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

The double edge of anti-CD40 siRNA therapy: It increases renal microcapillar density but favours the generation of an inflammatory milieu in the kidneys of ApoE -/- mice

Background: Chronic kidney disease (CKD) is associated with endothelial dysfunctions thus prompting links between microcirculation (MC), inflammation and major cardiovascular risk factors. Purpose of the study: We have previously reported that siRNA-silencing of CD40 (siCD40) reduced atherosclerosis (ATH) progression. Here, we have deepened on the effects of the siCD40 treatment by evaluating retrospectively, in stored kidneys from the siCD40 treated ApoE−/− mice, the renal microcirculation (measured as the density of peritubular capillaries), macrophage infiltration and NF-κB activation. Methods: Kidneys were isolated after 16 weeks of treatment with the anti-CD40 siRNA (siCD40), with a scrambled control siRNA (siSC) or with PBS (Veh. group). Renal endothelium, infiltrating macrophages and activated NF-κB in endothelium were identified by immunohistochemistry, while the density of stained peritubular capillaries was quantified by image analysis. Results: ATH was associated with a reduction in renal MC, an effect reversed by the anti-CD40 siRNA treatment (3.8 ± 2.7% in siCD40; vs. 1.8 ± 0.1% in siSC; or 1.9 ± 1.6% in Veh.; p < 0.0001). Furthermore, siCD40 treatment reduced the number of infiltrating macrophages compared to the SC group (14.1 ± 5.9 cells/field in siCD40; vs. 37.1 ± 17.8 cells/field in siSC; and 1.3 ± 1.7 cells/field in Veh.; p = 0.001). NF-κB activation also peaked in the siSC group, showing lower levels in the siCD40 and Veh. groups (63 ± 60 positive cells/section in siCD40; vs. 152 ± 44 positive cells/ section in siSC; or 26 ± 29 positive cells/section in veh.; p = 0.014). Lastly, serum creatinine was also increased in the siCD40 (3.4 ± 3.3 mg/dL) and siSC (4.6 ± 3.0 mg/dL) groups when compared with Veh. (1.1 ± 0.9 mg/dL, p = 0.1). Conclusions: Anti-CD40 siRNA therapy significantly increased the density of peritubular capillaries and decreased renal inflammation in the ATH model. These data provide a physiological basis for the development of renal diseases in patients with ATH. Furthermore, our results also highligth renal off-target effects of the siRNA treatment which are discussed

