter annotations (Figure S20b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Manual curation identified the promoter for an additional 196, mostly intergenic, pre-miRNAs, generating the largest miRNA promoter collection to date.

    “Manual curation furthermore identified the promoter for an additional 196, mostly intergenic, pre-miRNAs, thereby generating the—to our knowledge—largest miRNA promoter collection to date (Table S17, Figure S20a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Counts per million (cpm) normalization yielded the best reproducibility between different donors for the same cell type while maintaining the distinction in expression profile between different cell types.

    “First, we assessed various expression normalization strategies, and found that a counts per million (cpm) normalization (i.e., dividing the tag count of each miRNA by the total number of tags mapping to miRNA loci, and multiplying by 1,000,000) yielded the best reproducibility between different donors for the same cell type, while maintaining the distinction in expression profile between different”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Nearly 11% of robust and 5% of permissive human candidate miRNAs had a significant (P < 0.05) Drosha CAGE peak.

    “Nearly 11% of robust and 5% of permissive human candidate miRNAs had a significant (P < 0.05) Drosha CAGE peak (Figure S13; Figure S14 for mouse).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The promoter identification pipeline predicted promoters for 539 robust, 623 permissive, and 3,951 candidate pre-miRNAs in human, and for 358 robust, 446 permissive, and 994 candidate pre-miRNAs in mouse.

    “This pipeline predicted promoters for 539 robust, 623 permissive, and 3,951 candidate pre-miRNAs in human (Table S15), and for 358 robust, 446 permissive, and 994 candidate pre-miRNAs in mouse (Table S16).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    miRNAs such as miR-100-5p and miR-29a-3p were broadly expressed but specifically depleted in particular cell types, leukocytes and pluripotent stem cells, respectively.

    “In contrast, miRNAs such as miR-100-5p and miR-29a-3p were broadly expressed but specifically depleted in particular cell types (leukocytes and pluripotent stem cells, respectively; Figure 2d).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Cell ontology analysis using the CAGE expression pattern of each human pri-miRNA across 338 cell ontology clusters, encompassing 636 CAGE libraries, showed enriched expression of mir-202 in gonad, mir-208a in heart, and multiple miRNAs in brain including mir-488, mir-556, and mir-885.

    “A cell ontology analysis was performed using the CAGE expression pattern of each human pri-miRNA (Tables S15 and S17) across 338 cell ontology clusters (Table S20), encompassing 636 CAGE libraries. This showed enriched expression of mir-202 in gonad, of mir-208a, known to be a key regulator of cardiac function36, in heart, as well as of multiple miRNAs in brain, including mir-488, mir-556, and mir”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The median distance between the FANTOM5 annotated miRNA promoter and the associated RAMPAGE 5' end was 1 nucleotide, closer than any existing miRNA promoter annotations.

    “The median distance between the FANTOM5 annotated miRNA promoter and the associated RAMPAGE 5′ end was 1 nucleotide, and was thereby closer than any of the existing miRNA promoter annotations (Figure S20c).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The smallest labeled cluster in the miRNA expression network contains six nodes.

    “The smallest labeled cluster contains six nodes; for clarity, smaller clusters have not been labeled but can be identified by groups of nodes of the same color.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Candidate miRNAs tended to be restricted to specific cell types, with 80% of the robust candidate set and 96% of the permissive candidate set having a higher cell type specificity index than the median value for robust known miRNAs.

    “Candidate miRNAs tended to be restricted to specific cell types, with 80% of the robust candidate set and 96% of the permissive candidate set having a higher cell type specificity index than the median value for robust known miRNAs (Table S13).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mature miRNA expression levels correlated with the CAGE expression levels of the associated promoter, with comparable correlation values for intergenic (average Spearman r = 0.27; n = 180; P = 1 × 10−30) and intronic (average Spearman r = 0.25; n = 362; P = 2 × 10−53) miRNAs.

    “The expression levels of mature miRNAs correlated with the CAGE expression levels of the associated promoter, with comparable correlation values for intergenic (average Spearman r = 0.27; n = 180; P = 1 × 10−30, Mann-Whitney U test, one-sided) and intronic (average Spearman r = 0.25; n = 362; P = 2 × 10−53, Mann-Whitney U test, one-sided) miRNAs (Figure 3c and S27; Table S19).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The extension allowed assessment of miRNA expression in samples with only a CAGE library, covering an additional 49 primary cell types, 245 cell lines, 138 tissue types, and 13 time courses in human, and an additional 48 primary cell types, 1 cell line, 234 tissue types, and 12 time courses in mouse.

