Additional file 2: of Expanding preconception carrier screening for the Jewish population using high throughput microfluidics technology and next generation sequencing

Examples of primer design and supporting reads for the large-rearranged mutations. A. Deletion of ~5Kb in the GALT gene, leading to Galactosemia. This mutation is composed of four breakpoints, leading to two large deletions and one small insertion, and resulting in the loss of almost the entire gene (adapted from Coffee et al. [27]). Vertical arrows depict the breakpoints, and horizontal arrows mark the primers used for capture. Primers 1 F +1R are used to capture the amplicon created in the 5′ deleted region, and the 2 F + 2R primers are used to capture the amplicon created in the 3′ indel region. B. Insertion of a 353 bp Alu element into the MAK gene leads to Retinitis Pigmentosa (found by Tucker et al. [28]). (PNG 69 kb

Additional file 4: Figure S3. of A-to-I RNA editing in the rat brain is age-dependent, region-specific and sensitive to environmental stress across generations

A-to-I RNA editing in oocytes, AMY and PFC of adult female rats. Editing sites where % editing are high are presented in the top part of the figure; sites where % editing are low (0–4%) are presented in the bottom part. N’s, PFC, 11, AMY 12, Oocytes 5–12. (TIFF 584 kb

Additional file 1: Figure S1. of A-to-I RNA editing in the rat brain is age-dependent, region-specific and sensitive to environmental stress across generations

A Flow chart of sample preparation using the Illumina-based Htr2c-direcetd NGS. (PDF 92 kb

Additional file 2: Table S1. of A-to-I RNA editing in the rat brain is age-dependent, region-specific and sensitive to environmental stress across generations

RNA editing sites detected with mmPCR-seq. Table S2. Primer sequences used for mmPCR-seq, Real-Time PCR and Htr2c-directed NGS. Table S3. % RNA editing in PFC and AMY of neonatal (P0) vs. adult (P60) rats. Table S4. Age-dependent changes in Htr2c isoform prevalence in PFC and AMY. Table S5. % RNA editing in neonatal (P0) and adult (P60) PFC vs. AMY. Table S6. Htr2c and ADARs correlations with significant non-synonymous editing sites. Table S7. Changes in Htr2c isoform prevalence in PFC vs. AMY at P0 and P60. Table S8. The effects of PRS on RNA editing at learning- and stress-related genes in F0, F1 and F2. Table S9. Statistical analysis of editing differences between oocytes, PFC and AMY. (XLSX 92 kb

Fmrp Interacts with Adar and Regulates RNA Editing, Synaptic Density and Locomotor Activity in Zebrafish

<div><p>Fragile X syndrome (FXS) is the most frequent inherited form of mental retardation. The cause for this X-linked disorder is the silencing of the fragile X mental retardation 1 (<i>fmr1</i>) gene and the absence of the fragile X mental retardation protein (Fmrp). The RNA-binding protein Fmrp represses protein translation, particularly in synapses. In <i>Drosophila</i>, Fmrp interacts with the adenosine deaminase acting on RNA (Adar) enzymes. Adar enzymes convert adenosine to inosine (A-to-I) and modify the sequence of RNA transcripts. Utilizing the <i>fmr1</i> zebrafish mutant (<i>fmr1</i>-/-), we studied Fmrp-dependent neuronal circuit formation, behavior, and Adar-mediated RNA editing. By combining behavior analyses and live imaging of single axons and synapses, we showed hyperlocomotor activity, as well as increased axonal branching and synaptic density, in <i>fmr1</i>-/- larvae. We identified thousands of clustered RNA editing sites in the zebrafish transcriptome and showed that Fmrp biochemically interacts with the Adar2a protein. The expression levels of the <i>adar</i> genes and Adar2 protein increased in <i>fmr1</i>-/- zebrafish. Microfluidic-based multiplex PCR coupled with deep sequencing showed a mild increase in A-to-I RNA editing levels in evolutionarily conserved neuronal and synaptic Adar-targets in <i>fmr1</i>-/- larvae. These findings suggest that loss of Fmrp results in increased Adar-mediated RNA editing activity on target-specific RNAs, which, in turn, might alter neuronal circuit formation and behavior in FXS.</p></div

Targeted resequencing by mmPCR revealed differential RNA editing levels in <i>fmr1</i>-/- larvae.

