Transcriptome-wide Discovery of microRNA Binding Sites in Human Brain

In the paper

“In mice, these approaches have been applied in brain and liver tissues (Chi et al., 2009; Schug et al., 2013); however, human data are only available for cultured cells (Chi et al., 2009; Haecker et al., 2012; Hafner et al., 2010).”

What the sources say

• Partly supported

  The “Ago RNA targets in the mouse brain” section describes HITS-CLIP on mouse neocortex. The “Ago HITS-CLIP and miR-124” section also reports experiments in cultured HeLa cells. The paper does not establish the broader claim that human CLIP data were available only from cultured cells; it does not address all human data. (LLM, conf=0.87)

  cites Chi et al., 2009 sourceExperimental — accuracy not yet measured
• Supported

  Schug et al. is cited for the mouse-liver application, not the brain or human-data claims. The Methods sections on Animals and HITS-CLIP describe liver tissue harvested from mice and the HITS-CLIP assays performed on it. (LLM, conf=0.94)

  cites Schug et al., 2013 source
• Supported

  The Methods (PAR-CLIP) state that the study used cultured human embryonic kidney (HEK293) cells. Hafner et al. therefore supports the part of the citing sentence for which it is offered: an example of human CLIP-based data from cultured cells. (LLM, conf=0.91)

  cites Hafner et al., 2010 source

In the paper

“Notably, significantly enriched motifs among these lesser abundant seeds include those corresponding to miRNAs previously described as brain-enriched (e.g., miR-101, miR-153, and miR-485) or having synaptic (e.g., miR-138) or glial (e.g., miR-23) localization (Cohen et al., 2011; Farh et al., 2005; Lagos-Quintana et al., 2002; Sempere et al., 2004; Siegel et al., 2009; Smirnova et al., 2005), sign”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites Cohen et al., 2011 source
• ?Not checked — no full text of the sourcecites Farh et al., 2005 source
• Partly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites Lagos-Quintana et al., 2002 sourceExperimental — accuracy not yet measured

In the paper

“Furthermore, shifted binding by miR-221 was previously observed in a recent study employing the CLIP-based technique known as CLASH, an advanced approach for identifying noncanonical miRNA binding sites (Helwak et al., 2013).”

What the sources say

• Partly supported

  The Results section, “CLASH Directly Maps miRNA-Binding Sites” (page unavailable), describes CLASH as a method for identifying miRNA-target interactions; the Results also report noncanonical seed interactions. But the passages found for “miR-221” and “shifted binding” do not report shifted binding by miR-221, so the sentence’s specific observation is not supported by our copy. (LLM, conf=0.82)

  cites Helwak et al., 2013 sourceExperimental — accuracy not yet measured

In the paper

“Given that the average Ago-mRNA footprint is 45–62 nt (Chi et al., 2009), we extended the continuous regions with length less than 50 nt to 50 or 51 nt symmetrically from the 5′ and 3′ ends.”

What the sources say

• Supported

  In “Ago RNA targets in the mouse brain,” the authors report that Ago bound within 45–62 nt of cluster peaks at least 95% of the time and explicitly call this region the average Ago-mRNA footprint. The citing sentence accurately attributes that footprint range to the paper. (LLM, conf=0.94)

  cites Chi et al., 2009 source

In the paper

“miRNAs are excised from larger stem-loop-containing transcripts and are subsequently incorporated into argonaute (Ago) proteins. This generates functional entities capable of silencing target transcripts via translational repression and mRNA destabilization (Bazzini et al., 2012; Djuranovic et al., 2012).”

What the sources say

• Partly supported

  The Abstract reports that miR-430 represses translation and subsequently causes target mRNA decay, supporting the silencing part of the claim. Our copy does not describe miRNAs being excised from stem-loop-containing transcripts or incorporated into Argonaute proteins. (LLM, conf=0.88)

  cites Bazzini et al., 2012 sourceExperimental — accuracy not yet measured
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites Djuranovic et al., 2012 source

In the paper

“Our work revealed more than 7,000 Ago2 binding sites, many of which harbor sequences corresponding to the most abundant brain miRNAs.”

In the paper

“Furthermore, >25% of the Ago2 brain clusters in 3′ UTRs overlap with TargetScan-predicted conserved miRNA binding sites (>2-fold enrichment over background expectations, Pearson's chi-square test p < 2.2 × 10−16; Table S3) (Grimson et al., 2007).”

What the sources say

• Not checked yetcites Grimson et al., 2007 source

In the paper

“All four showed a repressive response to miR-23b treatment in cell culture, and, notably, a significant impact of the SNP on silencing was observed for three of the targets.”

In the paper

“Another exciting research avenue would be to test whether CDS-located miRNA binding sites residing within alternatively spliced exons confer isoform-specific silencing; indeed, a preliminary analysis of our data set indicates the presence of nearly 400 clusters within alternatively spliced cassette exons (data not shown).”