    “This allowed us to assess miRNA expression also in samples for which only a CAGE library was available, covering an additional 49 primary cell types, 245 cell lines, 138 tissue types, and 13 time courses in human, and an additional 48 primary cell types, 1 cell line, 234 tissue types, and 12 time courses in mouse.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The robust candidate set comprised 279 human and 33 mouse unique mature sequences, whereas the permissive candidate set provided an additional 5,826 human and 1,354 mouse mature sequences.

    “The robust candidate set comprised 279 (human) and 33 (mouse) unique mature sequences, whereas the permissive candidate set provided an additional 5,826 (human) and 1,354 (mouse) mature sequences.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The miRNA PCR assay could specifically detect the presence of the target miRNAs and measure a wide spectrum of expression levels.

    “Our results demonstrate that the miRNA PCR assay could specifically detect the presence of the target miRNAs, and measure a wide spectrum of expression levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Pluripotent stem cells were characterized by cell-type-specific miRNAs, whereas cell-type-specific depletion of broadly expressed miRNAs was predominantly found in leukocytes.

    “Pluripotent stem cells were characterized by cell-type-specific miRNAs, whereas cell-type-specific depletion of broadly expressed miRNAs was predominantly found in leukocytes.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The correlation between mature miRNA and pri-miRNA expression was substantially higher for highly differentially expressed miRNAs (average Spearman r = 0.59; n = 159; P = 1 × 10−35), and exceeded correlations found for previously published miRNA promoter annotations.

    “The correlation was substantially higher for highly differentially expressed miRNAs (average Spearman r = 0.59; n = 159; P = 1 × 10−35, Mann-Whitney U test, one-sided), and exceeded correlations found for previously published14,33–35 miRNA promoter annotations (Figure S20d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Sequencing depth increased from nearly 1,300 reads per sRNA library in the first miRNA atlas to nearly 4.4 million reads per library in FANTOM5, allowing accurate measurement of expression even of miRNAs not highly expressed.

    “Advances in sequencing technology led to an increase in sequencing depth from nearly 1,300 reads per sRNA library in the first miRNA atlas38 to nearly 4.4 million reads per library in FANTOM5, allowing an accurate measurement of the expression even of miRNAs not highly expressed.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Cross-mapping was corrected by discarding all mappings to unannotated loci if the short RNA sequence could be mapped to an annotated locus instead.

    “We corrected for cross-mapping as described previously48, discarding all mappings to unannotated loci if the short RNA sequence could be mapped to an annotated locus instead.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Analysis of CAGE tags suggested that the RNA fragments at the Drosha cleavage site do not have a 7-methylguanosine cap, but may instead be uncapped or have a non-canonical cap.

    “Analysis of these CAGE tags suggested that these RNA fragments do not have a 7-methylguanosine cap, but may instead be uncapped or, alternatively, have a non-canonical cap.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The MARA analysis allowed prediction of miRNA expression levels based on regulatory motifs found in the miRNA promoter region, indicating that transcriptional regulation plays a central role in governing miRNA expression levels.

    “The MARA analysis allowed us to predict miRNA expression levels based on the regulatory motifs found in the miRNA promoter region, indicating that transcriptional regulation plays a central role in governing miRNA expression levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Candidate miRNAs not reported previously were highly specific to cell type, and may therefore be missed in miRNA profiling studies in tissues rather than in specific cell types.

    “The candidate miRNAs not reported previously were in particular highly specific to cell type, and may therefore be missed in miRNA profiling studies in tissues rather than in specific cell types.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Each human miRNA in the robust set was manually curated by two annotators.

    “Each human miRNA in the robust set was manually curated by two annotators.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    RNA samples used for TaqMan miRNA assay were used after confirmation of RNA quality by measuring RIN value and 260/280 and 260/230 ratios.

    “were used after confirmation of the RNA quality by measuring the RNA Integrity Number (RIN) value using a TapeStation and the 260/280 and 260/230 ratios using NanoDrop.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Short RNA libraries were prepared following the Illumina TruSeq Small RNA Sample Preparation protocol using the same RNA samples from which CAGE libraries were produced, as well as one additional RNA sample without a matching CAGE library.

    “Short RNA libraries were prepared following the Illumina TruSeq Small RNA Sample Preparation protocol (catalog number RS-200-0012, RS-200-0024, RS-200-0036, RS-200-0048) using the same RNA samples from which CAGE libraries were produced previously17,18, as well as one additional RNA sample without a matching CAGE library.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Compared to existing miRNA expression atlases, the FANTOM5 atlas covers the widest range of normal primary cells, enabling detailed analyses of miRNA expression and their contribution to establishing and maintaining cell type identity.

