Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP

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

Several small molecules and antisense oligos that target miRNA biogenesis as well as miRNA mimics are in clinical trials as candidate therapies for diseases such as non-small cell lung cancer, keloid, chronic hepatitis C, cutaneous T-cell lymphoma and Alport's syndrome.

“Currently several small molecules and antisense oligos that target miRNA biogenesis as well as miRNA mimics themselves are in clinical trials as candidate therapies for diseases such as non-small cell lung cancer, keloid, chronic hepatitis C, cutaneous T-cell lymphoma and Alport's syndrome (Rupaimoole and Slack, 2017;Zhu et al., 2020).”

• cites Rupaimoole and Slack, 2017 sourceNot checked — no full text of the source
• cites Zhu et al., 2020 sourcePartly supported

  The Introduction (page unavailable) reports more than 100 ongoing RNA-targeted therapy trials, and the Conclusions (page unavailable) include ASOs, miRNA mimics and small molecules among therapies under clinical or preclinical evaluation. The paper does not establish that several therapies targeting miRNA biogenesis are in clinical trials for the five named diseases; its Conclusions say the discussed small-molecule miRNA regulators need further research before entering clinical studies. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

The repertoire of miRNA targets is a key determinant of the biological role of a given miRNA.

“Thus, the repertoire of miRNA targets is a key determinant of the biological role of a given miRNA (Ebert and Sharp, 2012).”

• cites Ebert and Sharp, 2012 sourceSupported

Classified as probably not a substantial claim.

miRNAs exert their repressive regulatory function by guiding RISC to complementary target sites in the 3′ UTR of target mRNAs, resulting in mRNA degradation, translation inhibition, or sequestration.

“Generally, miRNAs exert their repressive regulatory function by guiding the RNA-induced silencing complex (RISC) to complementary target sites in the 3′ untranslated region (UTR) of target mRNAs resulting in mRNA degradation, translation inhibition, or sequestration (Bartel, 2018).”

• cites Bartel, 2018 sourcePartly supported

  The miRNA Target Recognition section describes pairing to sites in target mRNAs’ 3′ UTRs, and miRNA Regulatory Mechanisms describes Argonaute-mediated mRNA decay and reduced translation. The latter calls decay and translational repression the two repressive consequences of this regulatory mode; our copy does not support sequestration as a third consequence. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

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

Various algorithms have been developed to predict miRNA:mRNA interactions throughout the transcriptome based on sequence complementarity.

“Building upon this principle of sequence complementarity, various algorithms have been developed to predict miRNA:mRNA interactions throughout the transcriptome (Agarwal et al., 2015;Krek et al., 2005).”

• cites Agarwal et al., 2015 sourceSupported
• cites Krek et al., 2005 sourceNot checked — no full text of the source

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

Many miRNAs display tissue-, cell type-, or condition-specific expression patterns and play key roles in the regulation of developmental programs.

“Many miRNAs also display tissue-, cell type-, or conditionspecific expression patterns and play key roles in the regulation of developmental programs (DeVeale et al., 2021;Manakov et al., 2009).”

• cites DeVeale et al., 2021 sourceNot checked — no full text of the source
• cites Manakov et al., 2009 sourcePartly supported

  The Results (miRNA Expression Analysis; page not provided) report many miRNAs changing expression during the development of mouse neuronal cultures. The Background and Discussion link miRNAs to brain development and suggest an important role in neuronal differentiation, but the study does not establish tissue- or cell-type-specific expression patterns or key roles across developmental programs generally. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

A suite of chimeric CLIP methods including CLASH, modified iPAR-CLIP, and CLEAR-CLIP were developed to enable direct experimental mapping of microRNA:target interactions.

“To enable this direct experimental mapping of microRNA:target interactions, a suite of chimeric CLIP methods including CrossLinking And Sequencing of Hybrids (CLASH), modified iPAR-CLIP, and CLEAR-CLIP (Broughton et al., 2016;Grosswendt et al., 2014;Helwak et al., 2013;Moore et al., 2015) were developed.”

• cites Broughton et al., 2016 sourceNot checked yet

  Not checked in the free run.

• cites Grosswendt et al., 2014 sourceSupported
• cites Helwak et al., 2013 sourceSupported

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

Recent efforts have shown success at identifying candidate miRNA target sites in vitro using purified RISC complex pre-loaded with a miRNA of interest, followed by high-throughput RNA binding assays.

“Recent efforts have shown success at identifying candidate miRNA target sites in vitro using purified RISC complex pre-loaded with a miRNA of interest, followed by highthroughput RNA binding assays (Becker et al., 2019;McGeary et al., 2019).”

• cites Becker et al., 2019 sourceSupported
• cites McGeary et al., 2019Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

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

Early methods including RIP and CLIP of AGO proteins provided the first broad view of the miRNA interaction landscape.

“Early methods including RNA Immunoprecipitation (RIP) and CrossLinking and ImmunoPrecipitation (CLIP) of AGO proteins provided the first broad view of the miRNA interaction landscape, revealing principles of miRNA regulation mechanisms as well as an overview of mRNAs regulated by miRNAs (Chi et al., 2009;Hafner et al., 2010).”