Nucleotide Complementarity Features in the Design of Effective Artificial miRNAs

L'importance du miARN dans la régulation des gènes a bien été établie. Cependant, le mécanisme précis du processus de reconnaissance des cibles n'est toujours pas complètement compris. Parmi les facteurs connus, la complémentarité en nucléotides, l'accessibilité des sites cibles, la concentration en espèces d'ARN et la coopérativité des sites ont été jugées importantes. En utilisant ces règles connues, nous avons précédemment conçu des miARN artificiels qui inhibent la croissance des cellules cancéreuses en réprimant l'expression de plusieurs gènes. De telles séquences guides ont été délivrées dans les cellules sous forme de shARN. Le VIH étant un virus à ARN, nous avons conçu et testé des ARN guides qui inhibent sa réplication en ciblant directement le génome viral et les facteurs cellulaires nécessaires au virus dans le cadre de mon premier projet. En utilisant une version mise à jour du programme de conception, mirBooking, nous devenons capables de prédire l'effet de concentration des espèces à ARN avec plus de précision. Les séquences guides conçues fournissaient aux cellules une résistance efficace à l'infection virale, égale ou meilleure que celles ciblant directement le génome viral par une complémentarité quasi-parfaite. Cependant, les niveaux de répression des facteurs viraux et cellulaires ne pouvaient pas être prédits avec précision. Afin de mieux comprendre les règles de reconnaissance des cibles miARN, les règles de couplage des bases au-delà du « seed » ont été approfondies dans mon deuxième projet. En concevant des séquences guides correspondant partiellement à la cible et en analysant le schéma de répression, nous avons établi un modèle unificateur de reconnaissance de cible par miARN via la protéine Ago2. Il montre qu'une fois que le « seed » est appariée avec l'ARN cible, la formation d'un duplex d'ARN est interrompue au niveau de la partie centrale du brin guide mais reprend plus loin en aval de la partie centrale en suivant un ordre distinct. L'implémentation des règles découvertes dans un programme informatique, MicroAlign, a permis d'améliorer la conception de miARN artificiels efficaces. Dans cette étude, nous avons non seulement confirmé la contribution des nucléotides non-germes à l'efficacité des miARN, mais également défini de manière quantitative la manière dont ils fonctionnent. Le point de vue actuellement répandu selon lequel les miARN peuvent cibler efficacement tous les gènes de manière égale, avec uniquement des correspondances de semences, peut nécessiter un réexamenThe importance of miRNA in gene regulation has been well established; however, the precise mechanism of its target recognition process is still not completely understood. Among the known factors, nucleotide complementarity, accessibility of the target sites, and the concentration of the RNA species, and site cooperativity were deemed important. Using these known rules, we previously designed artificial miRNAs that inhibit cancer cell growth by repressing the expression of multiple genes. Such guide sequences were delivered into the cells in the form of shRNAs. HIV is an RNA virus. We designed and tested guide RNAs that inhibit its replication by directly targeting the viral genome and cellular factors that the virus requires in my first project. Using an updated version of the design program, mirBooking, we become capable to predict the concentration effect of RNA species more accurately. Designed guide sequences provided cells with effective resistance against viral infection. The protection was equal or better than those that target the viral genome directly via near-perfect complementarity. However, the repression levels of the viral and cellular factors could not be precisely predicted. In order to gain further insights on the rules of miRNA target recognition, the rules of base pairing beyond the seed was further investigated in my second project. By designing guide sequences that partially match the target and analysing the repression pattern, we established a unifying model of miRNA target recognition via Ago2 protein. It shows that once the seed is base-paired with the target RNA, the formation of an RNA duplex is interrupted at the central portion of the guide strand but resumes further downstream of the central portion following a distinct order. The implementation of the discovered rules in a computer program, MicroAlign, enhanced the design of efficient artificial miRNAs. In this study, we not only confirmed the contribution of non-seed nucleotides to the efficiency of miRNAs, but also quantitatively defined the way through which they work. The currently popular view that miRNAs can effectively target all genes equally with only seed matches may require careful re-examination

Transcriptional and Post-Transcriptional Regulation of SPAST, the Gene Most Frequently Mutated in Hereditary Spastic Paraplegia

Hereditary spastic paraplegias (HSPs) comprise a group of neurodegenerative disorders that are characterized by progressive spasticity of the lower extremities, due to axonal degeneration in the corticospinal motor tracts. HSPs are genetically heterogeneous and show autosomal dominant inheritance in ∼70–80% of cases, with additional cases being recessive or X-linked. The most common type of HSP is SPG4 with mutations in the SPAST gene, encoding spastin, which occurs in 40% of dominantly inherited cases and in ∼10% of sporadic cases. Both loss-of-function and dominant-negative mutation mechanisms have been described for SPG4, suggesting that precise or stoichiometric levels of spastin are necessary for biological function. Therefore, we hypothesized that regulatory mechanisms controlling expression of SPAST are important determinants of spastin biology, and if altered, could contribute to the development and progression of the disease. To examine the transcriptional and post-transcriptional regulation of SPAST, we used molecular phylogenetic methods to identify conserved sequences for putative transcription factor binding sites and miRNA targeting motifs in the SPAST promoter and 3′-UTR, respectively. By a variety of molecular methods, we demonstrate that SPAST transcription is positively regulated by NRF1 and SOX11. Furthermore, we show that miR-96 and miR-182 negatively regulate SPAST by effects on mRNA stability and protein level. These transcriptional and miRNA regulatory mechanisms provide new functional targets for mutation screening and therapeutic targeting in HSP