In the paper

“Our data set harbors 88 putative miR-137 sites (Table S6; based on canonical TargetScan seeds), and 24 of these reside within schizophrenia-related genes (SZGene database, n = 13) and/or genes having correlated expression with prefrontal cortex abnormalities in psychiatric patients (n = 16) (Allen et al., 2008; Kim and Webster, 2010).”

What the sources say

• ?Not checked — no full text of the sourcecites Allen et al., 2008 source
• ?Not checked — no full text of the sourcecites Kim and Webster, 2010 source

In the paper

“Cloning and high-throughput sequencing of these Ago2-associated RNAs produced nearly 50 million reads for each sample, yielding an average of 200,000 reads mapping to known human mature miRNAs (miRBase) and 15 million uniquely mapped reads within the human genome (Figure 1D).”

In the paper

“Interestingly, the GATM gene SNP tested showed the most robust differential response, and the minor allele was strongly repressed.”

In the paper

“We identified 646 shared human and mouse Ago HITS-CLIP regions, representing a significant enrichment relative to background clusters (∼5-fold, Pearson's chi-square test, p < 2.2 × 10−16; Figure 5A and Table S3).”

In the paper

“Note that rs9468 and rs629301 were identified in Ago2 brain clusters with Bonferroni adjusted p value < 25%, while all others overlap with Bonferroni adjusted p value < 5% clusters.”

In the paper

“When miRNA read counts were grouped into their respective seed families, the top five most abundant Ago2-associated seed sequences (Let-7, miR-125, miR-124, miR-9, and miR-29) comprised nearly 55% of total Ago2 “seed load” present in the samples (Figures 1E and S1B), and the top 20 seeds accounted for 75%.”

In the paper

“This analysis produced 101 sites across 56 genes, many of these sites corresponding to brain-enriched miRNAs (Figure 5D).”

In the paper

“In total, we detected nearly 1,900 unique miRNAs (Table S2), including an abundance of known brain-enriched miRNAs that were present at relative levels comparable to previous studies profiling human brain miRNAs by small RNA sequencing and microarray (Hu et al., 2011; Shao et al., 2010).”

What the sources say

• ?Not matched

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

  cites Hu et al., 2011
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites Shao et al., 2010 source

In the paper

“Upon the removal of PCR duplicates on the basis of redundant genomic coordinates, each sample generated roughly 1 million uniquely mapped reads, providing considerable depth for HITS-CLIP analysis.”

In the paper

“As with previous CLIP-based studies, we observed an inherent bias toward identifying Ago2 binding sites in higher-expressed transcripts (data not shown); on average, our studies ascertained roughly two sites per target gene.”

In the paper

“Notably, >80% of the clusters are ≤ 60 nt in length, and only 6% are >100 nt (Table S3).”

In the paper

“Others have shown that mutations in this region may disrupt splicing inclusion of exon 10, which can contribute to certain tauopathies, including Alzheimer's disease and frontotemporal dementia with Parkinsonism (Hasegawa et al., 1999).”

What the sources say

• ?Not checked — no full text of the sourcecites Hasegawa et al., 1999 source

In the paper

“Our data indicate that it woulddisrupt a potential miR-124 seed, and the coincident expression of TUBA1A and miR-124 in developing brain suggests that their interaction may be required for proper corticogenesis (Coksaygan et al., 2006; Maiorano and Mallamaci, 2009).”

What the sources say

• ?Not checked — no full text of the sourcecites Coksaygan et al., 2006 source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites Maiorano and Mallamaci, 2009 source

In the paper

“To call HITS-CLIP peaks with information from multiple replicates, we used Fisher's method to combine p values of replicates.”

In the paper

“In addition to a previously validated interaction of miR-137 with TCF4, our data support seven additional functional targets for miR-137 within genes associated with psychiatric disease (Figure 6A).”

In the paper

“miRNAs typically bind to target mRNA 3′ UTRs containing short stretches of complementarity to the “seed” region of the miRNA, corresponding to positions 2–8 (Lewis et al., 2005).”

What the sources say

• ?Not checked — no full text of the sourcecites Lewis et al., 2005 source

In the paper

“The p values were transformed to FDRs with the Benjamini-Hochberg procedure (Benjamini and Hochberg, 1995).”

What the sources say

• ?Not checked — no full text of the sourcecites Benjamini and Hochberg, 1995 source

In the paper

“These data are consistent with prior reports citing that highly abundant miRNAs often constitute the vast majority of cellular miRNA pools and are generally the chief miRNAs mediating significant target suppression within cells (Chi et al., 2009; Hafner et al., 2010; Mullokandov et al., 2012).”

