Computational discovery of animal small RNA genes and targets

Though recently discovered, small RNAs appear to play a wealth of regulatory roles, being involved in degradation of target mRNAs, translation silencing of target genes, chromatin remodeling and transposon silencing. Presented here are the computational tools that I developed to annotate and characterize small RNA genes and to identify their targets. One of these tools is oligomap, a novel software for fast and exhaustive identi�cation of nearly-perfect matches of small RNAs in sequence databases. Oligomap is part of an automated annotation pipeline used in our laboratory to annotate small RNA sequences. The application of these tools to samples of small RNAs obtained from mouse and human germ cells together with subsequent computational analyses lead to the discovery of a new class of small RNAs which are now called piRNAs. The computational analysis revealed that piRNAs have a strong uridine preference at their 5' end, that unlike miRNAs, piRNAs are not excised from fold-back precursors but rather from long primary transcripts, and that the genome organization of their genes is conserved between human and mouse even though piRNAs on the sequence level are poorly conserved. In vertebrates, the most studied class of small regulatory RNAs are the miRNAs which bind to mRNAs and block translation. A computational framework is introduced to identify miRNA targets in mammals, ies, worms and �sh. The method uses extensive cross species conservation information to predict miRNA binding sites that are under evolutionary pressure. A downstream analysis of predicted miRNA targets revealed novel properties of miRNA target sites, one of which is a positional bias of miRNA target sites in long mammalian 3' untranslated regions. Intersection of our predictions with biochemical pathway annotation data suggested novel functions for some of the miRNAs. To gain further insights into the mechanism of miRNA targeting, I studied microarray data obtained in siRNA experiments. SiRNAs have been shown to produce o�- targets that resemble miRNA targets. This analysis suggests the presence of additional determinants of miRNA target site functionality (beyond complementarity between the miRNA 5' end and the target) in the close vicinity (about 150 nucleotides) of the miRNA-complementary site. Finally, as part of a study aiming to reduce siRNA o�-target e�ects by introducing chemical modi�cations in the siRNA, I performed microarray data analysis of siRNA transfection experiments. Presented are the methods used to quantify o�- target activity of siRNAs carrying di�erent types of chemical modi�cations. The analysis revealed that o�-targets caused by the passenger strand of the siRNA can be reduced by 5'-O-methylation

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

Functional characterisation of pncr003;2L, a small open reading frame gene conserved from drosophila to humans

Small open reading frame genes (smORFs) are a new class of genes, which emerged from the revision of the idea that open reading frames have to be longer than 100 codons to be protein coding and functional. Although bio-informatics evidence suggests that thousands of smORF genes could exist in any given genome, proof of their functional relevance can only be obtained through their functional characterization. This work represents such a study for a Drosophila smORF (pncr003;2L), which was initially misannotated as a non-coding RNA because of its lack of a canonical long open reading frame. Here I show that pncr003;2L codes for two small peptides of 28 and 29 aa, expressed in somatic and cardiac muscles. After generating a null condition for this gene, I use the adult Drosophila heart as a system to assess the function of pncr003;2L. With this system, I show that the small pncr003;2L peptides regulate heart contractions by modulating Ca2+ cycling in cardiac muscles, with either lack or excess of function of these peptides leading to cardiac arrhythmias, and abnormal calcium dynamics. Finally, through an extensive homology study, I show that these small peptides share a great amount of structural and functional homology with the peptides encoded by the vertebrate smORFs sarcolipin (sln) and phospoholamban (pln), which act as major regulators of the Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA), the channel responsible for calcium uptake into the ER following muscle contraction. These results highlight the importance of the pncr003;2L smORF and the Drosophila system, for the study of cardiac pathologies, but most importantly, they show that this family of peptides, conserved across evolution, represent an ancient system for the regulation of calciumtrafficking in muscles. This work corroborates the prevalence, and relevance of this novel class of genes, and shows that closer attention should be given to smORFs in order to determine the full extent of their biological contributio

