Disruption in A-to-I Editing Levels Affects C. elegans Development More Than a Complete Lack of Editing

A-to-I RNA editing, catalyzed by ADAR proteins, is widespread in eukaryotic transcriptomes. Studies showed that, in C. elegans, ADR-2 can actively deaminate dsRNA, whereas ADR-1 cannot. Therefore, we set out to study the effect of each of the ADAR genes on the RNA editing process. We performed comprehensive phenotypic, transcriptomics, proteomics, and RNA binding screens on worms mutated in a single ADAR gene. We found that ADR-1 mutants exhibit more-severe phenotypes than ADR-2, and some of them are a result of non-editing functions of ADR-1. We also show that ADR-1 significantly binds edited genes and regulates mRNA expression, whereas the effect on protein levels is minor. In addition, ADR-1 primarily promotes editing by ADR-2 at the L4 stage of development. Our results suggest that ADR-1 has a significant role in the RNA editing process and in altering editing levels that affect RNA expression; loss of ADR-1 results in severe phenotypes

Diminished Telomeric 3′ Overhangs Are Associated with Telomere Dysfunction in Hoyeraal-Hreidarsson Syndrome

BACKGROUND:Eukaryotic chromosomes end with telomeres, which in most organisms are composed of tandem DNA repeats associated with telomeric proteins. These DNA repeats are synthesized by the enzyme telomerase, whose activity in most human tissues is tightly regulated, leading to gradual telomere shortening with cell divisions. Shortening beyond a critical length causes telomere uncapping, manifested by the activation of a DNA damage response (DDR) and consequently cell cycle arrest. Thus, telomere length limits the number of cell divisions and provides a tumor-suppressing mechanism. However, not only telomere shortening, but also damaged telomere structure, can cause telomere uncapping. Dyskeratosis Congenita (DC) and its severe form Hoyeraal-Hreidarsson Syndrome (HHS) are genetic disorders mainly characterized by telomerase deficiency, accelerated telomere shortening, impaired cell proliferation, bone marrow failure, and immunodeficiency. METHODOLOGY/PRINCIPAL FINDINGS:We studied the telomere phenotypes in a family affected with HHS, in which the genes implicated in other cases of DC and HHS have been excluded, and telomerase expression and activity appears to be normal. Telomeres in blood leukocytes derived from the patients were severely short, but in primary fibroblasts they were normal in length. Nevertheless, a significant fraction of telomeres in these fibroblasts activated DDR, an indication of their uncapped state. In addition, the telomeric 3' overhangs are diminished in blood cells and fibroblasts derived from the patients, consistent with a defect in telomere structure common to both cell types. CONCLUSIONS/SIGNIFICANCE:Altogether, these results suggest that the primary defect in these patients lies in the telomere structure, rather than length. We postulate that this defect hinders the access of telomerase to telomeres, thus causing accelerated telomere shortening in blood cells that rely on telomerase to replenish their telomeres. In addition, it activates the DDR and impairs cell proliferation, even in cells with normal telomere length such as fibroblasts. This work demonstrates a telomere length-independent pathway that contributes to a telomere dysfunction disease

Addressing climate change with behavioral science: a global intervention tournament in 63 countries

Effectively reducing climate change requires marked, global behavior change. However, it is unclear which strategies are most likely to motivate people to change their climate beliefs and behaviors. Here, we tested 11 expert-crowdsourced interventions on four climate mitigation outcomes: beliefs, policy support, information sharing intention, and an effortful tree-planting behavioral task. Across 59,440 participants from 63 countries, the interventions’ effectiveness was small, largely limited to nonclimate skeptics, and differed across outcomes: Beliefs were strengthened mostly by decreasing psychological distance (by 2.3%), policy support by writing a letter to a future-generation member (2.6%), information sharing by negative emotion induction (12.1%), and no intervention increased the more effortful behavior—several interventions even reduced tree planting. Last, the effects of each intervention differed depending on people’s initial climate beliefs. These findings suggest that the impact of behavioral climate interventions varies across audiences and target behaviors

