Lower Expression of Genes near microRNA in C. elegans Germline

Background: MicroRNAs (miRNAs) are recently discovered short non-protein-coding RNA molecules. miRNAs are increasingly implicated in tissue-specific transcriptional control and particularly in development. Because there is mounting evidence for the localized component of transcriptional control, we investigated if there is a distance-dependent effect of miRNA. Results: We analyzed gene expression levels around the 84 of 113 know miRNAs for which there are nearby gene that were measured in the data in two independent C. elegans expression data sets. The expression levels are lower for genes in the vicinity of 59 of 84 (71%) miRNAs as compared to genes far from such miRNAs. Analysis of the genes with lower expression in proximity to the miRNAs reveals increased frequency matching of the 7 nucleotide "seed"s of these miRNAs. Conclusion: We found decreased messenger RNA (mRNA) abundance, localized within a 10 kb of chromosomal distance of some miRNAs, in C. elegans germline. The increased frequency of seed matching near miRNA can explain, in part, the localized effects

Diversity in parasitic nematode genomes: the microRNAs of Brugia pahangi and Haemonchus contortus are largely novel

<b>BACKGROUND:</b> MicroRNAs (miRNAs) play key roles in regulating post-transcriptional gene expression and are essential for development in the free-living nematode Caenorhabditis elegans and in higher organisms. Whether microRNAs are involved in regulating developmental programs of parasitic nematodes is currently unknown. Here we describe the the miRNA repertoire of two important parasitic nematodes as an essential first step in addressing this question. <b>RESULTS:</b> The small RNAs from larval and adult stages of two parasitic species, Brugia pahangi and Haemonchus contortus, were identified using deep-sequencing and bioinformatic approaches. Comparative analysis to known miRNA sequences reveals that the majority of these miRNAs are novel. Some novel miRNAs are abundantly expressed and display developmental regulation, suggesting important functional roles. Despite the lack of conservation in the miRNA repertoire, genomic positioning of certain miRNAs within or close to specific coding genes is remarkably conserved across diverse species, indicating selection for these associations. Endogenous small-interfering RNAs and Piwi-interacting (pi)RNAs, which regulate gene and transposon expression, were also identified. piRNAs are expressed in adult stage H. contortus, supporting a conserved role in germline maintenance in some parasitic nematodes. <b>CONCLUSIONS:</b> This in-depth comparative analysis of nematode miRNAs reveals the high level of divergence across species and identifies novel sequences potentially involved in development. Expression of novel miRNAs may reflect adaptations to different environments and lifestyles. Our findings provide a detailed foundation for further study of the evolution and function of miRNAs within nematodes and for identifying potential targets for intervention

microRNAs of parasitic helminths – identification, characterization and potential as drug targets

microRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation. They were first identified in the free-living nematode Caenorhabditis elegans, where the miRNAs lin-4 and let-7 were shown to be essential for regulating correct developmental progression. The sequence of let-7 was subsequently found to be conserved in higher organisms and changes in expression of let-7, as well as other miRNAs, are associated with certain cancers, indicating important regulatory roles. Some miRNAs have been shown to have essential functions, but the roles of many are currently unknown. With the increasing availability of genome sequence data, miRNAs have now been identified from a number of parasitic helminths, by deep sequencing of small RNA libraries and bioinformatic approaches. While some miRNAs are widely conserved in a range of organisms, others are helminth-specific and many are novel to each species. Here we review the potential roles of miRNAs in regulating helminth development, in interacting with the host environment and in development of drug resistance. Use of fluorescently-labeled small RNAs demonstrates uptake by parasites, at least in vitro. Therefore delivery of miRNA inhibitors or mimics has potential to alter miRNA activity, providing a useful tool for probing the roles of miRNAs and suggesting novel routes to therapeutics for parasite control

Antagonistic and cooperative AGO2-PUM interactions in regulating mRNAs.

Approximately 1500 RNA-binding proteins (RBPs) profoundly impact mammalian cellular function by controlling distinct sets of transcripts, often using sequence-specific binding to 3' untranslated regions (UTRs) to regulate mRNA stability and translation. Aside from their individual effects, higher-order combinatorial interactions between RBPs on specific mRNAs have been proposed to underpin the regulatory network. To assess the extent of such co-regulatory control, we took a global experimental approach followed by targeted validation to examine interactions between two well-characterized and highly conserved RBPs, Argonaute2 (AGO2) and Pumilio (PUM1 and PUM2). Transcriptome-wide changes in AGO2-mRNA binding upon PUM knockdown were quantified by CLIP-seq, and the presence of PUM binding on the same 3'UTR corresponded with cooperative and antagonistic effects on AGO2 occupancy. In addition, PUM binding sites that overlap with AGO2 showed differential, weakened binding profiles upon abrogation of AGO2 association, indicative of cooperative interactions. In luciferase reporter validation of candidate 3'UTR sites where AGO2 and PUM colocalized, three sites were identified to host antagonistic interactions, where PUM counteracts miRNA-guided repression. Interestingly, the binding sites for the two proteins are too far for potential antagonism due to steric hindrance, suggesting an alternate mechanism. Our data experimentally confirms the combinatorial regulatory model and indicates that the mostly repressive PUM proteins can change their behavior in a context-dependent manner. Overall, the approach underscores the importance of further elucidation of complex interactions between RBPs and their transcriptome-wide extent