Investigation of the role of putative inorganic carbon transporters in the carbon dioxide concentrating mechanisms of Chlamydomonas reinhardtii

Photosynthetic microalgae optimize the utilization of inorganic carbon by active uptake and concentration of inorganic carbon (Ci) around the carbon-fixing enzyme Rubisco. This process, mostly induced under limiting carbon conditions is called the Carbon Concentrating Mechanism or CCM. The photosynthetic green alga Chlamydomonas reinhardtii serves as an excellent model organism for the study of eukaryotic CCMs. However, unlike the prokaryotic cyanobacterial CCM, which has a well-characterized Ci uptake system, the Ci uptake and transport system of the C. reinhardtii CCM is not well understood. Since CO2 is a small neutral molecule, it is believed to be able to passively diffuse into the cell with or without any assistance from membrane bound transport proteins. However, the charged HCO3- ion needs transporters to facilitate its uptake across each membrane barrier. The hydrophobic barriers posed by the plasma membrane, chloroplast envelope and thylakoid membranes in the path of the charged HCO3- ion’s ultimate destination to the thylakoid lumen has led to the proposed existence of one or more transport proteins at each of these membrane locations. The roles of the C. reinhardtii NAR gene family, showing sequence homology to the Formate/Nitrite transporter family, were investigated for any changes with respect to the induction of the CCM. NAR1.2 was found to be the most interesting NAR protein with respect to the CCM. NAR1.2 was localized to the chloroplast envelope and is believed to be part of the chloroplastic Ci uptake system. The previously identified putative Ci transporter, LCI1 was localized to the plasma membrane. The expression of this protein in a LCI1 deficient background showed a significant increase in Ci uptake and Ci affinity of cells even in the absence of a functional CCM. However, the RNAi mediated knockdown of the protein failed to show any growth deficiencies or changes in photosynthetic rates at different pH levels. LCI1 is a transporter that is part of the plasma membrane Ci uptake system. In the quest for mutations in either previously identified or novel Ci transporters, an insertional mutagenesis project was also undertaken. The sequenced wild-type strain, CC-503, was found to be a natural mutant for the periplasmic carbonic anhydrase, CAH1

Doctor of Philosophy

dissertationAdenosine deaminases that act on RNA (ADARs) catalyze adenosine-to-inosine (A-to-I) conversion within double-stranded RNA (dsRNA). Since inosine prefers to base-pair with cytidine, it is read by cellular machinery like the ribosome as guanosine. Thus, A-to-I RNA editing can alter the translation of edited codons in cellular mRNAs. However, genome-wide A-to-I editing studies have demonstrated that editing in coding regions is exceedingly rare in most organisms. Instead, ADAR editing is abundant in noncoding sequences associated with protein-coding genes, particularly in introns and untranslated regions (UTRs). By extension, dsRNA structures must also be prevalent in such noncoding regions. These observations raise questions as to the purpose of RNA editing and dsRNA structure in gene-associated sequences. In this dissertation, I explore the physiological functions of ADAR enzymes and their noncoding dsRNA substrates in the nematode Caenorhadbitis elegans. First, I describe in Chapter 2 how ADARs prevent processing and silencing of cellular dsRNAs by the antiviral RNA interference (RNAi) machinery. Using RNAseq, I defined ADAR-edited dsRNAs, or editing-enriched regions (EERs), expressed during four stages of C. elegans development. I found that, in adr-1;adr-2 mutants, EERs gave rise to abundant ~23 nucleotide (nt) small interfering RNAs (siRNAs), and were involved in silencing their associated genes by an RNAi-dependent mechanism. Additionally, disruption of the 26G endogenous siRNA (endo-siRNA) pathway in adr-1;adr-2 mutant backgrounds caused a synthetic phenotype that was rescued by deleting factors involved in antiviral RNAi. These results suggest that ADARs limit RNAi activity against cellular dsRNAs, presenting a striking functional parallel to mammalian ADAR1, which prevents aberrant innate immune signaling by the antiviral dsRNA sensor MDA5. Though the work in Chapter 2 suggests that gene-associated dsRNAs can effect transcriptional silencing, in Chapter 3, I detail analyses suggesting that dsRNA-associated genes in fact exhibit higher-than-expected expression. I used three computational methods to define genome-wide loci in C. elegans encoding dsRNA structures, observing their enrichment on autosome distal arms. Despite that genes in distal arm regions are overall less highly expressed and less likely to be essential than genes in autosome centers, dsRNAs in these regions were enriched within essential and highly expressed genes. These analyses could not explicitly determine if gene expression patterns correlated with dsRNA formation or another property common to these loci. However, they suggest that dsRNA structures may function as important gene regulatory elements. In Chapter 4, I propose additional experiments to test contributions of dsRNA structure to gene expression regulation