What the sources say

• Not checked yetcites Chi et al., 2009 source
• Not checked yetcites Hafner et al., 2010 source
• Not checked yetcites Mullokandov et al., 2012 source

In the paper

“Note that the mouse Ago ternary map consists of all sites (i.e., uncurated in terms of robustness) identified by Ago HITS-CLIP in P13 mouse neocortex (refer to Chi et al., 2009, for a description).”

What the sources say

• Not checked yetcites Chi et al., 2009 source

In the paper

“a quality assessment of the Ago2 HITS-CLIP libraries was performed by the University of Iowa DNA Facility with the High Sensitivity DNA Assay on the Agilent Bioanalyzer DNA-1000.”

In the paper

“Interestingly, miR-23b is downregulated in and considered to be a broad suppressor of autoimmune diseases, including lupus, rheumatoid arthritis, and multiple sclerosis (Zhu et al., 2012).”

What the sources say

• ?Not checked — no full text of the sourcecites Zhu et al., 2012 source

In the paper

“We used Bonferroni adjusted p value < 0.05 as the cutoff in order to define statistically significant positions.”

In the paper

“Biological replicates were increased to n = 6 by repeating the transfections and downstream analyses.”

In the paper

“SNCA encodes alpha-synuclein, a component of the pathogenic aggregates observed in Parkinson's disease, and one of these SNPs (rs17016074) increases transcript levels by >30% (Sotiriou et al., 2009), whereas the other resides within a functional miR-153 binding site (Doxakis, 2010).”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites Sotiriou et al., 2009 source
• ?Not checked — no full text of the sourcecites Doxakis, 2010 source

In the paper

“The combined p value for each position was adjusted by Bonferroni correction in order to account for multiple hypothesis testing.”

In the paper

“During the past decade, microRNAs (miRNAs; ∼19–22 nucleotides) have emerged as key posttranscriptional regulators of gene expression, having been implicated in nearly all biological processes (Krol et al., 2010).”

What the sources say

• ?Not checked — no full text of the sourcecites Krol et al., 2010 source

In the paper

“Notably, SNCA duplications or triplications cause familial Parkinson's disease (Chartier-Harlin et al., 2004), and changes in alpha-synuclein levels have been hypothesized to contribute to more common forms of Parkinson's.”

What the sources say

• ?Not checked — no full text of the sourcecites Chartier-Harlin et al., 2004 source

In the paper

“Luminescent readings were measured for each sample in duplicate using a GloMax Microplate Reader (Promega), and results were calculated as the quotient of Renilla/Firefly luciferase activities with subsequent normalization to the pre-miR-Neg1 control for each 3′ UTR tested.”

In the paper

“However, several prior reports have noted that, for the most part, endogenous miRNAs are equivalently sorted into Ago1 and Ago2 (Dueck et al., 2012).”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites Dueck et al., 2012 source

In the paper

“These results validate the data set's utility for revealing clinically relevant information and warrant future studies aimed at investigating these interactions in specific brain-cell types.”

In the paper

“HEK 293 cells grown in 24-well plates were cotransfected in triplicate with 10 ng of luciferase reporter plasmids and 50 uM of commercially available synthetic pre-miRNAs corresponding to miR-137, miR-23b, or a negative control (miR-Neg1).”

In the paper

“Ago proteins (namely, Ago1 and Ago2) are the core components of miRNA-silencing complexes (Krol et al., 2010), and this powerful technique allows for sequencing of both the miRNAs and the mRNA target regions to which they bind.”

What the sources say

• ?Not checked — no full text of the sourcecites Krol et al., 2010 source

In the paper

“The expression levels from different samples were further normalized by quantile normalization in order to control for batch effect.”

In the paper

“Although the associated GWAS study implicates this gene in chronic kidney disease, rare GATM mutations have been shown to cause creatine deficiencies in brain, leading to cognitive impairment (Item et al., 2001).”

What the sources say

• ?Not checked — no full text of the sourcecites Item et al., 2001 source

In the paper

“For the miRNA inhibition study, Hafner et al. (2010) performed a pooled depletion of 25 highly expressed miRNAs in human embryonic kidney (HEK) 293 cells.”

What the sources say

• Not checked yetcites Hafner et al. (2010) source

In the paper

“In brain, miRNAs play vital roles in cell-fate specification, neurite projection, and synaptic plasticity, and growing evidence suggests that even slight aberrations in miRNA activities can be detrimental to neuronal function (Im and Kenny, 2012).”

What the sources say

• Not checked yetcites Im and Kenny, 2012 source

In the paper

“Official gene symbols for genes containing human and mouse shared Ago brain clusters were input as the “target set,” and genes containing human Ago2 brain clusters were input as “background set” in order to control for bias of HITS-CLIP approaches toward higher expressed transcripts.”