Rational design of biosafe crop resistance to a range of nematodes using RNA interference

Double stranded RNA (dsRNA) molecules targeting two genes have been identified that suppress economically important parasitic nematode species of banana. Proteasomal Alpha Subunit 4 (pas-4) and Actin-4 (act-4) were identified from a survey of sequence databases and cloned sequences for genes conserved across four pests of banana, Radopholus similis, Pratylenchus coffeae, Meloidogyne incognita and Helicotylenchus multicinctus. These four species were targeted with dsRNAs containing exact 21 nucleotide matches to the conserved regions. Potential off-target effects were limited by comparison to Caenorhabditis, Drosophila, rat, rice and Arabidopsis genomes. In vitro act-4 dsRNA treatment of R. similis suppressed target gene expression by 2.3 fold, nematode locomotion by 66 ± 4% and nematode multiplication on carrot discs by 49 ± 5%. The best transgenic carrot hairy root lines expressing act-4 or pas-4 dsRNA reduced transcript message abundance of target genes in R. similis by 7.9 fold and 4 fold and nematode multiplication by 94 ± 2% and 69 ± 3%, respectively. The same act-4 and pas-4 lines reduced P. coffeae target transcripts by 1.7 and 2 fold and multiplication by 50 ± 6% and 73 ± 8%. Multiplication of M. incognita on the pas-4 lines was reduced by 97 ± 1% and 99 ± 1% while target transcript abundance was suppressed 4.9 and 5.6 fold. There was no detectable RNAi effect on non-target nematodes exposed to dsRNAs targeting parasitic nematodes. This work defines a framework for development of a range of non-protein defences to provide broad resistance to pests and pathogens of crops

Mammalian gene regulation through the 3' UTR

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references.The untranslated region (UTR) at the 30 end of a mammalian mRNA is typically rich with regulatory motifs that influence the stability, localization, translation and other properties of the message. We explored two classes of motifs, microRNA (miRNA) complementary sites and cleavage and polyadenylation (poly(A)) signals, and provide evidence that specific sequence contextual features are important for their recognition. MiRNAs are 22 nt, non-coding RNAs that function as post-transcriptional gene regulators in animals and plants. They typically interact with target mRNAs through base-pairing predominantly between bases 2-8 (the 'seed' region) at the 50 end of the miRNA and complementary sites in the target 30 UTR ('seed matches'). These interactions result in target mRNA translational repression or deadenylation, or both. Through analysis of mRNA expression data following miRNA or siRNA overexpression or inhibition, we uncovered novel targeting determinants that influence mRNA levels. These include the presence of distinct seed match types and sequence context, in particular that increased AU content and conservation were independently associated with greater target down-regulation. Our results demonstrate that mRNA fold change increases multiplicatively (i.e., log-additively) with seed match count. We integrated these features into a target scoring scheme, TargetRank, and demonstrated the effectiveness of our rankings in predicting in vivo target responses. Mammalian genes frequently have multiple, competing poly(A) sites, and the features influencing site selection remain poorly understood. Poly(A) site recognition occurs co-transcriptionally and given that transcription is highly influenced by the tight packaging of genomic DNA into chromatin, we investigated the potential impact of nucleosome positioning on poly(A) site usage. Using recent, public, Illumina sequencing data from human nucleosome boundaries, we found evidence that greater nucleosome density in regions flanking but not overlapping poly(A) sites is associated with more frequent usage.by Cydney Brooke Nielsen.Ph.D

Computational methods for analyzing small RNAs and their interaction partners with large-scale techniques

