Veksler-Lublinsky, Isana et al. (2017) International Worm Meeting "Comparative genomics analysis unveils genetic factors governing the pathogenicity of P. aeruginosa towards C. elegans."

Veksler-Lublinsky, Isana, Vasquez-Rifo, Alejandro, Ausubel, Fred, Ambros, Victor, Cheng, Zhenyu

[International Worm Meeting, 2017]

The pathogenic potential of bacteria against animal host, such as Caenorhabditis elegans (C. elegans), can vary drastically between distinct strains of the same bacterial species. The remarkable fluidity of bacterial genomes, shaped by horizontal gene transfer (HGT) amongst populations of bacteria in their natural habitats, is thought to contribute to inter-strain variation in pathogenic behavior towards natural hosts, including nematodes. Pseudomonas aeruginosa (P. aeruginosa) is a free-living bacterium that is a pathogen of a broad range of eukaryotic hosts, including nematodes, insects, mammals and plants. Wild isolates of P. aeruginosa can lethally infect adult C. elegans worms, yet the pathogenicity (virulence) of P. aeruginosa strains against wild type C. elegans varies widely among distinct strains. Using a comparative genomics and phylogenetic approach, we investigated the genetic determinants that drive evolutionary changes in P. aeruginosa pathogenicity towards C. elegans. Our results suggest an evolutionary model that links the presence/absence of CRISPR with gain/loss of known virulence genes and potential anti-virulence genes, and accounts for differences in pathogenicity amongst P. aeruginosa strains against C. elegans.

Lutzky, Stav et al. (2021) International Worm Meeting "Functional interplay between microRNAs, RNA binding proteins, and alternative polyadenylation in Caenorhabditis elegans."

Veksler-Lublinsky, Isana, Lutzky, Stav

[International Worm Meeting, 2021]

Regulation of gene expression is fundamental for proper development, homeostasis, and adaptation to the environment for all living organisms. Post-transcriptional gene regulation, which takes place between the transcription and the translation of a gene, is largely controlled by two classes of regulators, microRNAs (miRNAs) and RNA-binding proteins (RBPs). MiRNAs are short non-coding RNA molecules that hybridize to complementary sequences on target mRNAs, usually located in the 3'UTR region, and repress their translation or mediate their degradation. RBPs also perform their function by binding to mRNAs; however, their binding sites can be located in various regions including 5'UTRs, coding sequences, and 3'UTRs. Unlike miRNAs' repressive role, the regulatory activity of RBPs may be positive or negative, depending on the protein, the mRNA, and the biological context. In recent years, a wide repertoire of functional connections between miRNAs and RBPs has been discovered, uncovering a new level of complexity in gene expression regulation. These connections include mutual regulation of the same target mRNAs by both miRNAs and RPBs. Since both miRNAs and RBPs bind to the 3'UTR region of mRNAs, its length may add a layer of complexity to the interplay between miRNAs and RBPs. 3'UTR length is regulated by alternative polyadenylation (APA). By changing the position of polyadenylation, APA can generate transcripts with multiple 3′ UTR isoforms, each containing distinct regulatory elements (e.g., miRNA and RBP binding sites). In this study, we used C.elegans UTRome.org data that provides 3'UTR variants across different tissues, to study the coordination between miRNAs and RBPs in tissue-specific gene regulation that happens through binding to 3'UTRs. We performed a comprehensive analysis to explore regulation by individual miRNA and RBPs, as well as mutual regulation by both miRNAs and RBPs. We identified miRNA-RBP pairs that show statistically significant differential co-targeting across tissues. Our results provide evidence for complex gene regulatory networks that involve multiple factors such as miRNAs and RBPs.

Duan, Ye et al. (2021) International Worm Meeting "Critical contribution of 3' non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA."

