The ERI-6/7 Helicase Acts at the First Stage of an siRNA Amplification Pathway That Targets Recent Gene Duplications

Endogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt) endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7–dependent siRNAs require Dicer. We identify that the eri-6/7–dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3–4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7–dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA–associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation

Genes misregulated in C. elegans deficient in Dicer, RDE-4, or RDE-1 are enriched for innate immunity genes

We describe the first microarray analysis of a whole animal containing a mutation in the Dicer gene. We used adult Caenorhabditis elegans and, to distinguish among different roles of Dicer, we also performed microarray analyses of animals with mutations in rde-4 and rde-1, which are involved in silencing by siRNA, but not miRNA. Surprisingly, we find that the X chromosome is greatly enriched for genes regulated by Dicer. Comparison of all three microarray data sets indicates the majority of Dicer-regulated genes are not dependent on RDE-4 or RDE-1, including the X-linked genes. However, all three data sets are enriched in genes important for innate immunity and, specifically, show increased expression of innate immunity genes

Comprehensive mapping of whisker-evoked responses reveals broad, sharply tuned thalamocortical input to layer 4 of barrel cortex

Cortical neurons are organized in columns, distinguishable by their physiological properties and input-output organization. Columns are thought to be the fundamental information-processing modules of the cortex. The barrel cortex of rats and mice is an attractive model system for the study of cortical columns, because each column is defined by a layer 4 (L4) structure called a barrel, which can be clearly visualized. A great deal of information has been collected regarding the connectivity of neurons in barrel cortex, but the nature of the input to a given L4 barrel remains unclear. We measured this input by making comprehensive maps of whisker-evoked activity in L4 of rat barrel cortex using recordings of multiunit activity and current source density analysis of local field potential recordings of animals under light isoflurane anesthesia. We found that a large number of whiskers evoked a detectable response in each barrel (mean of 13 suprathreshold, 18 subthreshold) even after cortical activity was abolished by application of muscimol, a GABAA agonist. We confirmed these findings with intracellular recordings and single-unit extracellular recordings in vivo. This constitutes the first direct confirmation of the hypothesis that subcortical mechanisms mediate a substantial multiwhisker input to a given cortical barrel

Dicer's helicase domain is required for accumulation of some, but not all, C. elegans endogenous siRNAs

Years after the discovery that Dicer is a key enzyme in gene silencing, the role of its helicase domain remains enigmatic. Here we show that this domain is critical for accumulation of certain endogenous small interfering RNAs (endo-siRNAs) in Caenorhabditis elegans. The domain is required for the production of the direct products of Dicer, or primary endo-siRNAs, and consequently affects levels of downstream intermediates, the secondary endo-siRNAs. Consistent with the role of endo-siRNAs in silencing, their loss correlates with an increase in cognate mRNA levels. We find that the helicase domain of Dicer is not necessary for microRNA (miRNA) processing, or RNA interference following exposure to exogenous double-stranded RNA. Comparisons of wild-type and helicase-defective strains using deep-sequencing analyses show that the helicase domain is required by a subset of annotated endo-siRNAs, in particular, those associated with the slightly longer 26-nucleotide small RNA species containing a 5′ guanosine

Fragment Length of Circulating Tumor DNA

<div><p>Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134–144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the <i>BRAF</i> V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132–145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the <i>EGFR</i> T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.</p></div

Extraction of cell-free DNA fractions for evaluating mutant allele frequency within specific fragment lengths.

<p>In <b>A</b>, an image of an 8% polyacrylamide gel loaded with a truncated library prepared from the cell-free DNA of a lung cancer patient (LC10, middle column). On either side is a custom-designed ladder made from phage lambda containing double-stranded DNA of 229, 240, and 262 bp in length. Six adjacent samples were excised from the gel corresponding to the colored boxes. In <b>B</b>, densitometry of the full-length libraries made from each fraction and the original library are shown. Colors of each curve and peak correspond to the colors in <b>A</b> (the library is shown in black). In <b>C</b>, the mutant allele frequency as determined by digital droplet PCR is shown for the library and each fraction. Colors for mutant allele frequency (%) correspond to the colors in <b>A</b> and <b>B</b>. Note that the purple fraction (peak fragment length of 320 bp) represented the largest increase (9.1-fold) in mutant allele frequency compared to the library (peak fragment length of 348 bp). Fractions containing longer fragment lengths than the library (e.g., blue fraction: peak fragment lengths of 361 bp) demonstrated a reduction in mutant allele frequency.</p

In lung cancer patients, mutant alleles occurred more commonly in shorter fragments of cell-free DNA.