Identification of a putative <em>Litomosoides sigmodontis</em> phosphate permease, <em>Ls-ppe-1</em>, and its role in the interaction between filarial nematodes and their Wolbachia endosymbionts & Establishment of RNA interference in the rodent filaria <em>Litomosoides sigmodontis</em>, a model of human filariasis

Filariasis is a vector-borne disease that affects 200 million individuals in the tropics, a large number of them seriously, where the disease leads to blindness (onchocerciasis) or elephantiasis (lymphatic filariasis). The Wolbachia, which are mutualistic endobacteria of most filarial nematodes, are essential for embryogenesis and larval development into adults and were thus identified as new target for anti-filarial drug development. Still, little is known about the molecular details of the symbiosis between Wolbachia and its filarial host but nematode genes that respond to an anti-wolbachial treatment may play important roles in the symbiosis. Therefore, the aim of this thesis was first to screen for such genes that might be involved in the symbiosis, and second to establish RNAi in the rodent filaria Litomosoides sigmodontis to study the function of filarial genes. Differential display PCR was used to detect several candidate genes that are up-regulated upon antibiotic treatment. One of these genes, Ls-ppe-1, was confirmed to be up-regulated over the treatment time and transcripts remained elevated one month after the treatment had been stopped, demonstrating a long-term alteration in the transcriptional state of the gene. The role of Ls-ppe-1 in the symbiosis may relate to a possible direct or indirect involvement in nucleotide metabolism. In contrast to other endosymbionts that have undergone gene reduction, Wolbachia has retained the ability to synthesize nucleotides (in addition to other metabolites). Potentially the phosphate permease provides the Wolbachia with phosphate as part of the mutualistic symbiosis The mitochondrial HSP60 was found to be up-regulated in immunohistochemical stained sections of O. volvulus after the depletion of Wolbachia by doxycycline treatment. The mRNA levels of filarial hsp60 in O. volvulus, quantified by real time PCR, were significantly increased after antibiotic treatment compared to untreated control worms. The results lead to the hypothesis that the increased expression of HSP60 is due to a disruption of the homeostasis of metabolic pathways for which Wolbachia may be needed. RNAi in adult worms of the rodent filaria L. sigmodontis was established and optimized. The actin gene was successfully knocked-down, whereas the transcript levels of Ls-hsp60 and Ls-gst2, used as controls to measure off-target effects, were not reduced. Soaking of nematodes with dsRNA coding for the C. elegans yolk receptor (Ce-rme-2), which has no orthologues in filaria, did not affect Ls-act transcription, demonstrating that the reduction of Ls-act is specific and not due to toxicity of dsRNA or off-target effects. The inhibition of Ls-act persisted for 72 hours, the length of the observation time. Two additional phenotypes were seen: first, the adult nematodes observed 48 and 72 hours after the beginning of the experiment showed paralysis by being stretched out and having slower movements, second, the release of microfilariae was significantly inhibited after soaking with dsRNA. The use of lower concentrations of dsRNA than those previously reported for RNAi in B. malayi as well as the establishment of proper controls for off-target effects could make RNAi a possibility to study the function of filarial genes. However, there are issues with RNAi in filarial nematodes and other parasitic nematodes, namely that their genomes appear to lack the genes to allow spreading of the dsRNA signal from cell to cell, indicating that the process is still hit-or-miss, depending on the gene to be silenced and/or the method of delivery