<p><b>A.</b> Schematic representation of the three major steps in the amplification and quantification of multiple RNA editing sites by next-generation sequencing. 1. A microfluidic-based PCR using the Fluidigm Access Array platform on the IFC chip (sample and primer inlets, black arrows) generates 48 targeted amplicons from 48 different samples. Schematic representation of the “on-chip” PCR; target regions (blue lines) contain a targeted RNA editing site (red circle) amplified by PCR with forward and reverse target-specific primers (TSP-F/TSP-R) fused to common sequences (CS1/CS2). 2. “Off-chip” PCR generates mini-library tagging, and the addition of IT-adaptor sequences creates 48 fully tagged and sequencer-compatible mini-libraries. Fusion primers containing CS1 and CS2 (red line primers) and the Ion Torrent PGM adaptor sequences P1 (green) and Aseq (orange) are used to generate completed amplicons (blue lines flanked by red lines). Barcode sequences (yellow) for sample indexing are fused to the Aseq-CS2 primer. 3. Parallel sequencing of the combined libraries on Ion Torrent-PGM. All mini-libraries are pooled together. <b>B.</b> Dot plot represents all calculated values of A/G ratios [percentages (dots) and means (black horizontal lines)] in the set of target sites that met all selection criteria in WT (blue circles) and <i>fmr1</i>-/- (green circles) RNA. <b>C.</b> The ten editing sites that exhibited significant differential RNA editing levels between <i>fmr1</i>-/- and WT larvae (<i>n</i> = 20 batches of 10 larvae for each genotype, *<i>p</i><0.05, **<i>p</i><0.005). <b>D.</b> Representative RNA editing sites showed increased editing levels in the brains of <i>fmr1</i>-/- zebrafish. <i>gria3b</i> showed a 14% increase, <i>grik2</i> showed an 8% increase and <i>ache</i> showed an 18% increase (<i>gria3b</i> and <i>grik2</i>: WT, <i>n</i> = 4; <i>fmr1</i>-/-, <i>n</i> = 5; <i>ache</i>, <i>n</i> = 3 per genotype, one brain per sample, *<i>p</i><0.05, **<i>p</i><0.005). <b>E-F.</b> Genes with multiple editing sites located in close proximity in the same amplified target region, were analyzed to quantify the relative abundance of all possible protein combinations formed by the editing pattern. Grey bars represent differences in the relative abundance of mRNA transcripts between WT and <i>fmr1</i>-/- larvae. <b>E.</b> In <i>gria2a</i>, LR (Leucine, Arginine) represents the genomically encoded unedited version that exhibited a 2.6% difference in relative abundance (*<i>p</i><0.05). <b>F.</b> In <i>gria3a</i>, AV (Alanine, Valine) represents the double-edited form that exhibited a 1.6% difference in relative abundance (*<i>p</i><0.05). Values are represented as means ± SEM. Statistical significance was determined by two-sample <i>t</i>-test assuming unequal variances.</p

Fmrp-Adar interaction in zebrafish.

<p><b>A.</b> Phylogenetic tree of zebrafish and human Adar proteins. Sequences are labeled with gene names, chromosomal locations, and accession numbers. To standardize and simplify the nomenclature, we named the genes Adar1-3, as indicated on the right side of each clade. Similarity values of each Adar member appear on top of each clade. <b>B.</b> Sequence conservation and motif distribution of Adar proteins in zebrafish and humans. Protein domains: adenosine deaminase domain (deaminase, white), double-stranded RNA binding motif (dsRBM, black) and zDNA binding domain (z_alpha, light grey). <b>C-R.</b> <i>In situ</i> hybridization showing lateral (<b>C</b>, <b>E</b>, <b>F</b>, <b>H</b>, <b>I</b>, <b>K</b>, <b>L</b>, <b>N</b>, <b>O</b>, <b>Q</b>) and dorsal (<b>D</b>, <b>G</b>, <b>J</b>, <b>M</b>, <b>P</b>, <b>R</b>) views of the spatial expression pattern of all four <i>adar</i> genes in 2 dpf <b>(C-D, F-G, I-J, L-M)</b> and 6 dpf <b>(E, H, K, N)</b> WT larvae. Expression is detected primarily in the nervous system. <b>O-R.</b> Selected regions (black frames in <b>L</b> and <b>M</b>) show <i>adar2b</i> (<b>O-P)</b> and <i>adar3</i> (<b>Q-R)</b> expression in the spinal cord of 2 dpf WT embryo. <b>S.</b> HEK-293T cells were transiently transfected with the zebrafish proteins Adar2a and Fmrp fused to EGFP and MYC, respectively (EGFP-Adar2a and MYC-Fmrp). Co-immunoprecipitation was used to detect Adar2a and Fmrp interaction. Actin was used as a negative control. The cell lysate was immunoprecipitated with anti-actin, anti-MYC, or anti-EGFP. Proteins were purified from the complexes and separated by SDS-PAGE. <b>T.</b> Western blot shows the protein content following the transfection prior to the immunoprecipitation. The proteins were detected with specific antibodies against MYC, EGFP, and actin. <b>U.</b> Computational sequence homology predicted the number of RNA recognition elements (RREs) in the CDS of <i>adar</i> genes that are recognized by Fmrp. <b>V.</b> RNA immunoprecipitation (RIP) assays show that Fmrp binds <i>adar1</i>. PCR amplification of <i>adar1</i> on RNA extracted from a RIP experiment conducted with anti-Actin and anti-MYC antibodies, and on total RNA extracted from HEK293T cells. <b>W.</b> RT-PCR assays showed that the mRNA expression levels of all four <i>adar</i> genes increased in 6 dpf <i>fmr1</i>-/- larvae (grey bars) when compared with WT larvae (white bars). Values are represented as means ± SEM. *<i>p</i><0.05, **<i>p</i><0.005, two-way <i>t</i>-test assuming unequal variances. <b>X.</b> Adar2 protein expression was analyzed by Western blot with specific antibodies against Adar2 and actin as a loading control. Elevated Adar2 protein levels of approximately 30% are present in <i>fmr1</i>-/- brains.</p

Increased expression of Fmrp-target genes in <i>fmr1</i>-/- zebrafish.