<p>In <b>A-C</b>, histograms of overall cell-free DNA fragment length from the entire 16-gene capture panel determined by sequencing compared between five healthy controls (blue lines) and individual tumor patients (red line). Plasma concentration of cell-free DNA and presence (+) /absence (-) of <i>EGFR</i> and <i>KRAS</i> amplifications are also described for each tumor patient. There was a strong left shift (i.e., shorter fragment lengths) and periodicity in LC5 compared to controls (<b>A</b>). In <b>B</b>, there was a subtle shift towards shorter fragment length that was most apparent at longer lengths (black arrow) where fewer inserts from the tumor patient (LC1) were present compared to the healthy controls. In <b>C</b>, no difference between the tumor patient (LC10) and the healthy controls was observed. In <b>D</b>, the length of fragments containing the WT or mutant <i>EGFR</i> allele is shown for healthy controls (blue dots) and tumor patients with the mutant L858R allele (orange dots). The solid bars indicate the mean fragment length for each sample. In <b>E</b>, a histogram of the fragment lengths of the mutant L858R allele from LC5 (orange line) vs. the WT allele in healthy controls (blue lines) demonstrates a higher prevalence of mutant allele at shorter fragment lengths. The black dashed-line identifies the fragment length that corresponds to the most inserts in the tumor patient. Note that the mutant allele more commonly occurs at shorter fragment lengths while the WT allele in healthy controls occurs more commonly at longer fragment lengths. In <b>F</b>, the fragment length associated with <i>EGFR</i> for the WT allele in the healthy controls (blue dots) and tumor patients with the mutant T790M allele (red dots) is displayed. The solid bars correspond to mean fragment length for each sample. In <b>G</b>, a histogram of the fragment length of the mutant allele (L858R) from LC9 (red line) vs. the WT allele in healthy controls (blue lines) is shown. The black dashed-line identifies the fragment length that corresponds to the most inserts in the tumor patient. Note that the WT allele in healthy controls more commonly occurs at longer fragment lengths. In <b>H</b>, the <i>EGFR</i> fragment length associated with the WT allele (pink dots) and the mutant T790M allele (MA; red dots) in each of the tumor patients are depicted. The mutant allele more commonly occurred at a shorter fragment length compared to the length of the WT allele within the same patient.</p

The cell-free DNA and ctDNA from melanoma patients consisted of shorter fragments than the healthy controls and the WT allele fragments.

<p>In <b>A</b>, the relative fragment length of cell-free DNA obtained from melanoma patients with and without metastatic disease (± lymph node, LN; <b>A</b>) tended to be shorter by densitometry compared to cell-free DNA from four healthy controls (<b>A</b>, gray lines). In <b>B</b>, the fragment lengths derived from cell-free DNA deep-sequencing in a patient with melanoma (<b>A</b>, black arrow) were generally shorter than the fragment lengths present in the pool of healthy controls (green and purple lines, respectively). (<b>C)</b> In the melanoma patient, cell-free DNA fragment lengths containing the mutant allele (<i>BRAF</i> V600E, red line) were shorter compared to the fragment lengths containing the wild-type (WT) allele (blue line). In the shorter fragments there was general overlap between the mutant and WT allele sizes since the <i>BRAF</i> V600E mutation is heterozygous. <b>(D)</b> Fragment lengths between 110–140 bp had the highest proportion of the mutant allele (<b>D</b>, red solid line; the mutant allele frequency <100 bp was erratic due to few observations). In <b>D</b>, the solid black line represents the overall frequency for each range of fragment lengths in the melanoma patient and indicates that there may be insufficient amount of DNA for detecting mutant alleles below 100 bp. Of note, the WT allele from the healthy control occurred more commonly between 160–180 bp (<b>D</b>, blue dashed line).</p