• cites Chi et al., 2009 sourceSupported
• cites Hafner et al., 2010 sourcePartly supported

  The Results (‘Identification of miRNA targets by AGO and TNRC6 family PAR-CLIP’ and ‘mRNAs interacting with AGOs contain miRNA seed complementary sequences’; pages not supplied) show broad mapping of AGO-associated miRNA target sites and reveal targeting principles. The Introduction discusses RIP-Chip as a transcriptome-level method, but does not establish that AGO RIP provided the first broad view of miRNA interactions; this paper’s target mapping used PAR-CLIP. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

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

Chimeric CLIP-seq approaches incorporate a phosphorylation step and an additional ligation step to increase the frequency of chimeric fragments to as much as 5.3%, although it often remains less than 2%.

“chimeric CLIP-seq approaches (including CLASH, CLEAR-CLIP, and modified iPAR-CLIP) have modified this approach by incorporating both a phosphorylation step (using 3' phosphatase minus T4 Polynucleotide Kinase) as well as an additional ligation step (without adapters) to encourage proximity-based ligation and increase the frequency of chimeric fragments to as much as 5.3%, although it often remains”

• cites Bjerke and Yi, 2020 sourcePartly supported

  The Introduction (page unavailable) says CLEAR-CLIP adds a ligation step to improve the low frequency of chimeric reads in the original CLASH study. The passages do not substantiate the claimed phosphorylation step, the 5.3% or less-than-2% frequencies, or the inclusion of modified iPAR-CLIP. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites Gay et al., 2018 sourcePartly supported

  The Results describe 5′ phosphorylation with T4 polynucleotide kinase followed by intermolecular ligation, and report that approximately 0.3%–1% of filtered qCLASH reads were hybrids. The paper does not report a 5.3% hybrid rate or specify phosphatase-minus T4 PNK for that step; the cited passages also do not establish those details for CLEAR-CLIP or modified iPAR-CLIP. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Grosswendt et al., 2014 sourcePartly supported

  The Experimental Procedures section (page unavailable) describes an added T4 RNA ligase reaction in modified iPAR-CLIP, supporting that part of the claim. Our copy does not document the specified phosphorylation step or a 5.3% chimeric-fragment frequency; the Discussion instead reports a ligation efficiency of 0.14% of reads. The Results’ “less than 2%” refers to chimeras containing bacterial sequences, not the overall frequency of chimeric fragments. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

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

Recent work performing in vitro RNA binding assays using pre-formed miRNA-loaded Argonaute complexes has shown that these complexes readily bind RNA with similar targeting principles as in vivo-identified miRNA targets.

“However, recent work performing in vitro RNA binding assays using pre-formed miRNA-loaded Argonaute complexes has shown that these complexes readily bind RNA with similar targeting principles as in vivo-identified miRNA targets (Becker et al., 2019;McGeary et al., 2019)”

• cites Becker et al., 2019 sourceSupported
• cites McGeary et al., 2019Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

The most abundant miRNA was miR-17-5p, while miR-20a-5p, miR-92a-5p, miR-19b-3p, miR-18a-5p, and miR-19a-3p were second through sixth most abundant.

“the most abundant miRNA was miR-17-5p, which has previously been shown to regulate cell cycle progression in 293T cells (Cloonan et al., 2008), while miR-20a-5p, miR-92a-5p, miR-19b-3p, miR-18a-5p, and miR-19a-3p were second through sixth most abundant.”

• cites Cloonan et al., 2008 sourceSupported

May combine two claims.

Targets identified using CLASH or similar chimeric ligation approaches showed enrichment for miRNA-dependent changes in RNA decay and specific functional categories, confirming that these methods yield high-quality sets of miRNA targets.

“targets identified using CLASH or similar chimeric ligation approaches showed enrichment for miRNA-dependent changes in RNA decay and specific functional categories, confirming that these methods yield high-quality sets of miRNA targets (Moore et al., 2015).”

• cites Moore et al., 2015 sourcePartly supported

  The Results section, “CLEAR-CLIP-identified sites are functional” (page unavailable), reports that chimera-identified sites correlate with miRNA-dependent changes in transcript levels and polysome association. The Discussion also describes low false-target identification rates for CLEAR-CLIP. The copy does not establish enrichment for changes in RNA decay specifically or for particular functional categories. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

Chimeric CLIP can both refine computationally predicted targets and reveal new targets not captured by prediction alone.

“matching previous findings that chimeric CLIP can both refine computationally predicted targets as well as reveal new targets not captured by prediction alone (Bjerke and Yi, 2020).”

• cites Bjerke and Yi, 2020 sourceSupported

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

CLASH with PIWI protein has been used to reveal mechanisms of piRNA targeting, and CLASH using E. Coli Hfq to enrich for sRNA-mRNA interactions revealed principles of small RNAs in bacteria.

“CLASH with PIWI protein has been used to reveal mechanisms of piRNA targeting (Shen et al., 2018), and CLASH using E. Coli Hfq to enrich for sRNA-mRNA interactions revealed principles of small RNAs in bacteria (Iosub et al., 2020).”

• cites Shen et al., 2018 sourceSupported
• cites Iosub et al., 2020 sourceSupported

Classified as probably not a substantial claim.

MicroRNA sites have been validated in coding sequence (CDS) regions, albeit with typically weaker repressive activity.

“MicroRNA sites have also been validated in coding sequence (CDS) regions, albeit with typically weaker repressive activity (Baek et al., 2008;Grimson et al., 2007).”

• cites Baek et al., 2008 sourceSupported
• cites Grimson et al., 2007 sourceSupported

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

Early CLASH publications showed the utility of chimeric analysis in mapping snoRNA interactions in yeast, and more recently it was utilized to generate an extensive catalog of putative snoRNA interactions and reveal novel targets of orphan snoRNAs.