This thesis describes the computational tools and analyzes developed to characterize small regulatory RNAs and their interaction partners using large-scale techniques. Following an introduction into the emerging world of small regulatory RNAs, our methodology for analyzing small RNAs from deep-sequencing data is described (chapter 2). This methodology allows the classification of small RNAs obtained by sequencing and provides several downstream analysis tools such as expression profiling and miRNA sample comparison. It has been integrated with a miRNA target prediction method into a web server which allows one to explore tissue-specific miRNA targeting (chapter 3). In the fourth chapter, an experimental procedure for genome-wide identification of miRNA targets is outlined. With this procedure, we identified the mRNAs, that are targeted by the most abundant miRNAs in HEK293 cells. Importantly, the experimental protocol enabled us to identify the exact location of the miRNA-mRNA interaction site within the mRNA as well as the precise position of the mRNA-protein crosslink. The fifth and sixth chapter describe our studies of murine embryonic stem cells and oocytes that are devoid of Dicer. The murine specific miR-290 cluster has been identified as an important regulator in embryonic stem cells. The loss of pluripotency in Dicer-/- embryonic stem cells has been linked to primary and secondary targets of the miR-290 cluster. In contrast, our analysis of Dicer-/- oocytes revealed that the miRNA pathway plays only a minor part during oocyte maturation, and loss of Dicer affects mainly the endo-siRNA pathway. Finally, we reanalyzed piRNA sequence reads from various species (chapter 6). This analysis revealed an unexpected 19 nt long processing product which is generated during piRNA biogenesis

Anti-viral RNA silencing: do we look like plants ?

The anti-viral function of RNA silencing was first discovered in plants as a natural manifestation of the artificial 'co-suppression', which refers to the extinction of endogenous gene induced by homologous transgene. Because silencing components are conserved among most, if not all, eukaryotes, the question rapidly arose as to determine whether this process fulfils anti-viral functions in animals, such as insects and mammals. It appears that, whereas the anti-viral process seems to be similarly conserved from plants to insects, even in worms, RNA silencing does influence the replication of mammalian viruses but in a particular mode: micro(mi)RNAs, endogenous small RNAs naturally implicated in translational control, rather than virus-derived small interfering (si)RNAs like in other organisms, are involved. In fact, these recent studies even suggest that RNA silencing may be beneficial for viral replication. Accordingly, several large DNA mammalian viruses have been shown to encode their own miRNAs. Here, we summarize the seminal studies that have implicated RNA silencing in viral infection and compare the different eukaryotic responses

Post-transcriptional regulation of Estrogen Receptor-α by miR-17-92 interaction and LMTK3 phosphorylation in Breast Cancer

Estrogen receptor-α (ERα) is expressed in two-thirds of BCs and is a well-known prognostic and predictive marker. For this reason it is one of the most studied proteins in BC. To understand how ERα positive BC develops, it is crucial to investigate both how this protein is regulated and which genes are modulated by it. MicroRNAs (miRNAs) control gene expression post-transcriptionally by interacting through sequence complementarity to their target transcripts. Through a microarray approach, we identified the subset of miRNAs modulated by ERα, that include up-regulation of miRNAs derived from the processing of two paralogous primary (pri-) transcripts, pri-miR-17-92 and pri-miR-106a-363. Characterisation of the miR-17-92 locus confirmed that the ERα target protein c-MYC binds its promoter in an estrogen-dependent manner. These findings indicated that miRNAs derived from these pri-miRNAs (miR-18a, miR-19b and miR-20b) target and down-regulate ERα, whilst a subset of pri-miRNA-derived mature miRNAs inhibit protein translation of the ERα transcriptional p160 co-activator, AIB1. Therefore, different subsets of the miRNAs identified act as part of a negative autoregulatory feedback loop. We observed that levels of pri-miR-17-92 increase earlier than the mature miRNAs derived from it, implicating precursor cleavage modulation after transcription. Pri-mir-17-92 is immediately cleaved by Drosha to pre-miR-18a, indicating that its regulation occurs during the formation of the mature molecule from the precursors. Furthermore, we wanted to explore the new kinases that regulate the ERα activity. Thereby, we performed kinome screening (by RNAi technologies) to determine kinases that regulate ERα in MCF-7 BC cells and identified a novel kinase, LMTK3, which acts as positive regulator of ERα's transcriptional activity. This could be a new therapeutic target and/or a novel biomarker for BC, although further studies are required to validate this. Together, these studies identify new transcriptional and translational factors that regulate ERα expression in BC.Open Acces