<p><b>A.</b> The full mRNA sequence of the <i>fmr1</i> gene, including the CDS (black bars) and UTRs (white bars). A single C-to-T mutation at position 113 results in a premature stop codon and truncated protein (gray bars). <b>B-D.</b> Whole-mount ISH assays show the spatial expression of <i>mtor</i>, <i>sash1</i>, and <i>tln1</i> in 6 dpf WT larvae. Fb, forebrain; Mb, midbrain; Hb, hindbrain. <b>E.</b> Relative mRNA expression of <i>mtor</i>, <i>sash1</i>, and <i>tln1</i> in 6 dpf <i>fmr1</i>-/- <i>(</i>grey bars) and WT larvae (white bars). Values are represented as means ± SEM (*<i>p</i><0.05, two-way <i>t</i>-test assuming unequal variances). <b>F.</b> Western blots show an approximate five-fold increase in the expression of mTor protein levels in <i>fmr1</i>-/- zebrafish brain tissue.</p

Transcriptome RNA hyperediting (HE) clusters in zebrafish.

<p><b>A.</b> Out of the overall 12 possible mismatches between RNA and DNA, there was a cluster enrichment of A-to-G transitions (93%) compared with the other 11 mismatch possibilities (7%). Web-logo diagrams show the abundance of each nucleotide located 1 bp upstream (position -1) and 1 bp downstream (position +1) of every A/G mismatch found in zebrafish. Top panels show the results for both -1 and +1 positions in the zebrafish exons (top right) and whole transcriptome (top left). Consistent with the established motif, ‘G’ is the least represented nucleotide, with 8.4% in whole transcriptome and 11.4% in exons. Bottom panels show the results for both -1 and +1 positions surrounding the 42,500 editing sites comprising the RADAR dataset in humans (bottom left) and mice (bottom right). In position -1, similar to the case of zebrafish, ‘G’ is considerably under-representation with 8.4% and 4.4% in humans and mice, respectively. <b>B</b>. The distribution and number of A-to-I RNA hyperediting (HE) sites. The top chart represents all detected DNA-RNA mismatches. A-to-G mismatches are the majority (93%) of all mismatches. Middle and bottom charts show the genomic location of the detected HE clusters. <b>C.</b> Most of the detected RNA editing sites were found in repeats. Comparison between the distribution of the total and edited repeat families in the zebrafish genome showed an enrichment of the hAT family DNA repeats. While the hAT family occupies only 8% of total repeats in the entire zebrafish genome, it holds 26% of the total cluster containing sequences. <b>D.</b> <i>mfold</i> analysis of RNA secondary structure performed on the two most prominent DNA repeats (ANGEL and TDR19), which are members of the hAT family and account for over 11% of all sites detected. Structure analysis shows a long-stemmed dsRNA structure with palindrome traits that enable Adar binding and, consequently, RNA editing. Color code represents the strength of the nucleotide connection.</p

Hyperlocomotor activity and altered response to dark-to-light transition in <i>fmr1</i>-/- larvae.

<p><b>A.</b> Locomotor activity (cm/min) recording was performed in 6 dpf <i>fmr1</i>-/- larvae (black line) and WT larvae (grey line) throughout a daily cycle under a 14 h light/10 h dark cycle. <i>fmr1</i>-/- larvae are hyperactive during both day and night (WT, n = 30; <i>fmr1</i>-/-, n = 34, ***<i>p</i><0.0001). <b>B.</b> Average total activity (cm/min) during both day and night is presented for 6 dpf <i>fmr1</i>-/- and WT larvae. Values are represented as means ± SEM (*<i>p</i><0.05, two-way <i>t</i>-test assuming unequal variances). <b>C.</b> Larvae were kept under alternating 30-min light/dark cycles during the day. The <i>fmr1</i>-/- larvae were hyperactive compared with WT larvae during the light periods (WT, n = 177; <i>fmr1</i>-/-, n = 179). <b>D.</b> Total average activity under alternating 30-min light/dark cycles during the day during both light and dark periods, is presented for 6 dpf <i>fmr1</i>-/- larvae and WT larvae. Values are represented as means ± SEM (*<i>p</i><0.05, two-way <i>t</i>-test assuming unequal variances). <b>E.</b> Transition analysis demonstrating the differences in total average activity per genotype, calculated by comparing 5 min after and 5 min before light-to-dark and dark-to-light transitions. While the WT larvae showed reduced activity, the <i>fmr1</i>-/- larvae showed increased activity during the dark-to-light transitions (**<i>p</i><0.005). Values are represented as means ± SEM. Statistical significance was determined by using a two-way <i>t</i>-test assuming unequal variances.</p