“early CLASH publications showed the utility of the chimeric analysis approach in mapping snoRNA interactions in yeast (Kudla et al., 2011) and more recently was utilized to generate an extensive catalog of putative snoRNA interactions and reveal novel targets of orphan snoRNAs (Gumienny et al., 2017).”

• cites Kudla et al., 2011 sourceSupported
• cites Gumienny et al., 2017Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

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

TargetScan version 7.2 predicts 896 miR-1-3p targets.

“genes were also gropued based on predictions of 896 miR-1-3p targets and 1,820 miR-124-3p targets by TargetScan version 7.2 (Agarwal et al., 2015)”

• cites Agarwal et al., 2015 sourcePartly supported

  The Results section, “The TargetScan database (v7.0)” (page not provided), says the authors used their model to rank TargetScan version 7 predictions. Our copy does not report version 7.2 or a count of 896 miR-1-3p targets, so it supports the prediction method but not the specific count attributed to it. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

Requiring crosslinking of both miRNA and mRNA target would reduce post-lysis interactions, but low efficiency of UV crosslinking currently limits such efforts.

“Requiring crosslinking of both miRNA and mRNA target would reduce post-lysis interactions, but low efficiency of UV crosslinking currently limits such efforts (Moore et al., 2015).”

• cites Moore et al., 2015 sourcePartly supported

  The Results section (page unavailable) says ligation to non-cross-linked targets could falsely identify interactions and tests for post-lysis reassociation, supporting the concern about post-lysis artifacts. It does not propose requiring UV cross-linking of both the miRNA and mRNA target. The Discussion (page unavailable) identifies miRNA–target ligation efficiency, not low UV-cross-linking efficiency, as the limiting factor. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

Other small regulatory RNAs, including piRNAs and snoRNAs, play critical roles in regulating RNA processing through interaction with complementary sequences.

“there exist many other small regulatory RNAs that play critical roles in regulating RNA processing through interaction with complementary sequences, including piRNAs, snoRNAs, and others (Gumienny et al., 2017;Shen et al., 2018).”

• cites Gumienny et al., 2017Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Shen et al., 2018 sourcePartly supported

  The Abstract and Discussion (pages not provided) show that piRNAs bind target RNAs through sequence pairing and participate in post-transcriptional regulation of germline gene expression. The paper does not establish that piRNAs regulate RNA processing specifically, and the passages do not address snoRNAs. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

TargetScan version 7.2 predicts 1,820 miR-124-3p targets.

“genes were also gropued based on predictions of 896 miR-1-3p targets and 1,820 miR-124-3p targets by TargetScan version 7.2 (Agarwal et al., 2015)”

• cites Agarwal et al., 2015 sourcePartly supported

  The Results section, “The TargetScan database (v7.0)” (page not provided), shows predicted miR-124 sites, supporting the use of TargetScan for miR-124 predictions. The Materials and methods section identifies the version studied as 7.0; our copy does not substantiate the claimed version 7.2 or the count of 1,820 targets. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

The same probe-capture approach can be used to recover miRNA interactions for a targeted gene of interest, revealing both distinct miRNA targeting as well as co-targeting by several miRNAs from the same seed family.

“We further demonstrate that the same probe-capture approach can be used to recover miRNA interactions for a targeted gene of interest, revealing both distinct miRNA targeting as well as co-targeting by several miRNAs from the same seed family.”

May combine two claims.

An average of 2.9% of peaks overlapped microRNAs, but weighting peaks by information content in IP versus input revealed that an average of 38.8% of total peak information was at microRNAs.

“Although only an average of 2.9% of peaks overlapped microRNAs due to their limited number, weighting peaks by information content in IP versus input revealed that an average of 38.8% of total peak information was at microRNAs, confirming substantial enrichment (Fig. 1C).”

May combine two claims.

Omitting the SDS-PAGE and nitrocellulose membrane transfer steps in chimeric AGO2 CLIP dramatically increases recovery of miRNA chimeric reads and simplifies the workflow, while maintaining high signal-to-noise ratio.

“Further, we find that in the case of chimeric AGO2 CLIP, omitting the SDS-PAGE and nitrocellulose membrane transfer steps dramatically increases recovery of miRNA chimeric reads and simplifies the workflow, and rigorously validate that this approach maintains high signal-to-noise ratio.”

May combine two claims.

Even with increased unique fragments in no-gel chimeric eCLIP, recovery of chimeras per miRNA is still correlated to miRNA abundance, making necessary sequencing to generate robust maps for a miRNA of interest cost-prohibitive for most miRNAs.

“However, as the recovery of chimeras per miRNA was still correlated to miRNA abundance, even with the increased number of unique (non-PCR duplicate) fragments in no-gel chimeric eCLIP the necessary sequencing to generate robust maps for a miRNA of interest would be cost-prohibitive for most miRNAs.”

May combine two claims.

Removal of the gel transfer steps required on average 6.3 fewer PCR cycles of amplification, suggesting a >70-fold increased experimental yield.

“We observed that removal of the gel transfer steps required on average 6.3 fewer PCR cycles of amplification, suggesting a >70-fold increased experimental yield (Fig. 2B).”

May combine two claims.

Location analysis of chimeric read alignments indicated an enrichment for expected target regions except for miR-4284, with an average of 33% of chimeric reads mapped to 3' UTRs and additional 21% to CDS.