Characterization of the MIR-44 Family of Micrornas in the C. Elegans Germline

While numerous individual miRNAs have been detected in the germline, the functions of most other specific miRNAs remain largely unknown. Functions of miRNAs have been difficult to determine as miRNAs often modestly repress target mRNAs and are suggested to sculpt or fine tune gene expression to allow for the robust expression of cell fates. Analysis of newly generated mir-44 family mutants has identified a group of miRNAs that modulate the pathway of germline sex determination in C. elegans. Mutants produce fewer sperm and display an earlier switch to producing oocytes. In the germline, cell fate decisions are made for germline sex determination during C. elegans hermaphrodite larval development when sperm are generated in a short window before the switch to oocyte production. To understand the genetic relationship between mir-44/45 and the genes that regulate the switch from producing sperm to oocytes, I examined several components of this pathway. The results suggest that mir-44/45 regulates proper fog-1 expression through fbf-1 and fem-3 to promote sperm specification. Our research indicates that the mir-44 family promotes sperm cell fate decision at the time of the developmental switch from spermatogenesis to oogenesis. In addition to the function of the mir-44 family in hermaphrodite sex determination, I have also identified a function of mir-44/45 in regulating the process of spermiogenesis and sperm transfer in males. Mutant male sperm frequently fail to activate and during mating mir-44/45 sperm often do not transfer following interaction with hermaphrodites. The combination of these results in males and hermaphrodites indicate that the mir-44 family of microRNAs functions to regulate multiple processes in C. elegans development and germline function

Identification of Novel (\u3cem\u3eR\u3c/em\u3eNAi \u3cem\u3eDe\u3c/em\u3eficient) Genes in \u3cem\u3eC. elegans\u3c/em\u3e: A Dissertation

RNA interference or RNAi was first discovered as an experimental approach that induces potent sequence-specific gene silencing. Remarkably, subsequent studies on dissecting the molecular mechanism of the RNAi pathway reveal that RNAi is conserved in most eukaryotes. In addition, genes and mechanisms related to RNAi are employed to elicit the regulation of endogenous gene expression that controls a variety of important biological processes. To investigate the mechanism of RNAi in the nematode C. elegans, we performed genetic screens in search of RNAi deficient mutants (rde). Here I report the summary of the genetic screens in search of rde mutants as well as the identification of two novel genes required for the RNAi pathway, rde-3 and rde-8. In addition, we demonstrate that some of the rde genes, when mutated, render the animals developmentally defective, suggesting that these rde genes also function in developmental gene regulation. This work presents novel insights on the components of the RNAi pathway and the requirement of these components in the regulation of endogenous gene expression

Uridylation and adenylation of RNAs.

The posttranscriptional addition of nontemplated nucleotides to the 3' ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated addition of uridine or adenosine to the 3' ends of RNAs occurs in different organisms ranging from algae to humans, and on different kinds of RNAs, such as histone mRNAs, mRNA fragments, U6 snRNA, mature small RNAs and their precursors etc. These modifications may lead to different outcomes, such as increasing RNA decay, promoting or inhibiting RNA processing, or changing RNA activity. Growing pieces of evidence have revealed that such modifications can be RNA sequence-specific and subjected to temporal or spatial regulation in development. RNA tailing and its outcomes have been associated with human diseases such as cancer. Here, we review recent developments in RNA uridylation and adenylation and discuss the future prospects in this research area

Small RNAs and Argonautes Provide a Paternal Epigenetic Memory of Germline Gene Expression to Promote Thermotolerant Male Fertility: A Dissertation

