The TRIM-NHL protein NHL-2 is a co-factor in the nuclear and somatic RNAi pathways in C. elegans

Proper regulation of germline gene expression is essential for fertility and maintaining species integrity. In the C. elegans germline, a diverse repertoire of regulatory pathways promote the expression of endogenous germline genes and limit the expression of deleterious transcripts to maintain genome homeostasis. Here we show that the conserved TRIM-NHL protein, NHL-2, plays an essential role in the C. elegans germline, modulating germline chromatin and meiotic chromosome organization. We uncover a role for NHL-2 as a co-factor in both positively (CSR-1) and negatively (HRDE-1) acting germline 22G-small RNA pathways and the somatic nuclear RNAi pathway. Furthermore, we demonstrate that NHL-2 is a bona fide RNA binding protein and, along with RNA-seq data point to a small RNA independent role for NHL-2 in regulating transcripts at the level of RNA stability. Collectively, our data implicate NHL-2 as an essential hub of gene regulatory activity in both the germline and soma

Comparative functional characterization of the CSR-1 22G-RNA pathway in Caenorhabditis nematodes

As a champion of small RNA research for two decades, Caenorhabditis elegans has revealed the essential Argonaute CSR-1 to play key nuclear roles in modulating chromatin, chromosome segregation and germline gene expression via 22G-small RNAs. Despite CSR-1 being preserved among diverse nematodes, the conservation and divergence in function of the targets of small RNA pathways remains poorly resolved. Here we apply comparative functional genomic analysis between C. elegans and Caenorhabditis briggsae to characterize the CSR-1 pathway, its targets and their evolution. C. briggsae CSR-1-associated small RNAs that we identified by immunoprecipitation-small RNA sequencing overlap with 22G-RNAs depleted in cbr-csr-1 RNAi-treated worms. By comparing 22G-RNAs and target genes between species, we defined a set of CSR-1 target genes with conserved germline expression, enrichment in operons and more slowly evolving coding sequences than other genes, along with a small group of evolutionarily labile targets. We demonstrate that the association of CSR-1 with chromatin is preserved, and show that depletion of cbr-csr-1 leads to chromosome segregation defects and embryonic lethality. This first comparative characterization of a small RNA pathway in Caenorhabditis establishes a conserved nuclear role for CSR-1 and highlights its key role in germline gene regulation across multiple animal species

Visualization of replication initiation and elongation in Drosophila

Chorion gene amplification in the ovaries of Drosophila melanogaster is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, we found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2–7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. We utilized this system to demonstrate that the pre–replication complex component, double-parked protein/cell division cycle 10–dependent transcript 1, is not only necessary for proper MCM2–7 localization, but, unexpectedly, is present during elongation

Distinct Argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline

Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that in the germ-line, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germ-line nuage structures called P-granules. WAGO-1 silences certain genes, transposons, pseudogenes and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest novel regulatory functions for small RNAs

Persistent Complex Bereavement Disorder Symptom Domains Relate Differentially to PTSD and Depression: A Study of War-Exposed Bosnian Adolescents.

Persistent Complex Bereavement Disorder (PCBD) is a newly proposed diagnosis placed in the Appendix of the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) as an invitation for further research. To date, no studies have examined the dimensionality of PCBD or explored whether different PCBD criteria domains relate in similar, versus differential, ways to other psychological conditions common to war-exposed bereaved youth, including symptoms of Posttraumatic Stress Disorder (PTSD) and depression. We evaluated the dimensionality of proposed PCBD B and C symptom domains, and their respective relations with measures of PTSD and depression, in 1142 bereaved Bosnian adolescents exposed to the 1992-1995 Bosnian civil war. Instruments included the UCLA PTSD Reaction Index, the Depression Self-Rating Scale, and the UCLA Grief Screening Scale (a prototype measure of PCBD symptoms). We investigated potential differences in grief, PTSD, and depression scores as a function of cause of death. We then examined hypothesized differential relations between PCBD B and C symptom domain subscales and selected external correlates, specifically measures of depression and the four-factor emotional numbing model of PTSD. Results of both analyses provide preliminary evidence of a multidimensional structure for PCBD in this population, in that the PCBD Criterion C subscale score covaried more strongly with each of the four PTSD factors and with depression than did PCBD Criterion B. We conclude by discussing theoretical, methodological, clinical, and policy-related implications linked to the ongoing study of essential features of PCBD

Hispanic ethnicity and Caucasian race: Relations with posttraumatic stress disorder\u27s factor structure in clinic-referred youth.

The severity of posttraumatic stress disorder (PTSD) symptoms is linked to race and ethnicity, albeit with contradictory findings (reviewed in Alcántara, Casement, & Lewis-Fernández, 2013; Pole, Gone, & Kulkarni, 2008). We systematically examined Caucasian (n = 3,767) versus non-Caucasian race (n = 2,824) and Hispanic (n = 2,395) versus non-Hispanic ethnicity (n = 3,853) as candidate moderators of PTSD\u27s 5-factor model structural parameters (Elhai et al., 2013). The sample was drawn from the National Child Traumatic Stress Network\u27s Core Data Set, currently the largest national data set of clinic-referred children and adolescents exposed to potentially traumatic events. Using confirmatory factor analysis, we tested the invariance of PTSD symptom structural parameters by race and ethnicity. Chi-square difference tests and goodness-of-fit values showed statistical equivalence across racial and ethnic groups in the factor structure of PTSD and in mean item-level indicators of PTSD symptom severity. Results support the structural invariance of PTSD\u27s 5-factor model across the compared racial and ethnic groups. Furthermore, results indicated equivalent item-level severity across racial and ethnic groups; this supports the use of item-level comparisons across these groups

Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway

Argonaute (AGO) proteins interact with distinct classes of small RNAs to direct multiple regulatory outcomes. In many organisms, including plants, fungi, and nematodes, cellular RNA-dependent RNA polymerases (RdRPs) use AGO targets as templates for amplification of silencing signals. Here, we show that distinct RdRPs function sequentially to produce small RNAs that target endogenous loci in Caenorhabditis elegans. We show that DCR-1, the RdRP RRF-3, and the dsRNA-binding protein RDE-4 are required for the biogenesis of 26-nt small RNAs with a 5′ guanine (26G-RNAs) and that 26G-RNAs engage the Piwi-clade AGO, ERGO-1. Our findings support a model in which targeting by ERGO-1 recruits a second RdRP (RRF-1 or EGO-1), which in turn transcribes 22G-RNAs that interact with worm-specific AGOs (WAGOs) to direct gene silencing. ERGO-1 targets exhibit a nonrandom distribution in the genome and appear to include many gene duplications, suggesting that this pathway may control overexpression resulting from gene expansion