Folate levels modulate oncogene‐induced replication stress and tumorigenicity

Chromosomal instability in early cancer stages is caused by replication stress. One mechanism by which oncogene expression induces replication stress is to drive cell proliferation with insufficient nucleotide levels. Cancer development is driven by alterations in both genetic and environmental factors. Here, we investigated whether replication stress can be modulated by both genetic and non‐genetic factors and whether the extent of replication stress affects the probability of neoplastic transformation. To do so, we studied the effect of folate, a micronutrient that is essential for nucleotide biosynthesis, on oncogene‐induced tumorigenicity. We show that folate deficiency by itself leads to replication stress in a concentration‐dependent manner. Folate deficiency significantly enhances oncogene‐induced replication stress, leading to increased DNA damage and tumorigenicity in vitro. Importantly, oncogene‐expressing cells, when grown under folate deficiency, exhibit a significantly increased frequency of tumor development in mice. These findings suggest that replication stress is a quantitative trait affected by both genetic and non‐genetic factors and that the extent of replication stress plays an important role in cancer development.SynopsisOncogene‐induced replication stress is shown here as a quantitative trait enhanced by non‐genetic factors such as the essential dietary nutrient folate. The combination of oncogene expression and folate deficiency enhances replication‐induced genomic instability and cancer development in vivo.Folate deficiency by itself leads to replication stress in a concentration‐dependent manner that can be rescued by nucleoside supplementation.The extent of oncogene‐induced replication stress can be enhanced by an additional source of stress, resulting in enhanced DNA damage.Activation of the DNA damage response pathways by ATM and ATR is enhanced by the combination of oncogene expression and folate deficiency.Tumorigenicity potential in vitro and tumor development in vivo caused by oncogene expression are significantly enhanced by folate deficiency.Oncogene‐induced replication stress is shown here as a quantitative trait enhanced by non‐genetic factors such as the essential dietary nutrient folate. The combination of oncogene expression and folate deficiency enhances replication‐induced genomic instability and cancer development in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/113743/1/emmm201404824.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/113743/2/emmm201404824-sup-0001-Appendix.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/113743/3/emmm201404824.reviewer_comments.pd

Reduced telomeric overhang signal in the HHS-affected cells.

<p>(A) Genomic DNA samples prepared from S2 and control LCLs (PDL of 44 and 50, respectively; 2 and 4 µg) or fibroblasts (PDL of 14 and 16 for S2 and C, respectively; 2 µg) were digested with MboI and AluI and electrophoresed in a 0.7% agarose gel. The average length of the 3′ overhang was estimated by in-gel hybridization of native DNA to a C-rich telomeric probe (native panels). The DNA was subsequently denatured <i>in situ</i> and re-hybridized to the same probe to measure the total TTAGGG repeat signal (denatured panels). (B) The histograms below the images represent the quantified native (overhang) signals normalized to the denatured (total) signals and presented as percentage of the normalized overhang signals of the controls.</p

TIF formation in HHS-affected fibroblasts with normal telomere length.

<p>(A) Control (C) and HHS-affected (S2) primary fibroblast cultures (established at the ages of 30 and 17 years, and grown to PDL of 14 and 10, respectively) were immunostained for TRF1 (green) and γ-H2AX (red), and with DAPI for the nuclei (blue), as indicated above the images. The bottom panels show enlarged images that include several telomeres. The images of the affected and control cells were obtained and processed in the same way, side by side. (B) The number of TIFs (defined as colocalized TRF1 and γ-H2AX foci) was counted in randomly-chosen 67 affected and 58 control cells. The graph shows the percentage of cells with at least five such foci. (C) Genomic DNA was prepared from these cultures and the average length of telomeres estimated by Southern analysis.</p

The HHS-affected cells express normal hTR levels and assemble active telomerase.

<p>Total RNA was prepared from blood leukocytes (A) and LCLs (B) derived from the affected siblings (red), unaffected parents (dark gray), or unaffected individuals (C – control; light gray). (A,B) hTR levels were measured by SYBR-green real-time RT-PCR, normalized to the levels of the U93 small nucleolar RNA, and presented as values relative to the controls. S2a indicates a later passage of the S2 LCL. Normalization to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA did not significantly change the results (data not shown). (C) Whole-cell extracts were prepared from LCLs and assayed for telomerase activity by TRAP assay, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005666#s4" target="_blank">Materials and Methods</a>. The amounts of the extracts used in total protein were 10, 30, and 100 ng for the left panel, and 100 ng for the right panel. The PDL of the LCLs at which the samples were taken are: S2a, 38; C, 40; P1, 20; P2, 28; and S2, 25.</p

Genealogical tree of the HHS-affected family.

<p>Open circles and squares represent unaffected females and males, respectively. Black circles and squares represent affected females and males. A gray square indicates a family member who died from pulmonary fibrosis. Tilted lines indicate mortality, and the ages of mortality are indicated underneath.</p

Impaired proliferation of cell cultures prepared from an HHS-affected patient.

<p>EBV-infected lymphoblastoid (A) and primary fibroblast (B) cultures were grown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005666#s4" target="_blank">Materials and Methods</a>. The cumulative population doubling level (PDL) of the HHS-affected (S2) and control (C) cultures is drawn as a function of days in culture.</p