Ubiquitin fold modifier 1 (UFM1) is a small, metazoan-specific, ubiquitin-like protein modifier that is essential for embryonic development. Although loss-of-function mutations in UFM1 conjugation are linked to endoplasmic reticulum (ER) stress, neither the biological function nor the relevant cellular targets of this protein modifier are known. Here, we show that a largely uncharacterized ribosomal protein, RPL26, is the principal target of UFM1 conjugation. RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. Biochemical analysis and structural modeling establish that UFMylated RPL26 and the UFMylation machinery are in close proximity to the SEC61 translocon, suggesting that this modification plays a direct role in cotranslational protein translocation into the ER. These data suggest that UFMylation is a ribosomal modification specialized to facilitate metazoan-specific protein biogenesis at the ER.

The booming nanotech industry has raised public concerns about the environmental health and safety impact of engineered nanomaterials (ENMs). High-throughput assays are needed to obtain toxicity data for the rapidly increasing number of ENMs. Here we present a suite of high-throughput methods to study nanotoxicity in intact animals using Caenorhabditis elegans as a model. At the population level, our system measures food consumption of thousands of animals to evaluate population fitness. At the organism level, our automated system analyzes hundreds of individual animals for body length, locomotion speed, and lifespan. To demonstrate the utility of our system, we applied this technology to test the toxicity of 20 nanomaterials under four concentrations. Only fullerene nanoparticles (nC60), fullerol, TiO2, and CeO2 showed little or no toxicity. Various degrees of toxicity were detected from different forms of carbon nanotubes, graphene, carbon black, Ag, and fumed SiO2 nanoparticles. Aminofullerene and UV irradiated nC60 also showed small but significant toxicity. We further investigated the effects of nanomaterial size, shape, surface chemistry, and exposure conditions on toxicity. Our data are publicly available at the open-access nanotoxicity database www.QuantWorm.org/nano.

Phenotypic assays are crucial in genetics; however, traditional methods that rely on human observation are unsuitable for quantitative, large-scale experiments. Furthermore, there is an increasing need for comprehensive analyses of multiple phenotypes to provide multidimensional information. Here we developed an automated, high-throughput computer imaging system for quantifying multiple Caenorhabditis elegans phenotypes. Our imaging system is composed of a microscope equipped with a digital camera and a motorized stage connected to a computer running the QuantWorm software package. Currently, the software package contains one data acquisition module and four image analysis programs: WormLifespan, WormLocomotion, WormLength, and WormEgg. The data acquisition module collects images and videos. The WormLifespan software counts the number of moving worms by using two time-lapse images; the WormLocomotion software computes the velocity of moving worms; the WormLength software measures worm body size; and the WormEgg software counts the number of eggs. To evaluate the performance of our software, we compared the results of our software with manual measurements. We then demonstrated the application of the QuantWorm software in a drug assay and a genetic assay. Overall, the QuantWorm software provided accurate measurements at a high speed. Software source code, executable programs, and sample images are available at www.quantworm.org. Our software package has several advantages over current imaging systems for C. elegans. It is an all-in-one package for quantifying multiple phenotypes. The QuantWorm software is written in Java and its source code is freely available, so it does not require use of commercial software or libraries. It can be run on multiple platforms and easily customized to cope with new methods and requirements.

DNA damage activates a robust transcriptional stress response, but much less is known about how DNA damage impacts translation. The advent of genome editing with Cas9 has intensified interest in understanding cellular responses to DNA damage. Here, we find that DNA double-strand breaks (DSBs), including those induced by Cas9, trigger the loss of ribosomal protein RPS27A from ribosomes via p53-independent proteasomal degradation. Comparisons of Cas9 and dCas9 ribosome profiling and mRNA-seq experiments reveal a global translational response to DSBs that precedes changes in transcript abundance. Our results demonstrate that even a single DSB can lead to altered translational output and ribosome remodeling, suggesting caution in interpreting cellular phenotypes measured immediately after genome editing.

Although DSBs are known to initiate transcriptional changes, less is understood 56 about the role of translation in the DNA damage response. A purely transcriptional 57 4 reaction to a genetic insult leaves a gap in response, potentially exposing a cell to the 58 impact of damaged DNA during a critical time window in which damage had raised an 59 alarm but newly transcribed mRNAs have not accumulated. While transcriptional 60 changes can modulate protein abundance hours or days after a genomic insult, 61 translational control can enact regulatory programs within minutes of an environmental 62 stress (Andreev et al., 2015;Sidrauski et al., 2015). 63We thus sought to characterize how cells respond to DNA damage at the 64 translation level, and in particular, how cells respond to a single double-strand break 65 during Cas9-mediated genome editing. We serendipitously found that cells temporarily 66 deplete core ribosomal proteins, RPS27A and RPL40, in response to dsDNA damage. 67 RPS27A and RPL40 are regulated post-transcriptionally and in a p53-independent 68 manner, and their depletion persists days after the initial genomic lesion with Cas9. We 69 also found that both non-specific double-strand breaks as well as single, targeted 70 double-strand breaks reduce translation via eukaryotic initiation factor 2 alpha (eIF2α) 71 phosphorylation, and that modulating the downstream effects of eIF2α phosphorylation 72 during Cas9 editing leads to different repair outcomes. Ribosome profiling and RNA-seq 73 data from Cas9-edited cells suggest that cells mount a translation response to dsDNA 74 damage that precedes transcriptional changes. Our data demonstrate that Cas9-75 mediated genome editing can trigger temporary ribosome remodeling and translational 76 shutdown in response to DNA double-strand breaks. 77 5 Results 78Ribosome proteins RPS27A and RPL40 are downregulated after genome editing 79 with Cas9 80 While investigating changes in ubiquitin gene expression after DNA damage, we 81 serendipitously observed that the two ribosomal proteins encoded as fusion proteins 82 with ubiquitin, RPS27A (eS31) and RPL40 (eL40), are downregulated after Cas9-guide 83 RNA (gRNA) ribonucleoprotein (RNP) nucleofection ( Figure 1A). This downregulation 84 was apparent as late as 48-72 hours after nucleofection, even though at this point Cas9 85 was largely absent from the cell (Figure 1B) and genomic formation of indels was 86 completed (Figure 1C). We found that RPS27A levels recovered 96 hours after 87 nucleofection and RPL40 levels were beginning to increase within 72 hours ( Figure 88 1A), suggesting that the cell resets protein expression three to four days after editing 89 ( Figure S1A). 90 Downregulation of RPS27A and RPL40 depended on the DNA double-strand 91 break, as catalytically inactive dCas9 did not provoke a similar response (Figure 1A). 92The guide RNA used in this experiment targeted a non-coding region of the JAK2 gene 93 (sgIntron), and JAK2 levels remain unchanged after Cas9 nucleofection (Figure S1B). 94Our data the...