Complexity of murine cardiomyocyte miRNA biogenesis, sequence variant expression and function

microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ∼40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5' start but common at the 3' end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5' isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5' isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5' most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy.This work was supported by by the Victor Chang Cardiac Research Institute and grants 573726, 573731 and 514904 from the National Health & Medical Research Council awarded to TP

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist

Complexity of Murine Cardiomyocyte miRNA Biogenesis, Sequence Variant Expression and Function

<div><p>microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ∼40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5′ start but common at the 3′ end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5′ isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5′ isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5′ most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy.</p> </div

Novel miR*, novel non-canonical miRNAs and novel antisense miRNA.

†<p>Novel miR* that are processed within the expected window of the mature strand are labelled “generic”. Entries are ranked by tag abundance.</p>‡<p>(pre-)miRNA with known function and/or expression in the heart as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030933#pone.0030933-Matkovich1" target="_blank">[29]</a>.</p>§<p>All antisense hairpins have at least one tag aligned to the opposing side of the stem.</p>||<p>miRBase v16 annotated miRNAs removed from miRBase v17.</p>∧<p>reported in miRbase v18.</p

5′ and 3′ isomiRs in HL-1 cardiomyocytes.

<p>Thresholds were set at expression ≥150 tags (only miRNA mapped to one loci shown). Entries are ranked by tag abundance and truncated after the top 20 entries.</p><p>†(pre-)miRNA with known function and/or expression in the heart as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030933#pone.0030933-Matkovich1" target="_blank">[29]</a>.</p

Diversity of HL-1 cardiomyocyte miRNA processing.

<p>(<b>A</b>) Schematic of hairpin miRNA precursor showing proportion of tags derived from either 5′ (blue) or 3′ (green) arm (in % of total hairpin-mapped tags; shown at center of boxed regions). For each arm, the proportion of tags representing 5′ start or 3′ end positions that vary from miRBase annotation is also shown (in % of tags per arm; shown at ends of boxed regions). Red lines indicate typical Dicer and Drosha processing sites. (<b>B</b>) 5′ strand bias of each miRNA is plotted against the sum of tags per hairpin. Color scheme represents miRBase version 16 annotation as mature species on 5′ or 3′ arms (red and orange circles, respectively), or as a 5p/-3p pair (blue circles). (<b>C</b>) Examples of miRNAs with unexpected strand bias. Schematics of hairpin indicate abundance of tag mapping to opposing arms (on left), while Venn diagrams indicate number and overlap of predicted mRNA targets (Targetscan) for each of these miRNA (on right; top: all predicted targets, bottom: only targets with specific gene function annotation (Ingenuity) as listed; #: significant enrichment of gene function term, p<0.01). (<b>D</b>) The proportion of tags representing 5′ isomiRs for each miRNA is plotted against tag abundance. Color scheme represents position of miRNA on 5′ (blue) or 3′ (green) arm. (<b>E</b>) As in (D) but showing prevalence of 3′ isomiRs. (<b>F</b>) Examples of miRNAs with high 5′ isomiR proportion. Tag sequences representing canonical miRNA (black) and major 5′ isomiR (red) are depicted on top, while analyses of mRNA targets (as in D) are shown below. (<b>G</b>) Examples of distinct miRNA 3′ end variability. Distribution of tag 3′ ends for miR-499 is from the heart biopsy, while HL-1 cell data is shown for miR-181a, miR-15a and miR-301a (miRBase-annotated 3′ end positions are boxed). Panels (B, D and E) depict only generic miRNAs (tags with 5′ start position +/− 3 nt of miRbase v16 annotation or novel miR* directly juxtaposed to a known miRNA) with an expression level of ≥150 tags; the dashed lines show tag proportion thresholds of 80% and 20% used throughout this study to categorize miRNAs.</p

isomiRs of miR-133a with different targeting properties.

<p>(<b>A</b>) Major mature miR-133a species and their abundance in HL-1 cardiomyocytes. Sequence tags are grouped into those with canonical (black, ‘can’) and +1 (red, ‘iso’) 5′ start sites. Brackets denote sequences used as miRNA mimics in panels D and E. (<b>B</b>) Venn diagrams indicate number and overlap of predicted mRNA targets (Targetscan) for canonical and +1 5′ isomiR variants of miR-133a (top: all predicted targets, bottom: only targets with roles in cardiovascular disease (Ingenuity; #: significant enrichment of gene function term, p<0.01). (<b>C</b>) Schematic of reporter constructs made to contain three copies of predicted miR-133a binding sites from <i>Pgam1</i> or <i>Ctgf</i> mRNA 3′UTRs. Base pairing potential between sites and miR-133a isomiRs is also shown. (<b>D</b>) <i>Ctgf</i> or <i>Pgam1</i> R-luc reporters were transfected into HeLa cells with mimics of the major variants of the canonical (can/23 nt) and +1 5′ variants (iso/22 nt) of miR-133a, an irrelevant control, or no mimic at all, and luciferase activity measured 24 hours later. The fold change of expression is calculated as no mimic/mimic and results are averages of four independent experiments with standard error. (<b>E</b>) Transfections as in (D) comparing mimics of different lengths (can/23 nt vs can/22 nt, iso/22 nt vs. iso/23 nt; for sequence see panel A). Repression is given as a percentage of that seen with the respective major variant and results are averages of at least three independent experiments with standard error.</p

New miRNA variants mapping to known hairpin precursors.

<p>(<b>A</b>) Examples of generic miR* species discovered in HL-1 cells not currently annotated in miRBase. Position within the hairpin structure is consistent with canonical processing. (The additional case of miR-721* is shown in panel C). (<b>B</b>) We refer to a set of miRNA tags outside the canonical processing region of hairpins as an extreme isomiR (e-miRs). Position of the major e-miR on the mir-711 hairpin suggests it as a novel case of Ago2-mediated processing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030933#pone.0030933-Cifuentes1" target="_blank">[54]</a>. (<b>C</b>), The miR 721 hairpin gives rise to an abundant e-miR; expression of the annotated mature miR-721 and its generic miR* counterpart is much lower. <i>Left,</i> hairpin fold as listed in miRbase; <i>right</i>, hypothetical alternative fold consistent with canonical processing of miR-721 together with the novel e-miR-721*. (<b>D</b>) Venn diagrams indicate number and overlap of predicted mRNA targets (Targetscan) for the three miRNA variants mapping to the miR-721 hairpin (top: all predicted targets, bottom: only targets with roles in cardiovascular disease (Ingenuity). miRNAs already annotated in miRBase are marked by open boxes, while novel species are highlighted by filled boxes.</p