“Next, location analysis of chimeric read alignments again indicated an enrichment for expected target regions except for miR-4284, with an average of 33% of chimeric reads mapped to 3' UTRs and additional 21% to CDS (Fig. 1G).”

May combine two claims.

We observed a high correlation in IP versus input enrichment between with-gel and no-gel libraries considering either all 250,339 clusters identified by CLIPper in the no-gel libraries (Pearson correlation 0.72) or all 353,280 clusters identified in the with-gel libraries (Pearson correlation 0.80).

“Expanding this analysis transcriptome-wide among non-chimeric reads, we observed a high correlation in IP versus input enrichment between with-gel and no-gel libraries considering either all 250,339 clusters identified by CLIPper in the no-gel libraries (Pearson correlation 0.72, P.Value < 2.2×10 -16 ) (Fig. 2E) or all 353,280 clusters identified in the with-gel libraries (Pearson correlation 0.80”

May combine two claims.

The enrichment for 3' UTR and CDS regions and increase in frequency of seed matching k-mers was preserved between with-gel and no-gel chimeric reads.

“The enrichment for 3' UTR and CDS regions and increase in frequency of seed matching k-mers was also preserved between with-gel and no-gel chimeric reads.”

May combine two claims.

For the top 20 miRNAs, an average of 58.7% of clusters contained 6-mers complementary to the cognate miRNA 6-mer seed region in positions [2:7], versus an average of 1.7% for non-seed 6-mers, and for 19 of the top 20 the miRNA seed was the most commonly observed 6-mer.

“Motif analysis indicated that for the top 20 miRNAs an average of 58.7% of clusters contained 6-mers complementary to the cognate miRNA 6-mer seed region in positions [2:7] (versus an average of 1.7% for non-seed 6-mers), and for 19 of the top 20 the miRNA seed was the most commonly observed 6-mer (Fig. 1F).”

May combine two claims.

We observed a high correlation between with-gel and no-gel approaches for both miRNA-only (Pearson correlation 0.950) and miRNA-chimeric read counts (Pearson correlation 0.953).

“We observed a high correlation between with-gel and no-gel approaches for both miRNA-only (Pearson correlation 0.950, P.Value < 2.2×10 -16 ) (Sup.Fig. 2A) and miRNA-chimeric read counts (Pearson correlation 0.953, P.Value < 2.2×10 -16 ) (Fig. 2C).”

May combine two claims.

In the top 25 miRNAs were three miRNAs from the mir-106b-25 cluster, of which two (miR-106b-5p and miR-93-5p) share seed sequence with miR-17-5p and a third (miR-25-3p) shares seed sequence with miR-92a-3p.

“In the top 25 miRNAs were also three miRNAs from the mir-106b-25 cluster, of which two (miR-106b-5p and miR-93-5p) share seed sequence with miR-17-5p and a third (miR-25-3p) shares seed sequence with miR-92a-3p.”

May combine two claims.

Information at non-chimeric peaks overlapping miRNAs (and the frequency of miRNA-only reads generally) was higher in no-gel experiments, replicated even when size-separating large (>30nt) and small (<30) library fractions.

“However, we did note that information at non-chimeric peaks overlapping miRNAs (as well as the frequency of miRNA-only reads generally) was higher in the no-gel experiments (Sup.Fig. 2D), and we replicated this observation even when explicitly sizeseparating large (>30nt) and small (<30) library fractions (Sup.Fig. 2E)”

May combine two claims.

For the two replicates, we identified a total of 403,532 and 428,936 unique chimeric reads, including 132,801 and 146,966 3' UTR chimeras, a dramatic increase over non-chimeric eCLIP or prior chimeric CLIP approaches.

“Confirming the ability of eCLIP to improve the recovery of unique (non-PCR duplicate) fragments, for the two replicates we identified a total of 403,532 and 428,936 unique chimeric reads (2.5% and 2.5% of uniquely mapped deduplicated reads, or 0.28% and 0.30% of 144 and 145×10 6 initial sequenced reads respectively for the two replicates), including 132,801 and 146,966 3' UTR chimeras (Fig. 1D), a”

May combine two claims.

miR-4284 had chimeras that predominantly mapped to mitochondrial transcripts and lacked seed matches, suggesting that this may not reflect a bona fide microRNA in 293T cells.

“One exception, miR-4284, had chimeras that predominantly mapped to mitochondrial transcripts and lacked seed matches, suggesting that this may not reflect a bona fide microRNA in 293T cells.”

May combine two claims.

The number of per-miRNA chimeric reads in total chimeric eCLIP correlates with non-chimeric miRNA abundance, leading to low coverage beyond the first few miRNAs.

“the number of per-miRNA chimeric reads in total chimeric eCLIP correlates with non-chimeric miRNA abundance (Sup Fig. 1C-D, Sup Fig. 2B-C), leading to low coverage beyond the first few miRNAs (Fig. 1E).”

May combine two claims.

miR-25-3p clusters showed high enrichment for 3' UTR (30.4%) and CDS (34.8%) regions, and 46.0% contained the canonical miR-25 seed sequence.

“These clusters showed high enrichment for 3' UTR (30.4%) and CDS (34.8%) regions (Fig. 3C), and motif analysis indicated that 46.0% of clusters contained the canonical miR-25 seed sequence”

May combine two claims.

In each case, read density within clusters was correlated with standard chimeric eCLIP, and clusters showed specific enrichment for seed region sequences, indicating that probe-enriched targets retain high signal-to-noise.

