′ Untranslated regions (3 ′ UTRs) of mRNAs emerged as central regulators of cellular function because they contain important but poorly characterized cis-regulatory elements targeted by a multitude of regulatory factors. The model nematode Caenorhabditis elegans is ideal to study these interactions because it possesses a well-defined 3 ′ UTRome. To improve its annotation, we have used a genome-wide bioinformatics approach to download raw transcriptome data for 1088 transcriptome data sets corresponding to the entire collection of C. elegans trancriptomes from 2015 to 2018 from the Sequence Read Archive at the NCBI. We then extracted and mapped high-quality 3 ′-UTR data at ultradeep coverage. Here, we describe and release to the community the updated version of the worm 3 ′ UTRome, which we named 3 ′ UTRome v2. This resource contains high-quality 3 ′-UTR data mapped at single-base ultraresolution for 23,084 3 ′-UTR isoform variants corresponding to 14,788 protein-coding genes and is updated to the latest release of WormBase. We used this data set to study and probe principles of mRNA cleavage and polyadenylation in C. elegans. The worm 3 ′ UTRome v2 represents the most comprehensive and high-resolution 3 ′-UTR data set available in C. elegans and provides a novel resource to investigate the mRNA cleavage and polyadenylation reaction, 3 ′-UTR biology, and miRNA targeting in a living organism.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease characterized by progressive muscle degeneration. The condition is driven by nonsense and missense mutations in the dystrophin gene, leading to instability of the sarcolemma and skeletal muscle necrosis and atrophy. Resulting changes in muscle-specific gene expression that take place in dystrophin’s absence remain largely uncharacterized, as they are potentially obscured by the chronic inflammation elicited by muscle damage in humans. Caenorhabditis elegans possess a mild inflammatory response that is not active in the muscle, and lack a satellite cell equivalent. This allows for the characterization of the transcriptome rearrangements affecting disease progression independently of inflammation and regeneration. In effort to better understand these dynamics, we have isolated and sequenced body muscle-specific transcriptomes from C. elegans lacking functional dystrophin at distinct stages of disease progression. We have identified an upregulation of genes involved in mitochondrial function early in disease progression, and an upregulation of genes related to muscle repair in later stages. Our results suggest that in C. elegans, dystrophin may have a signaling role early in development, and its absence may activate compensatory mechanisms that counteract muscle degradation caused by loss of dystrophin. We have also developed a temperature-based screening method for synthetic paralysis that can be used to rapidly identify genetic partners of dystrophin. Our results allow for the comprehensive identification of transcriptome changes that potentially serve as independent drivers of disease progression and may in turn allow for the identification of new therapeutic targets for the treatment of DMD.

MicroRNAs (miRNAs) are known to modulate gene expression, but their activity at the tissue-specific level remains largely uncharacterized. To study their contribution to tissue-specific gene expression, we developed novel tools to profile putative miRNA targets in the Caenorhabditis elegans intestine and body muscle. We validated many previously described interactions and identified ∼3500 novel targets. Many of the candidate miRNA targets curated are known to modulate the functions of their respective tissues. Within our data sets we observed a disparity in the use of miRNA-based gene regulation between the intestine and body muscle. The intestine contained significantly more putative miRNA targets than the body muscle highlighting its transcriptional complexity. We detected an unexpected enrichment of RNA-binding proteins targeted by miRNA in both tissues, with a notable abundance of RNA splicing factors. We developed in vivo genetic tools to validate and further study three RNA splicing factors identified as putative miRNA targets in our study (asd-2, hrp-2, and smu-2), and show that these factors indeed contain functional miRNA regulatory elements in their 3′UTRs that are able to repress their expression in the intestine. In addition, the alternative splicing pattern of their respective downstream targets (unc-60, unc-52, lin-10, and ret-1) is dysregulated when the miRNA pathway is disrupted. A reannotation of the transcriptome data in C. elegans strains that are deficient in the miRNA pathway from past studies supports and expands on our results. This study highlights an unexpected role for miRNAs in modulating tissue-specific gene isoforms, where post-transcriptional regulation of RNA splicing factors associates with tissue-specific alternative splicing.