During each life cycle, gametes must preserve and pass on both genetic and epigenetic information, making the germline both immortal and totipotent. In the male germline the dramatic morphological transformation of a germ cell through meiosis, into a sperm competent for fertilization, while retaining this information is an incredible example of cellular differentiation. This process of spermatogenesis is inherently thermosensitive in numerous metazoa ranging from worms to man. Here, I describe the role of two redundant AGO-class paralogs, ALG-3/4, and their small RNA cofactors, in promoting thermotolerant male fertility in Caenorhabditis elegans. alg-3/4 double mutants exhibit temperature dependent sterility resulting from defective spermiogenesis, the postmeiotic differentiation of haploid spermatids into spermatozoa competent for fertilization. The essential Argonaute CSR-1 functions with ALG-3/4 to positively regulate target genes required for spermiogenesis by promoting transcription via a small RNA positive feedback loop. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal loaded into CSR-1 to maintain transcriptionally active chromatin at genes required for spermiogenesis and to provide an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. The ALG-3/4 small RNA pathway also initiates silencing small RNA signals loaded into WAGO vii Argonautes, which function to posttranscripitonally silence their target mRNAs. Silencing WAGO/small RNA-complexes are present in sperm and presumably transmitted to offspring upon fertilization. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic activating and silencing signals in the form of Argonaute/small-RNA complexes, constituting a memory of gene expression in preceding generations

Parallel Genetics of Gene Regulatory Sequences in Caenorhabditis elegans

Wie regulatorische Sequenzen die Genexpression steuern, ist von grundlegender Bedeutung für die Erklärung von Phänotypen in Gesundheit und Krankheit. Die Funktion regulatorischer Sequenzen muss letztlich in ihrer genomischen Umgebung und in entwicklungs- oder gewebespezifischen Zusammenhängen verstanden werden. Da dies eine technische Herausforderung ist, wurden bisher nur wenige regulatorische Elemente in vivo charakterisiert. Hier verwenden wir Induktion von Cas9 und multiplexed-sgRNAs, um hunderte von Mutationen in Enhancern/Promotoren und 3′ UTRs von 16 Genen in C. elegans zu erzeugen. Wir quantifizieren die Auswirkungen von Mutationen auf Genexpression und Physiologie durch gezielte RNA- und DNA-Sequenzierung. Bei der Anwendung unseres Ansatzes auf den 3′ UTR von lin-41, bei der wir hunderte von Mutanten erzeugen, stellen wir fest, dass die beiden benachbarten Bindungsstellen für die miRNA let-7 die lin-41-Expression größtenteils unabhängig voneinander regulieren können, mit Hinweisen auf eine mögliche kompensatorische Interaktion. Schließlich verbinden wir regulatorische Genotypen mit phänotypischen Merkmalen für mehrere Gene. Unser Ansatz ermöglicht die parallele Analyse von genregulatorischen Sequenzen direkt in Tieren.How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. The function of regulatory sequences must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. We quantify the impact of mutations on expression and physiology by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3′ UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression largely independently of each other, with indications of a compensatory interaction. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of gene regulatory sequences directly in animals

Biogenesis and Stability of Germline Small RNAs in C. elegans.

Across the animal kingdom, small, noncoding RNAs preserve and promote fertility by engaging Argonaute effector proteins to silence deleterious genetic elements. Generated in germline and inherited into progeny, endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) regulate vast suites of gametic and zygotic genes, yet remarkably little is known about how they are regulated. With an expanded repertoire of small RNA classes, Caenorhabditis elegans provides an ideal model for investigating how animals drive epigenetic inheritance of fertility-preserving germline small RNAs. The conserved methyltransferase HEN1 methylates small RNAs to prevent their degradation. Methylation of germline small RNAs enhances accumulation, promoting robust inheritance into progeny. All plant small RNAs are methylated, but animal HEN1 methylates only some small RNAs. The mechanisms of selective methylation were unknown. I identified the functional C. elegans ortholog of HEN1 and demonstrated that it methylates all piRNAs but only select subclasses of endo-siRNAs. I further found that particular endo-siRNAs are methylated in maternal, but not paternal, germlines. Through genetic and biochemical analyses, I showed that small RNA methylation status is likely dictated by the associated Argonaute. This established selective expression of divergent Argonautes as a novel mechanism for differentially stabilizing germline small RNAs, with significant implications for preferential inheritance of maternal epigenetic information. piRNAs are essential for animal fertility, but their expression mechanisms are poorly characterized. In collaboration with bioinformatician Mallory Freeberg, I showed that C. elegans male and female germlines express distinct piRNA subsets that evolve independently and differ in inheritance. A common sequence motif lies upstream of nematode piRNA loci. We discovered that this motif varies significantly between male and female piRNAs. Using a novel transgenic approach, I established that C. elegans piRNAs represent thousands of tiny, autonomous transcriptional units, rivaling coding genes in number. I further demonstrated that the upstream motif is required for piRNA expression and that variation at a single nucleotide position within this motif orchestrates selective male versus female germline enrichment and inheritance of piRNAs. These and additional included studies define novel factors and mechanisms involved in regulation of germline small RNAs and transgenerational transmission of their crucial epigenetic information.PHDHuman GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111471/1/acbilli_1.pd