“In each case, the read density within clusters identified from chimeric reads was correlated with that observed from standard chimeric eCLIP (Sup.Fig. 4G-H), and clusters showed specific enrichment for sequences matching the seed region of the cognate miRNA (Fig. 4J, Sup.Fig. 4I), indicating that the more extensive set of probe-enriched targets retains the high signal-to-noise observed for non-en”

May combine two claims.

In mouse liver, the number of clusters was dramatically increased in probe-capture experiments, and seed matching sites were over-represented in clusters.

“the number of clusters was dramatically increased in probe-capture experiments (Sup.Fig. 4M-N), and seed matching sites for miRNAs were over-represented in clusters called from chimeric reads (Sup.Fig. 4O-P).”

May combine two claims.

Counts of chimeric reads per miRNA were highly correlated between gene-enriched (no-gel) chimeric eCLIP and matched with-gel non-enriched chimeric eCLIP (Pearson correlation 0.65 and 0.75 for ULK1 and APP respectively), indicating probe enrichment did not dramatically bias miRNA representation.

“counts of chimeric reads per miRNA were highly correlated between gene-enriched (no-gel) chimeric eCLIP and matched with-gel non-enriched chimeric eCLIP (Pearson correlation 0.65 and 0.75 for ULK1 and APP respectively) (Fig. 5C-D), indicating that the probe enrichment did not dramatically bias miRNA representation among gene specific chimeras.”

May combine two claims.

3' UTRs of downregulated genes contained miR-1 and miR-124 seed matches more often than 3' UTRs of upregulated genes, and genes containing complemented seed sites in 3' UTRs showed the strongest shift towards gene repression.

“3' UTRs of downregulated genes contained miR-1 and miR-124 seed matches more often than 3' UTRs of upregulated genes (Sup.Fig. 6G), and genes containing complemented seed sites in 3' UTRs showed the strongest shift towards gene repression for the two transfected miRNAs (Fig. 6D and Sup.Fig. 6H)”

May combine two claims.

Both small RNA-seq and total chimeric eCLIP confirmed reproducible and specific enrichment of overexpressed miR-1 and miR-124 and incorporation into mRNA-bound AGO2, with dramatic up-regulation in small RNA-seq (446- and 311-fold) and chimeric eCLIP (48,021- and 1,239-fold).

“Both small RNA-seq and total chimeric eCLIP confirmed reproducible and specific enrichment of the overexpressed miRNA (Sup.Fig. 6A-C) as well as incorporation into mRNA-bound AGO2 (Sup.Fig. 6D-E), with both miR-1 and miR-124 showing dramatic up-regulation in both small RNA-seq (average 446-and 311-fold respectively versus mock transfection) and chimeric eCLIP (48,021-and 1,239-fold respectively) (”

May combine two claims.

The 5 targeted miRNAs went from 4.9% in standard chimeric eCLIP to 93.6% in the enriched pool, indicating high specificity.

“Out of all identified chimeras, the 5 targeted miRNAs went from 4.9% in standard chimeric eCLIP to 93.6% in the enriched pool (Fig. 4F), indicating a high specificity in recovering the desired miRNAs.”

May combine two claims.

At least 100,000 chimeras were obtained for 89 of the 94 targeted miRNAs; extrapolating the non-enriched library to similar total depth would yield 100,000 chimeras for only the most abundant 32.

“we obtained at least 100,000 chimeras for 89 of the 94 targeted miRNAs; extrapolating sequencing the non-enriched library to similar total depth (~736 million reads) would yield 100,000 chimeras for only the most abundant 32 (Fig. 3B).”

May combine two claims.

Clusters from the probe-enriched experiment recapitulated specific enrichment for miRNA seed sequence, and read density within clusters was highly correlated with standard chimeric eCLIP (R = 0.63, p = 9.8×10^-194), indicating maintained high signal-to-noise.

“Clusters identified from chimeric reads in the probe-enriched experiment recapitulated specific enrichment for the miRNA seed sequence (Fig. 4G) and the read density within clusters was highly correlated with (while providing far deeper coverage compared to) standard chimeric eCLIP (R = 0.63, p = 9.8×10 -194 ) (Sup.Fig. 4A), indicating that probe-capture enrichment maintained the same high signal-”

May combine two claims.

The frequency of chimeras for targeted miRNAs increased from 0.01% to 0.48% of sequenced reads, resulting in more than a 12-fold increase in genes with reproducible 3' UTR clusters.

“The frequency of chimeras for the targeted miRNAs was increased from 0.01% to 0.48% of sequenced reads (Fig. 4D) resulting in a more than a 12-fold increase in the number of genes with reproducible 3' UTR clusters (Fig. 4E).”

May combine two claims.

In the miR-17 family experiment, selected miRNAs accounted for an average of 26.3% of total chimeric reads without enrichment; probe capture increased representation by 3.8-fold to 98.9% of chimeric reads and by 32.4-fold among all sequenced reads (from 0.07% to 2.4%).

“Even though miRNAs that were selected in the miR-17 family experiment accounted for an average of 26.3% of total chimeric reads without enrichment, probe capture further increased representation of the selected miRNAs by 3.8-fold to 98.9% of chimeric reads and by 32.4-fold among all sequenced reads (from 0.07% to 2.4%) (Sup.Fig. 4B-D).”

May combine two claims.

In mouse liver tissue, probe-based enrichment increased representation of chimeras for the 5-miRNA pool from 9.6% to 69.2% of chimeras and from 0.2% to 6.4% of total reads; for miR-122-5p from 34.2% to 66.6% of chimeras and from 0.8% to 5.9% of total reads.