MicroRNAs (miRNAs) are known to modulate gene expression, but their activity at the tissue-specific level remains largely uncharacterized. In order to study their contribution to tissue-specific gene expression, we developed novel tools to profile miRNA targets in the C. elegans intestine and body muscle.We validated many previously described interactions, and identified ~3,500 novel targets. Many of the miRNA targets curated are known to modulate the functions of their respective tissues. Within our datasets we observed a disparity in the use of miRNA-based gene regulation between the intestine and body muscle. The intestine contained significantly more miRNA targets than the body muscle highlighting its transcriptional complexity. We detected an unexpected enrichment of RNA binding proteins targeted by miRNA in both tissues, with a notable abundance of RNA splicing factors.We developed in vivo genetic tools to validate and further study three RNA splicing factors identified as miRNA targets in our study (asd-2, hrp-2 and smu-2), and show that these factors indeed contain functional miRNA regulatory elements in their 3’UTRs that are able to repress their expression in the intestine. In addition, the alternative splicing pattern of their respective downstream targets (unc-60, unc-52, lin-10 and ret-1) is dysregulated when the miRNA pathway is disrupted.A re-annotation of the transcriptome data in C. elegans strains that are deficient in the miRNA pathway from past studies supports and expands on our results. This study highlights an unexpected role for miRNAs in modulating tissue-specific gene isoforms, where post-transcriptional regulation of RNA splicing factors associates with tissue-specific alternative splicing.

23Background: Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease 24 characterized by progressive muscle degeneration. The condition is driven by nonsense 25 and missense mutations in the dystrophin gene, but the resulting changes in muscle-26 specific gene expression that take place in dystrophin's absence remain uncharacterized, 27 as they are potentially obscured by the chronic inflammation elicited by muscle damage in 28 humans. C. elegans possess a mild inflammatory response that allows for the 29 characterization of the transcriptome rearrangements affecting disease progression 30 independently of inflammation. 31Results: In effort to better understand these dynamics we have isolated and sequenced 32 body muscle-specific transcriptomes from C. elegans lacking functional dystrophin at 33 distinct stages of disease progression. We have identified two consecutively altered gene 34 networks, which are also disrupted in the dystrophin deficient mdx mouse model. We 35 found an upregulation of genes involved in mitochondrial function early in disease 36 progression, and an upregulation of genes related to muscle fibre repair in later stages. 37 This suggests that dystrophin may have a signaling role early in development, and its 38 absence may activate compensatory mechanisms that counteract muscle degradation 39 caused by loss of dystrophin. We have also developed a temperature-based screening 40 method for synthetic paralysis that can be used to rapidly identify genetic partners of 41 dystrophin. 42 Conclusions: Our results allow for the comprehensive identification of transcriptome 43 rearrangements that potentially serve as independent drivers of disease progression and 44 may in turn allow for the identification of new therapeutic targets for the treatment of DMD. 45 BACKGROUND 46 47Duchenne muscular dystrophy (DMD) is an X-linked, recessive disease caused by 48 out of frame mutations in the dystrophin gene [1]. The dystrophin gene codes for a 49 structural protein found beneath the sarcolemma, where it is anchored both to the 50 dystrophin glycoprotein complex (DGC) and cytoskeletal actin, thus stabilizing the protein 51 complex and the integrity of the cell membrane [2]. In humans, the absence of functional 52 dystrophin results in progressive degeneration of the skeletal and cardiac muscles. The 53 hallmark symptoms of DMD extend beyond muscle degeneration to include respiratory 54 failure, cardiomyopathy, and pseudohypertrophy. The condition remains the most 55 commonly diagnosed type of muscular dystrophy, affecting approximately 1 in 3,500 male 56 births globally. 57 While the role of dystrophin in forming a physical connection between the 58 extracellular matrix (ECM) and cytoskeleton has been well characterized [3], a 59 comprehensive molecular definition of dystrophin's function is not fully understood. 60 Vertebrate models of DMD include the mdx mouse [4] and the golden retriever muscular 61 dystrophy canine [5]. Both models have contributed significantly towar...

The availability of rapid genome sequencing (rGS) for children in a critical‐care setting is increasing. This study explored the perspectives of geneticists and intensivists on optimal collaboration and division of roles when implementing rGS in neonatal and pediatric intensive care units (ICUs). We conducted an explanatory mixed methods study involving a survey embedded within an interview with 13 genetics and intensive care providers. Interviews were recorded, transcribed, and coded. Geneticists endorsed higher confidence in performing a physical exam and interpreting/communicating positive results. Intensivists endorsed highest confidence in determining whether genetic testing was appropriate, communicating negative results, and consenting. Major qualitative themes that emerged were: (1) concerns with both “genetics‐led” and “intensivist‐led” models with workflows and sustainability (2) shift the role of determining rGS eligibility to ICU medical professionals, (3) continued role of geneticists to assess phenotype, and (4) include genetic counselors (GCs) and neonatal nurse practitioners to enhance workflow and care. All geneticists supported shifting decisions regarding eligibility for rGS to the ICU team to minimize time cost for the genetics workforce. Exploring models of geneticist‐led phenotyping, intensivist‐led phenotyping for some indications, and/or inclusion of a dedicated inpatient GC may help offset the time burden of consenting and other tasks associated with rGS.