Duan, Ye, Ambros, Victor, Veksler-Lublinsky, Isana

[International Worm Meeting, 2021]

MicroRNAs (miRNA) are endogenous regulatory non-coding RNA that exist in all multi-cellular organisms. It is well established that base pairing to the miRNA seed region (g2-g8 from the 5' end) is essential for targeting. However, the in vivo biological function of 3' non-seed region (g9-g22) are less well understood, especially for evolutionarily conserved miRNAs such as let-7a, whose entire sequence is deeply conserved among bilaterians. Here we report the first systematic investigation of the biological functions and targeting principles of the 3' non-seed nucleotides of miRNA let-7a as an example of evolutionarily conserved miRNAs. We used CRISPR/Cas9 genome engineering in C. elegans to show that the 3' non-seed sequence of let-7a determines the specific and essential in vivo function that distinguish let-7a from its family paralogs. We also found that the identity of each single nucleotide at g11-g16 (referred to as the "critical non-seed region") is essential for the in vivo function of let-7a. We confirmed that the conserved NHL protein lin-41/TRIM71 is a major functional target that requires the critical non-seed pairing of let-7a. Furthermore, by phenotypic assays and ribosome profiling, we found that let-7a has additional in vivo targets that require pairing in the critical non-seed region, including heterochronic genes daf-12 and hbl-1. We show that the repression mechanisms for these targets can include either translational inhibition, mRNA destabilization or both. Outside the critical non-seed region, we found that nucleotides at g17-g22 are less critical for let-7a function; however, we found that g17-g22 pairing can contribute to full function for sites with mismatches in the g11-g16 region. Strikingly, despite the involvement in compensating for imperfect seed pairing, the 3' non-seed pairing of let-7a is nevertheless necessarily required even in the context of perfect seed complementarity, at least for full repressing efficacy of lin-41. Our research provided phenotypic and molecular evidence that for certain miRNA and targets, exemplified by the evolutionarily conserved let-7a::lin-41 interactions, the specific configuration of the 3' non-seed base-pairing critically influence miRNA function in vivo.

Panzade, Ganesh et al. (2021) International Worm Meeting "Annotation of isomiR dynamics across the C. elegans developmental stages"

Panzade, Ganesh, Veksler-Lublinsky, Isana, Zinovyeva, Anna

[International Worm Meeting, 2021]

microRNAs (miRNAs) are short, ~22 nucleotides, small RNAs that repress genes through mRNA destabilization and translational repression. During canonical miRNA biogenesis, several miRNA species, or isoforms, known as isomiRs, are produced from a single precursor miRNA. The isomiRs are thought to be generated through Drosha or Dicer cleavage at alternate positions on either the primary or the precursor miRNAs, generating truncated or extended 5' or 3' miRNA ends. Because mature miRNA sequence modifications can alter their gene target repertoire, we wished to investigate the extent of isomiR prevalence and dynamics across C. elegans developmental stages. We performed small RNAseq from staged animals to assess isomiR variability across developmental stages. Using isomiR-SEA (Urgese et al 2016) for isomiR identification and quantification, we provide an isomiR profiling map against the miRBase annotated miRNAs at all stages of C. elegans development. We found that many miRNAs display isomiR level variability at different developmental stages, suggesting that the functional specificity of isomiRs to the developmental stage may exist. Not surprisingly, 3' end miRNA alterations were more frequent than the potentially seed-altering 5' end extensions or truncations. Some miRNAs had isomiRs that were just as, or more abundant than their annotated canonical mature miRNAs. These isomiRs included those with 5' end truncations and deletions, predicted to target new, potentially distinct sets of genes. Overall, we will present annotation of isomiR dynamics across C. elegans developmental stages, which we hope can provide us with insights into miRNA biogenesis and the intriguing potential of isomiR function.

Ow, Maria C. et al. (2013) International Worm Meeting "Genome-wide study of stress-responsive microRNA and mRNA transcriptomes in C. elegans."

Ambros, Victor, Veksler-Lublinsky, Isana, Ow, Maria C.

[International Worm Meeting, 2013]

All organisms must cope with stress and have evolved various means to respond to detrimental changes in their environment. Because most of the pathways involved in stress response are conserved throughout evolution, studying stress response in C. elegans is likely to result in crucial insights in understanding the pathology and treatment of human disorders. MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that regulate gene expression by imperfectly base-pairing to the 3'UTR of their target mRNAs. MiRNAs are intimately involved in broad aspects of animal development. Their misregulation can result in the onset of a number of pathophysiological conditions, including heart disease, diabetes, and cancer. Given the prominent role of miRNAs in nearly all aspects of biology, we predict that they also play a role in stress response. In this study we employ genome-wide methods, including small RNA deep sequencing and mRNA transcript microarrays, to study the response of C. elegans to various stresses including oxidative stress, heat stress, and exposure to bacterial pathogens. We employ bioinformatics to determine stress-responsive miRNAs and mRNAs and identify stress-mediated gene regulatory patterns. Our analyses suggest the existence of regulatory patterns common to all stresses as well as stress-specific sub-systems.