“going from 9.6% to 69.2% of chimeras and from 0.2% to 6.4% of total reads for the 5-miRNA pool (Fig. 4F,K-L) and from 34.2% to 66.6% of chimeras and from 0.8% to 5.9% of total reads for miR-122-5p (Sup.Fig. 4J-L).”

May combine two claims.

Independent no-gel experiments showed similar sample-specific expression for these miRNAs, whereas mixed lysate samples had intermediate miRNA frequencies.

“Independent no-gel experiments showed similar sample-specific expression for these miRNAs, whereas intermediate miRNA frequencies were observed in mixed lysate samples (Sup.Fig. 2O).”

May combine two claims.

Across all 94 miRs, average annotation distribution was 39.9% 3' UTR and 31.9% CDS clusters, and the 2-7 seed match sequence was the most abundant 6-mer for 21 (top 10 for 47) of the 94 profiled miRNAs.

“Expanding to all 94 miRs showed a similar annotation distribution, with average 39.9% 3' UTR and 31.9%CDS clusters (Sup.Fig. 3C), and the 2-7 seed match sequence was the most abundant 6-mer for 21 (and in the top 10 for 47) out of the 94 profiled miRNAs (Fig. 3E).”

May combine two claims.

Individual target sites were well separated, identifying numerous putative miRNA target sites in 3' UTRs of ULK1 and APP.

“we observed that individual target sites were well separated from each other, identifying numerous putative miRNA target sites in 3' UTRs of ULK1 and APP (Fig. 5E-F).”

May combine two claims.

Many datasets showed strong enrichment for the 6-mer at the end of the PCR primer, and manual inspection confirmed these regions often contained stretches of homology to the miRNA that likely cause mis-priming.

“many datasets also showed strong enrichment for the 6-mer at the end of the PCR primer (Fig. 3E), and manual inspection confirmed that these regions often contained stretches of homology to the miRNA that likely cause mis-priming of the PCR primer.”

May combine two claims.

miR-1 contributed more than 66% of reads in small RNA-seq after overexpression, while miR-124 was more frequent in chimeras (more than 35%).

“miR-1 was more abundant after over-expression in small RNA-seq (contributing more than 66% of reads (Fig. 6B)), miR-124 was more frequent in chimeras (more than 35% (Fig. 6C))”

May combine two claims.

Other non-targeted let-7 members were also enriched but were distinguishable by sequencing.

“although other non-targeted let-7 members were also enriched, they were distinguishable by sequencing (Fig. 4H).”

May combine two claims.

In the let-7 family experiment, targeted miRNAs accounted for 0.01% before enrichment, increasing 49.4-fold to 0.4% following enrichment, with an increase from 3.2% of chimeric reads without enrichment to 82.2% after enrichment.

“Chimeric reads for the targeted miRNAs in the let-7 family experiment were less common, accounting for only 0.01% before enrichment but increasing 49.4-fold to 0.4% following enrichment, with an increase from 3.2% of chimeric reads without enrichment to 82.2% after enrichment (Fig. 4F,H-I).”

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

Preliminary analyses observed addition of non-templated nucleotides after reverse transcription that may complicate characterization of microRNA 5' end isomiRs from chimeric eCLIP data.

“in preliminary analyses we observed the addition of non-templated nucleotides after reverse transcription that may complicate this analysis (data not shown)”

May combine two claims.

The experiments profiled nearly six million non-PCR duplicate, uniquely mapped chimeric reads in HEK293T, including 2.6 million across 6 with-gel and 3.4 million across 12 no-gel experiments.

“the experiments described in our study profiled nearly six million non-PCR duplicate, uniquely mapped chimeric reads in HEK293T, including 2.6 million across 6 with-gel and 3.4 million across 12 no-gel experiments”

May combine two claims.

Coverage remained dependent on miRNA abundance, with miR-20a-5p and miR-17-5p yielding over 210,000 with-gel and 340,000 no-gel chimeras each; 46 miRNAs in merged with-gel and 52 in merged no-gel had at least 10,000 chimeras.

“coverage remained dependent on miRNA abundance, with miR-20a-5p and miR-17-5p yielding over 210,000 with-gel and 340,000 no-gel chimeras each; however, 46 miRNAs in the merged with-gel and 52 in the merged no-gel had at least 10,000 chimeras”

May combine two claims.

The number of CDS clusters was roughly similar to that of 3' UTR clusters, and genes with chimeric clusters only in CDS for miR-124 (146) and miR-1 (93) showed significant repression in miR overexpression (p = 2.3×10^-5 and p = 7.3×10^-4), albeit weaker than genes with 3' UTR only clusters.

“we observed that the number of CDS clusters was roughly similar to that of 3' UTR clusters (Fig. 6E), and that genes with chimeric clusters only in CDS for miR-124 (146) and miR-1 (93) showed significant repression in miR over-expression (p = 2.3×10 -5 a6d p = 7.3×10 -4 respectively), albeit weaker than the repression observed for genes with 3' UTR only clusters”

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

In pilot analyses, mapping to human miRNAs gave similar results as separate mapping to both human and rat miRNA annotations.

“in pilot analyses, mapping to human miRNAs gave similar results as separate mapping to both human and rat miRNA annotations”

May combine two claims.

These typically had hundreds to thousands of significant clusters located within 3' UTR or CDS of dozens to over a thousand unique genes, and clusters were enriched for miRNA seed matches, with the miRNA 2-7 seed being the most frequently observed 6-mer for 44 of the 46 miRNAs.