    “Compared to existing miRNA expression atlases29,38, the FANTOM5 atlas covers the widest range of normal primary cells, enabling detailed analyses of miRNA expression and their contribution to establishing and maintaining cell type identity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductreasoning

    A negative binomial distribution was used for Drosha CAGE peak significance, with the dispersion parameter estimated by fitting the distribution to the number of CAGE tags in any 8- or 9-basepair window on the human or mouse genome.

    “We therefore used the negative binomial distribution instead, with the dispersion parameter r estimated by fitting the distribution to the number of CAGE tags in any 8- or 9-basepair window on the human or mouse genome.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Comparing promoters of miRNAs, protein-coding genes, and long non-coding RNAs showed a similar prevalence of transcription factor binding sites in proximal promoter regions, suggesting that the basic mechanisms of transcriptional regulation are largely the same for these three classes of gene products.

    “Comparing the promoters of miRNAs, protein-coding genes, and long non-coding RNAs showed a similar prevalence of transcription factor binding sites in proximal promoter regions (data not shown), suggesting that the basic mechanisms of transcriptional regulation are largely the same for these three classes of gene products.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Two samples with fewer than 100,000 mapped tags were discarded from further analysis.

    “Two samples had fewer than 100,000 mapped tags and were discarded from further analysis.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    For human, an overall statistical significance value was calculated by combining the FANTOM5 and ENCODE statistical significance into a single P-value using Fisher's method.

    “For human, we calculated an overall statistical significance value by combining the FANTOM5 and ENCODE statistical significance into a single P-value using Fisher’s method.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The P-values listed in Tables S13, S15, and S17 for each miRNA for specific cell ontology clusters were not corrected for multiple testing.

    “The P-values listed in Tables S13, S15, and S17 for each miRNA for specific cell ontology clusters were not corrected for multiple testing.”
    Quote in the paperNot a substantial claim
47 of 47

  • The authors state this as an assumption.

    miR-153-3p and miR-345-5p are lowly expressed in myeloid cells.

    “Expression levels of miRNAs reported to be cell markers for other cell types and assumed to be lowly expressed in myeloid cells, including miR-153-3p (ref. 57) and miR-345-5p (ref. 58), were also analyzed in order to determine the detection limit of the assay.”
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Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • hsa-miR-181a-5p was identified among oligodendrocyte-enriched miRs.

    “We also identified hsa-miR-181a-5p, previously implicated in oligodendrocyte maturation 56 and suggested as a potential epigenetic biomarker of multiple sclerosis 57 .”

    From Human brain microRNAs exhibit cell-type specificity and age dependence (2026) · cites this paper as [56]

    The Results section on cell ontology analysis reports miRNA enrichment by cell type, but does not identify hsa-miR-181a-5p as oligodendrocyte-enriched. Searches of our copy for hsa-miR-181a-5p, miR-181 and oligodendrocytes found no passage addressing that specific claim; page numbers were unavailable. (LLM, conf=0.72)

    Experimental — accuracy not yet measured

  • For any given human cell type, the top five expressed miRNAs represent on average approximately 50% of the overall miRNA pool.

    “Recently, the work by the Functional Annotation of the Mammalian Genome (FANTOM5) consortium found that for any given human cell type, the top five expressed miRNAs represent on average ~50% of the overall miRNA pool (13).”

    From Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation (2018) · cites this paper as [13]

  • hsa-miR-181a-5p has been implicated in oligodendrocyte maturation and suggested as a potential epigenetic biomarker of multiple sclerosis.

    “previously implicated in oligodendrocyte maturation 56 and suggested as a potential epigenetic biomarker of multiple sclerosis 57”

    From Human brain microRNAs exhibit cell-type specificity and age dependence (2026) · cites this paper as [56]

  • Among ubiquitously expressed ncRNAs are let-7d-3p, miR-320-3p, Snord4a, Snord55, and endothelial miR-151-5p.

    “Among these are ncRNAs known to be expressed in a large number of cell types, such as let-7d-3p, miR-320-3p (25), ncRNAs the cell type specificity of which is still unknown, such as Snord4a and Snord55, as well as those known to be expressed in the cell types that are abundant in all tissues (like endothelial miR-151-5p)”

    From A mouse tissue atlas of small noncoding RNA (2020) · cites this paper as [25]

  • For the majority of identified brain ncRNA, the cell type of origin could not be called based on data generated within previous studies offering cell type resolution.

    “for the majority of these identified RNA we could not call the cell type of origin based in the data generated within previous studies offering cell type resolution (24, 25).”