Zinovyeva, Anna et al. (2015) International Worm Meeting "ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal."

Zinovyeva, Anna, Wohlschlegel, James, Vashisht, Ajay, Ambros, Victor, Veksler-Lublinsky, Isana

[International Worm Meeting, 2015]

MicroRNAs are small non-coding RNAs that, together with Argonaute proteins, regulate expression of genes essential for normal development and physiology. We have previously characterized mutations in a C. elegans microRNA-specific Argonaute ALG-1 that are antimorphic (alg-1(anti)) [1]. alg-1(anti) cause dramatically stronger microRNA-related phenotypes than a complete loss of ALG-1 and ALG-1(anti) miRISCs (microRNA induced silencing complexes) fail to undergo the functional transition from microRNA processing to target repression. To better understand this transition we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We found that the mutant ALG-1(anti) protein fails to interact with numerous wild type miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically over-accumulate microRNA* (passenger) strands. Interestingly, immunoprecipitated ALG-1(anti) complexes contained non-stoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of their corresponding mature microRNAs. We show that such aberrant loading of microRNA* strands into Argonaute is dependent on the microRNA identity, suggesting that specific microRNA features may allow wild type Argonaute to distinguish among different microRNAs. These findings suggest a previously unknown function for Argonaute in microRNA strand selection and passenger strand disposal.[1]. Zinovyeva et al (2014) PLoS Genet 10(4):e1004286.

Ren, Zhiji et al. (2015) International Worm Meeting "Staufen negatively modulates microRNA activity in Caenorhabditis elegans."

Ambros, Victor, Veksler-Lublinsky, Isana, Morrissey, David, Ren, Zhiji, Vasquez-Rifo, Alejandro

[International Worm Meeting, 2015]

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that post-transcriptionally regulate gene expression primarily through binding to the 3' untranslated region (3'UTR) of target mRNAs, which results in translation inhibition and/or mRNA degradation. MiRNAs are known to play important roles in various developmental and physiological processes. Staufen is a double-stranded RNA binding protein that has been shown in Drosophila and mammals to regulate mRNA localization, translation and decay. There is only one Staufen homolog identified in C. elegans (stau-1), and the mutants of stau-1 have been shown to exhibit enhanced exogenous RNAi phenotypes and slight germline defects.Here we demonstrate that loss-of-function mutations of stau-1 increase miRNA activity for several miRNA families. First, both stau-1(tm2266) and stau-1(q798) suppress the developmental timing defects of a let-7 family mutant, mir-48 mir-241(nDf51). In addition, stau-1(tm2266) suppresses the phenotype of a lsy-6 hypomorphic allele (ot150) in a set of bilaterally symmetric gustatory neurons (ASEL/R). Finally, stau-1(tm2266) suppresses the heterochronic phenotype of lin-4(e912); lin-14(n719) animals. Interestingly, stau-1(tm2266) does not suppress the heterochronic phenotype of the 3'UTR deletion mutant lin-14(n355n679), which removes all the predicated binding sites for lin-4 and let-7 family miRNAs. Furthermore, small RNA high-throughput sequencing analysis reveals that there is no dramatic change in most small RNA population between wild type and stau-1(tm2266), except several endogenous siRNAs in the WAGO pathway. The modulation of stau-1 on miRNA activity does not seem to be simply caused by the enhanced exogenous RNAi phenotype of stau-1 mutants since eri-1(mg366) does not suppress, but rather enhances the heterochronic phenotypes of mir-48 mir-241(nDf51) animals. Therefore, our results suggest that STAU-1 negatively modulates miRNA activity downstream of biogenesis, possibly by competing with miRNAs for binding to target mRNA 3'UTRs.