“These typically had hundreds to thousands of significant clusters located within the 3' UTR or CDS of dozens to over a thousand unique genes (Fig. 7A, Sup.Fig. 7B), and these clusters were enriched for miRNA seed matches as expected, with the miRNA 2-7 seed being the most frequently observed 6-mer for 44 of the 46 miRNAs (Fig. 7B).”

May combine two claims.

Merging replicates yielded 963,813 and 856,953 3' UTR chimeras for with-gel and no-gel respectively, and cluster identification identified 31,494 putative high-confidence miRNA interaction sites in with-gel and 36,559 in no-gel.

“yielding 963,813 and 856,953 3' UTR chimeras for with-gel and no-gel respectively, and performed cluster identification for each miRNA with at least 1,000 chimeric reads, enabling identification of 31,494 putative highconfidence miRNA interaction sites in with-gel and 36,559 in no-gel respectively (CLIPper p ≤ 10 - 5 ).”

May combine two claims.

TargetScan-predicted targets were more numerous than genes with chimeric eCLIP clusters and showed significant repression upon miRNA overexpression for both miR-124 and miR-1, although magnitude was similar to only the low-confidence chimeric eCLIP set.

“TargetScan-predicted targets were more numerous than genes with chimeric eCLIP clusters and showed significant repression upon miRNA over-expression for both miR-124 and miR-1 (Fig. 6F, Sup.Fig. 6I), although the magnitude was more similar to only the low-confidence (RPMC≥0 read) chimeric eCLIP set.”

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

862 miR-1-3p targets predicted by TargetScan overlapped GENCODE v29 gene identifiers.

“of which 862 of miR-1-3p and 1,769 of miR-124-3p targets overlapped GENCODE v29 gene identifiers.”

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

The miRNAs with >1000 average chimeric reads include miRNAs from two abundant families in HEK293T cells.

“Color indicates miRNAs from two abundant families in HEK293T cells.”

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

1,769 miR-124-3p targets predicted by TargetScan overlapped GENCODE v29 gene identifiers.

“of which 862 of miR-1-3p and 1,769 of miR-124-3p targets overlapped GENCODE v29 gene identifiers.”

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

Regulation via miRNA-mediated repression of gene expression is involved in nearly every physiological system and misregulation of miRNA biology is implicated in a broad spectrum of diseases.

“Regulation via miRNA-mediated repression of gene expression has been shown to be involved in nearly every physiological system and misregulation of miRNA biology has been implicated in a broad spectrum of diseases ranging from cancer to neurodegenerative diseases (Quinlan et al., 2017;Rupaimoole and Slack, 2017).”

• cites Quinlan et al., 2017 sourceNot checked — no full text of the source
• cites Rupaimoole and Slack, 2017 sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Active research and development in RNA-targeted therapies creates a need for tools that can accurately profile miRNA:mRNA target interactions in different cell cultures and tissues at scale.

“Active research and development in the area of RNA-targeted therapies creates a need for tools that can accurately profile miRNA:mRNA target interactions in different cell cultures and tissues at scale.”

Classified as probably not a substantial claim.

Computational approaches typically focus on sequence complementarity particularly in nucleotides 2-8 (the seed region) and sequence conservation across species.

“Computational approaches typically focus on a small set of key features, including sequence complementarity particularly in nucleotides 2-8 (commonly referred to as the 'seed' region of the miRNA), and sequence conservation across species.”

Probably not a substantial claim: may not pass S4 (based).

Successful mapping of chimeric miRNA:target reads requires at least 40 nt total length (including both a 20-22nt miRNA and a sufficient target sequence to uniquely map).

“Finally, as successful mapping of chimeric miRNA:target reads requires at least 40 nt total length (including both a 20-22nt miRNA and a sufficient target sequence to uniquely map)”

Probably not a substantial claim: may not pass S4 (based).

Many mammalian miRNAs have extremely high or low GC content, making design of primers with reasonable melting temperatures challenging.

“First, many mammalian miRNAs have extremely high or low GC content, making design of primers with reasonable melting temperatures challenging.”

Probably not a substantial claim: may not pass S2 (checkable).

Deep profiling of regulatory maps for individual miRNAs is needed to study the molecular mechanisms of how miRNA regulation alters physiology and disease.

“Thus, deep profiling of regulatory maps for individual miRNAs is needed to study the molecular mechanisms of how miRNA regulation alters physiology and disease.”

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

False-positive chimeras can be introduced due to the PCR primer annealing to similar sequences elsewhere in the transcriptome.

“Additionally, false-positive chimeras can be introduced due to the PCR primer annealing to similar sequences elsewhere in the transcriptome.”

Classified as probably not a substantial claim.

A method to specifically enrich the sequenced library for individual miRNAs of interest would enable deeper profiling for biologically relevant miRNAs in a particular cell line or tissue system.

“Thus, a method to specifically enrich the sequenced library for individual miRNAs of interest would enable us to make full use of this improved yield by allowing deeper profiling for biologically relevant miRNAs in a particular cell line or tissue system under study.”

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

AGO IP and computational analysis has the advantage of enabling prediction of interactions for all miRNAs in one experiment, but there remain many situations where experimental mapping of direct miRNA:target interactions would be preferred.

“Although the combination of AGO IP and computational analysis has the advantage of enabling prediction of interactions for all miRNAs in one experiment, there remains many situations where experimental mapping of direct miRNA:target interactions would be preferred.”