    From A mouse tissue atlas of small noncoding RNA (2020) · cites this paper as [25]

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Authors
The FANTOM Consortium, de Rie, D., Abugessaisa, I., Alam, T., Arner, E., Arner, P., Ashoor, H., Åström, G., Babina, M., Bertin, N., Burroughs, A. M., Carlisle, A. J., Daub, C. O., Detmar, M., Deviatiiarov, R., Fort, A., Gebhard, C., Goldowitz, D., Guhl, S., Ha, T. J., Harshbarger, J., Hasegawa, A., Hashimoto, K., Herlyn, M., Heutink, P., Hitchens, K. J., Hon, C. C., Huang, E., Ishizu, Y., Kai, C., Kasukawa, T., Klinken, P., Lassmann, T., Lecellier, C.-H., Lee, W., Lizio, M., Makeev, V., Mathelier, A., Medvedeva, Y. A., Mejhert, N., Mungall, C. J., Noma, S., Ohshima, M., Okada-Hatakeyama, M., Persson, H., Rizzu, P., Roudnicky, F., Sætrom, P., Sato, H., Severin, J., Shin, J. W., Swoboda, R. K., Tarui, H., Toyoda, H., Vitting-Seerup, K., Winteringham, L., Yamaguchi, Y., Yasuzawa, K., Yoneda, M., Yumoto, N., Zabierowski, S., Zhang, P. G., Wells, C. A., Summers, K. M., Kawaji, H., Sandelin, A., Rehli, M., Hayashizaki, Y., Carninci, P., Forrest, A. R. R., & de Hoon, M. J. L.
Affiliations
  • RIKEN (JP)
  • King Abdullah University of Science and Technology (SA)
  • Karolinska University Hospital (SE)
  • Charité – Universitätsmedizin Berlin (DE)
  • National University of Singapore (SG)
  • National Institutes of Health (US)
  • Roslin Institute (GB)
  • ETH Zurich (CH)
  • Kazan Federal University (RU)
  • University Hospital Regensburg (DE)
  • University of British Columbia (CA)
  • The Wistar Institute (US)
  • German Center for Neurodegenerative Diseases (DE)
  • The University of Queensland (AU)
  • The University of Melbourne (AU)
  • Institute of Medical Science, The University of Tokyo (JP)
  • The University of Western Australia (AU)
  • The Kids Research Institute Australia (AU)
  • Engelhardt Institute of Molecular Biology (RU)
  • Russian Academy of Sciences (RU)
  • Ohu University (JP)
  • Lund University (SE)
  • Norwegian University of Science and Technology (NO)
  • Tokyo University of Pharmacy and Life Sciences (JP)
  • Nihon University (JP)
  • RIKEN Center for Integrative Medical Sciences (JP)
  • Translational Research Institute (AU)
  • University of Copenhagen (DK)
Corresponding author
Forrest, Alistair R R
ORCID
Venue
Nature Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2017-08-21
Volume
35
Issue
9
Pages
872-878
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
DOI
10.1038/nbt.3947
PMC
PMC5767576
OpenAlex
W2748577514
Cited by
561
References
67
Citations by year
2026: 26 · 2025: 39 · 2024: 69 · 2023: 66 · 2022: 82 · 2021: 89 · 2020: 91 · 2019: 49 · 2018: 44 · 2017: 5
FWCI
17.36
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, other-oa)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, other-oa)
  • UWA Profiles and Research Repository (UWA) (submittedVersion, other-oa)
  • HAL (submittedVersion)
  • University of Regensburg Publication Server (University of Regensburg) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
  • Lund University Publications (Lund University) (submittedVersion)
  • DZNE Pub (submittedVersion)
  • King Abdullah University of Science and Technology Repository (King Abdullah University of Science and Technology) (submittedVersion)
Funders
  • Conquer Cancer Foundation
  • Ministry of Education, Culture, Sports, Science and Technology
  • Lundbeckfonden (R82-2010-6779)
  • Novo Nordisk
  • Russian Science Foundation (15-14-30002, 14-44-00022)
  • Novo Nordisk Fonden (NNF15OC0015894)
  • H. Lundbeck A/S
  • Biotechnology and Biological Sciences Research Council (BBS/E/D/20211552, BBS/E/D/20211550, BBS/E/D/20211551, BB/I024801/1, BBS/E/D/10002070, BBS/E/D/20221657)
  • Australian Research Council (DP160101960)
  • Japan Society for the Promotion of Science (16H02587)
  • RIKEN
  • National Cancer Institute (P30CA010815)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
Keywords
microRNA, miRNA promoters, mature miRNA, CAGE, FANTOM5, human, mice, sequence conservation, transcriptional regulation, non-coding RNA
MeSH terms
Animals, Cells, Cultured, Humans, Promoter Regions, Genetic, Gene Library, Gene Expression Profiling, MicroRNAs, Mice, Molecular Sequence Annotation, High-Throughput Nucleotide Sequencing

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

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