A Novel 3-Hydroxysteroid Dehydrogenase That Regulates Reproductive Development and Longevity

A multidisciplinary approach identifies novel biochemical activities involved in the synthesisof C. elegans bile acid-like steroids, which act as hormones that regulate sterol metabolism and longevity

Isolation and characterization of two closely related beta-carbonic anhydrases of Chlamydomonas reinhardtii

Aquatic photosynthetic organisms such as the green alga, C. reinhardtii respond to low carbon dioxide conditions by inducing a carbon dioxide concentrating mechanism (CCM). Important components of the CCM are carbonic anhydrases (CAs), zinc metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. C. reinhardtii now has nine carbonic anhydrases, three alpha-CAs and six beta-CAs. This dissertation describes the identification and characterization of two closely related beta-CAs, CAH7 and CAH8. These CAs are 63% identical. CAH7 and CAH8 encode proteins of 399 and 333 amino acids, respectively. Both of these CAs are constitutively expressed at the transcript and protein level. Preliminary results of immunolocalization studies localized CAH7 in the chloroplast while CAH8 was localized in the periplasm. Both the CAH7 and CAH8 open reading frames (ORF) were cloned in the overexpression vector pMal-c2x and expressed as recombinant proteins. Activity assay demonstrated that CAH7 and CAH8 are active carbonic anhydrases. The proposed roles for CAH7 and CAH8 are discussed. Previously, insertional mutants were generated to be able to isolate bicarbonate-transporters and other proteins that might be essential for a functional CCM. One of the generated insertional mutants is slc211, a mutant that requires high carnon dioxide for optimum growth. The mutant slc211 had an insertion in the novel gene designated as CIA7. RNA interference was successfully used to reduce the expression of CIA7. The resultant transformants had a growth phenotype similar to slc211 requiring high carbon dioxide for optimum growth. These results suggest that CIA7 is a gene that facilitates growth in C. reinhardtii under low carbon dioxide conditions. The possible functions of CIA7 are discussed

POS-1 Regulation of Endo-mesoderm Identity in C. elegans: A Dissertation

How do embryos develop with such poise from a single zygote to multiple cells with different identities, and yet survive? At the four-cell stage of the C. elegans embryo, only the blastomere EMS adopts the endo-mesoderm identity. This fate requires SKN-1, the master regulator of endoderm and mesoderm differentiation. However, in the absence of the RNA binding protein POS-1, EMS fails to fulfill its fate despite the presence of SKN-1. pos-1(-) embryos die gutless. Conversely, the RNA binding protein MEX-5 prevents ectoderm blastomeres from adopting the endo-mesoderm identity by repressing SKN-1. mex-5(-) embryos die with excess muscle at the expense of skin and neurons. Through forward and reverse genetics, I found that genes gld-3/Bicaudal C, cytoplasmic adenylase gld-2, cye-1/Cyclin E, glp-1/Notch and the novel gene neg-1 are suppressors that restore gut development despite the absence of pos-1. Both POS-1 and MEX-5 bind the 3’UTR of neg-1 mRNA and its poly(A) tail requires GLD-3/2 for elongation. Moreover, neg-1 requires MEX-5 for its expression in anterior ectoderm blastomeres and is repressed in EMS by POS-1. Most neg-1(-) embryos die with defects in anterior ectoderm development where the mesoderm transcription factor pha-4 becomes ectopically expressed. This lethality is reduced by the concomitant loss of med- 1, a key mesoderm-promoting transcription factor. Thus the endo-mesoderm identity of EMS is determined by the presence of SKN- 1 and the POS-1 repression of neg-1, whose expression is promoted by MEX-5. Together they promote the anterior ectoderm identity by repressing mesoderm differentiation. Such checks and balances ensure the vital plurality of cellular identity without the lethal tyranny of a single fate