Classified as probably not a substantial claim.

Experimental identification of direct miRNA interactions on a large scale has proven more challenging.

“Experimental identification of direct miRNA interactions on a large scale has proven more challenging.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

It would not be surprising that post-lysis interactions could occur.

“suggesting that it would not be surprising that such post-lysis interactions could occur.”

May combine two claims.

Both human and rat samples were optimally fragmented for successful ligation and incorporation into chimeric reads that had similar enrichment for 3' UTR regions.

“validating that both samples were optimally fragmented for successful ligation and incorporation into chimeric reads that had similar enrichment for 3' UTR regions”

May combine two claims.

PCR enrichment replaces the actual RNA fragment sequence with the primer sequence in the final read, causing loss of information about related miRNAs and miRNA 5' ends.

“the replacement of the actual RNA fragment sequence with the primer sequence in the final read (and subsequent loss of information about related miRNAs, including miRNA family members which often only contain a single nucleotide difference, and miRNA 5' ends) represents a clear limitation.”

May combine two claims.

A substantial rate of false positives is present even in standard with-gel chimeric approaches and is moderately increased in the no-gel approach.

“these results suggest that a substantial rate of false positives are present even in standard with-gel chimeric approaches and are moderately increased in the nogel approach.”

• AssumptionRests on an assumption: The 1.2% of human miRNA chimeras aligning to rat in human-only experiments reflects the baseline mapping error rate.
• AssumptionRests on an assumption: Half of potential post-lysis interactions pair a human miRNA with human target sequence and are undetectable by lysate mixing approaches.

May combine two claims.

Overlapping PCR-enriched datasets with standard AGO2 peaks can help remove false-positive signal and can help enable identification of which miRNA is causing a putative AGO2 interaction.

“suggesting that overlapping PCR-enriched datasets with standard AGO2 peaks can help remove false-positive signal and can help enable identification of which miRNA is causing a putative AGO2 interaction.”

May combine two claims.

Probe-capture chimeric-eCLIP can enrich for entire miRNA families while preserving the exact sequence of the specific miRNA bound to each target mRNA, enabling deep profiling of miRNA families with highly related sequences.

“Probe-capture chimeric-eCLIP can enrich for entire miRNA families while preserving the exact sequence of the specific miRNA bound to each target mRNA, enabling deep profiling of miRNA families with highly related sequences.”

• AssumptionRests on an assumption: Probe capture simply enriches for but does not alter the original chimeric RNA fragments.

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

MicroRNAs often regulate gene expression by inducing RNA degradation, and a common way to validate miRNA targets at scale is to show downregulation following miRNA overexpression.

“As microRNAs often regulate gene expression by inducing RNA degradation, a common way to validate miRNA targets at scale is to show downregulation following miRNA overexpression (Lim et al., 2005).”

• cites Lim et al., 2005 sourceNot checked — no full text of the source

May combine two claims.

The two-step PCR approach allows enrichment of a miRNA of interest and provides selection of chimeric rather than miRNA- or mRNA-only fragments.

“this approach allows enrichment not only of a miRNA of interest but also provides selection of chimeric rather than miRNA-or mRNA-only fragments.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

This resource represents a unique opportunity to further explore the regulatory roles and networks of miRNAs in the well-characterized 293T cell line.

“Thus, this resource represents a unique opportunity to further explore the regulatory roles and networks of miRNAs in the wellcharacterized 293T cell line.”

May combine two claims.

PCR-based enrichment is experimentally simpler and can yield robust profiling for many miRNAs, but the PCR primer replaces the sequence of the original miRNA, making it impossible to separate highly similar family members, leading to favor probe-capture for general use.

“Although PCR-based enrichment is experimentally simpler and can yield robust profiling for many miRNAs, the common amplification of transcriptomic sites with sequence complementary to the primer coupled with the fact that the PCR primer replaces the sequence of the original miRNA (making it impossible to separate highly similar family members) led us to favor the probe-capture approach for general”

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

In the implementation described, the study does not directly address the stoichiometry of binding between miRNAs and their targets, and it remains an open question whether certain interactions identified by probe-enriched chimeric eCLIP reflect a high enough fraction of expressed copies of that mRNA to drive functionally relevant expression changes.

“in the implementation we describe here we do not directly address the stoichiometry of binding between miRNAs and their targets, and it remains an open question whether certain interactions identified by probe-enriched chimeric eCLIP reflect a high enough fraction of expressed copies of that mRNA to drive functionally relevant expression changes.”

Classified as probably not a substantial claim.

In traditional CLIP, denaturing SDS-PAGE and nitrocellulose transfer removes non-crosslinked RNA, providing a mechanism for removing potential post-lysis interactions.

“In a traditional CLIP experiment, the denaturing SDS-PAGE and nitrocellulose transfer removes non-crosslinked RNA, providing a mechanism for removing potential post-lysis interactions.”

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

Animal experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California San Diego.

“Animal experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California San Diego.”

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

Chimeric eCLIP data has been deposited at the Gene Expression Omnibus (GEO ID GSE198251).

“Chimeric eCLIP data has been deposited at the Gene Expression Omnibus (GEO ID GSE198251 ; reviewer token yruvagicbjixnin).”

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

The primary data processing pipeline for chimeric eCLIP data is available at https://github.com/YeoLab/chim-eCLIP.

“The primary data processing pipeline for chimeric eCLIP data is available at https://github.com/YeoLab/chim-eCLIP.”