Additional file 1: of Massive A-to-I RNA editing is common across the Metazoa and correlates with dsRNA abundance

Supplementary Figures 1–8, Supplementary Table 2. (PDF 760 kb

Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme

<p>Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE.</p

Supplementary File 2

A spreadsheet with all the A-to-G modification sites detected in the coding regions of the squid, along with their number of supporting reads in all the tissues studied

Supplementary File 1

A text file in a fasta format with the constructed squid coding sequences

Systematic Identification of Rhythmic Genes Reveals <em>camk1gb</em> as a New Element in the Circadian Clockwork

<div><p>A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA–seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, <em>camk1gb</em>, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of <em>camk1gb</em> disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that <em>camk1gb</em> plays a role in linking the pineal master clock with the periphery.</p> </div

<i>camk1gb</i> spatio-temporal expression.

<p>A) Rhythmic expression of <i>camk1gb</i> exclusively in the pineal glands (indicated by black arrows) of 48–72 hpf embryos as detected by whole mount ISH under constant darkness. B) Rhythmic expression of <i>camk1gb</i> in the zebrafish embryo (right) is correlated with the RNA-seq (solid line, left vertical axis) and the microarray data (dashed line, right vertical axis) from the adult (left). Correlation coefficients between the whole mount ISH results and the data obtained by microarrays and RNA-seq were determined by Pearson correlation (r = 0.89 and 0.77, respectively). For whole mount ISH, statistical differences in mRNA levels were determined by one-way ANOVA followed by a Tukey test (P-value<0.05). Error bars represent SE (n = 10–15). CT = circadian time. Gray and black bars represent subjective day and subjective night, respectively.</p

Effect of <i>camk1gb</i> knockdown on pineal <i>aanat2</i> and <i>otx5</i> mRNA rhythms.

<p>Zebrafish embryos injected with either control morpholino (black line) or <i>camk1gb</i> morpholino (gray line) were subjected to DD during their third day of development and pineal <i>aanat2</i> and <i>otx5</i> mRNA levels were determined by whole mount ISH. A) Embryos were sampled at 4-hr intervals for <i>aanat2</i>. Statistical differences in <i>aanat2</i> mRNA levels between the control morpholino and <i>camk1gb</i> morpholino injected embryos were determined by two-tailed <i>t</i>-test with Bonferroni correction (* P-value<0.05, ** P-value<0.01, *** P-value<0.001). B) Whole-mount ISH for <i>aanat2</i> in the heads (dorsal views) of representative larvae from each group, at CT14 and CT18. Arrows indicate pineal <i>aanat2</i> mRNA expression. C) <i>otx5</i> expression at CT18. Error bars represent SE (n = 10–15). CT = circadian time.</p

Genes detected as circadian using both DNA microarray and RNA–seq.

<p>Genes detected as circadian using both DNA microarray and RNA–seq.</p

Clock-controlled rhythmic gene expression is disrupted in ΔCLK-expressing pineal glands.

<p>(A) Experimental procedure for transcriptome analysis. Adult fish were kept under DD and pineal glands were sampled at 12 time points (indicated by arrows) throughout two daily cycles. Black and gray bars correspond to subjective night and day, respectively. (B) The mRNA-seq analysis resulted in the identification of 29 circadian genes in the pineal gland of Tg(<i>aanat2</i>:EGFP-ΔCLK) fish compared with 290 circadian genes in the pineal gland of Tg(<i>aanat2</i>:EGFP) control fish.</p

Diverse effects of ΔCLK on expression profiles of clock-controlled genes in the pineal gland.

<p>Representative examples of expression profiles of CCGs in the pineal gland of control Tg(<i>aanat2</i>:EGFP) fish (control; blue trendline) compared with the expression profiles of these genes in the pineal gland of Tg(<i>aanat2</i>:EGFP-ΔCLK) fish (ΔCLK; red trendline). Black and gray bars denote subjective night and day, respectively. CT, circadian time. While the majority of CCGs became arrhythmic (A–I), a few maintained their circadian profile in the Tg(<i>aanat2</i>:EGFP-ΔCLK) pineal gland (J–L). For some of the CCGs that became arrhythmic in the Tg(<i>aanat2</i>:EGFP-ΔCLK) pineal gland the overall basal expression levels remained relatively intermediate or high (A–D), whereas for others the expression was down-regulated (E–H) or abolished (I).</p