Vieux, Karl-Frederic et al. (2021) International Worm Meeting "Screening by deep sequencing reveals mediators of miRNA tailing in C. elegans"

McJunkin, Katherine, Prothro, Katherine, Veksler-Lublinsky, Isana, Palmer, Cameron, Vieux, Karl-Frederic

[International Worm Meeting, 2021]

Most micro-RNAs (miRNA) result from the processing of longer primary transcripts transcribed from miRNA genes by the RNA polymerase Pol II. These transcripts harbor hairpin structures which are recognized and processed twice to yield mature miRNAs: (1) once in the nucleus by Drosha, with the support of Pasha (PASH -1 in C. elegans) and (2) again in the cytoplasm by Dicer. The resulting ~22nt mature miRNAs are effective regulators of gene expression. When loaded onto the RNA-induced silencing complex (RISC), mature miRNAs direct RISC activity to transcripts with complementary sequence elements to repress their expression. In turn, their levels must also be regulated. Despite this, the processes responsible for miRNA turnover remain poorly understood. In instances where they are turned over, miRNAs are frequently modified by the addition of untemplated nucleotides to their 3' end, but the role of this tailing is often unclear. Here we characterized the prevalence and functional consequences of microRNA tailing in vivo, using the C. elegans model. Our data showed that miRNA tailing in C. elegans consists mostly of mono-uridylation of mature miRNA species, with rarer mono-adenylation - likely added to microRNA precursors. Through a targeted RNAi screen, we discovered that the TUT4/TUT7 gene family member CID-1 is required for uridylation, whereas the GLD2 gene family member F31C3.2 is required for adenylation. Thus, the TUT4/TUT7 and GLD2 gene families have broadly conserved roles in miRNA modification. We also examined the role of tailing in turnover in the absence of ongoing miRNA production, using a temperature sensitive pash-1 strain. At permissive temperatures, PASH-1 is functional, and biogenesis is uninterrupted. When upshifted to 25°C, PASH-1 activity is abrogated, and mature miRNA production ceases. We determined the half-lives of miRNAs after acute inactivation of microRNA biogenesis, revealing that half-lives are generally long (median=20.7h), as observed in other systems. Despite an increased proportion of tailed species in older microRNAs, we detected no changes to microRNA abundance or decay dynamics upon disrupting tailing. Thus, tailing is not a global regulator of miRNA decay in C. elegans. Nonetheless, by identifying the responsible enzymes, this work lays the groundwork to explore whether tailing plays more specialized context- or miRNA-specific regulatory roles.

Hebbar, Shilpa et al. (2017) International Worm Meeting "Functional screen to identify modulators of miRNA activity."

Zinovyeva, Anna, Hebbar, Shilpa

[International Worm Meeting, 2017]

Regulation of gene expression is essential for normal physiology and development. One of the ways cells regulate gene expression is through a class or non-coding RNAs called microRNAs (miRNAs). miRNAs associate with Argonaute proteins to form miRNA Induced Silencing Complexes (miRISCs) to post-transcriptionally repress gene expression by targeting mRNA 3'UTRs through partial sequence complementarity. Proteins associated with C. elegans miRISCs were identified using Argonaute ALG-1 immunoprecipitation and 2'-O-methyl oligonucleotide-mediated miRNA-specific pulldown experiments followed by MUDPIT proteomics ss . We hypothesized that factors that co-precipitate with ALG-1 and/or miRNA-specific miRISCs play a role in regulating miRNA biogenesis or function. To identify which miRISC interactors are important for miRNA activity and/or processing, we are carrying out functional assays in several genetically sensitized backgrounds. Specifically, we are utilizing RNAi to knock down the putative miRISC interactors in animals with compromised miRNA activity (such as lsy-6(ot150), mir-48 mir-241 (nDf51), and mir-35-41 (nDf50) mutants). We hypothesize that enhancement or suppression of the phenotypes associated with reduction of miRNA function in these mutants will indicate potential role(s) of the candidate genes in miRNA regulation. We will report on our progress in identifying which physical interactors of ALG-1/miRISCs are important for modulating miRNA activity. ss Zinovyeva, A. Y., Veksler-Lublinsky, I., Vashisht, A. A., Wohlschlegel, J. A., & Ambros, V. R. (2015). Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal. Proceedings of the National Academy of Sciences, 112(38), E5271-E5280.