Transcriptomic analysis of subarachnoid cysts of Taenia solium reveals mechanisms for uncontrolled proliferation and adaptations to the microenvironment.

Subarachnoid neurocysticercosis (SANCC) is caused by an abnormally transformed form of the metacestode or larval form of the tapeworm Taenia solium. In contrast to vesicular parenchymal and ventricular located cysts that contain a viable scolex and are anlage of the adult tapeworm, the subarachnoid cyst proliferates to form aberrant membranous cystic masses within the subarachnoid spaces that cause mass effects and acute and chronic arachnoiditis. How subarachnoid cyst proliferates and interacts with the human host is poorly understood, but parasite stem cells (germinative cells) likely participate. RNA-seq analysis of the subarachnoid cyst bladder wall compared to the bladder wall and scolex of the vesicular cyst revealed that the subarachnoid form exhibits activation of signaling pathways that promote proliferation and increased lipid metabolism. These adaptions allow growth in a nutrient-limited cerebral spinal fluid. In addition, we identified therapeutic drug targets that would inhibit growth of the parasite, potentially increase effectiveness of treatment, and shorten its duration.

FUS regulates a subset of snoRNA expression and modulates the level of rRNA modifications.

FUS is a multifunctional protein involved in many aspects of RNA metabolism, including transcription, splicing, translation, miRNA processing, and replication-dependent histone gene expression. In this work, we show that FUS depletion results in the differential expression of numerous small nucleolar RNAs (snoRNAs) that guide 2'-O methylation (2'-O-Me) and pseudouridylation of specific positions in ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Using RiboMeth-seq and HydraPsiSeq for the profiling of 2'-O-Me and pseudouridylation status of rRNA species, we demonstrated considerable hypermodification at several sites in HEK293T and SH-SY5Y cells with FUS knockout (FUS KO) compared to wild-type cells. We observed a similar direction of changes in rRNA modification in differentiated SH-SY5Y cells with the FUS mutation (R495X) related to the severe disease phenotype of amyotrophic lateral sclerosis (ALS). Furthermore, the pattern of modification of some rRNA positions was correlated with the abundance of corresponding guide snoRNAs in FUS KO and FUS R495X cells. Our findings reveal a new role for FUS in modulating the modification pattern of rRNA molecules, that in turn might generate ribosome heterogeneity and constitute a fine-tuning mechanism for translation efficiency/fidelity. Therefore, we suggest that increased levels of 2'-O-Me and pseudouridylation at particular positions in rRNAs from cells with the ALS-linked FUS mutation may represent a possible new translation-related mechanism that underlies disease development and progression.

Collagen synthesis disruption and downregulation of core elements of TGF-ß, Hippo, and Wnt pathways in keratoconus corneas.

To understand better the factors contributing to keratoconus (KTCN), we performed comprehensive transcriptome profiling of human KTCN corneas for the first time using an RNA-Seq approach. Twenty-five KTCN and 25 non-KTCN corneas were enrolled in this study. After RNA extraction, total RNA libraries were prepared and sequenced. The discovery RNA-Seq analysis (in eight KTCN and eight non-KTCN corneas) was conducted first, after which the replication RNA-Seq experiment was performed on a second set of samples (17 KTCN and 17 non-KTCN corneas). Over 82% of the genes and almost 75% of the transcripts detected as differentially expressed in KTCN and non-KTCN corneas were confirmed in the replication study using another set of samples. We used these differentially expressed genes to generate a network of KTCN-deregulated genes. We found an extensive disruption of collagen synthesis and maturation pathways, as well as downregulation of the core elements of the TGF-ß, Hippo, and Wnt signaling pathways influencing corneal organization. This first comprehensive transcriptome profiling of human KTCN corneas points further to a complex etiology of KTCN.

KTCNlncDB-a first platform to investigate lncRNAs expressed in human keratoconus and non-keratoconus corneas.

Keratoconus (KTCN, OMIM 148300) is a degenerative eye disorder characterized by progressive stromal thinning that leads to a conical shape of the cornea, resulting in optical aberrations and even loss of visual function. The biochemical background of the disease is poorly understood, which motivated us to perform RNA-Seq experiment, aimed at better characterizing the KTCN transcriptome and identification of long non-coding RNAs (lncRNAs) that might be involved in KTCN etiology. The in silico functional studies based on predicted lncRNA:RNA base-pairings led us to recognition of a number of lncRNAs possibly regulating genes with known or plausible links to KTCN. The lncRNA sequences and data regarding their predicted functions in controlling the RNA processing and stability are available for browse, search and download in KTCNlncDB (http://rhesus.amu.edu.pl/KTCNlncDB/), the first online platform devoted to KTCN transcriptome.Database URL: http://rhesus.amu.edu.pl/KTCNlncDB/.

siRNA release from pri-miRNA scaffolds is controlled by the sequence and structure of RNA.

shmiRs are pri-miRNA-based RNA interference triggers from which exogenous siRNAs are expressed in cells to silence target genes. These reagents are very promising tools in RNAi in vivo applications due to their good activity profile and lower toxicity than observed for other vector-based reagents such as shRNAs. In this study, using high-resolution northern blotting and small RNA sequencing, we investigated the precision with which RNases Drosha and Dicer process shmiRs. The fidelity of siRNA release from the commonly used pri-miRNA shuttles was found to depend on both the siRNA insert and the pri-miR scaffold. Then, we searched for specific factors that may affect the precision of siRNA release and found that both the structural features of shmiR hairpins and the nucleotide sequence at Drosha and Dicer processing sites contribute to cleavage site selection and cleavage precision. An analysis of multiple shRNA intermediates generated from several reagents revealed the complexity of shmiR processing by Drosha and demonstrated that Dicer selects substrates for further processing. Aside from providing new basic knowledge regarding the specificity of nucleases involved in miRNA biogenesis, our results facilitate the rational design of more efficient genetic reagents for RNAi technology.

Sequence-non-specific effects generated by various types of RNA interference triggers.

RNA interference triggers such as short interfering RNA (siRNA) or genetically encoded short hairpin RNA (shRNA) and artificial miRNA (sh-miR) are widely used to silence the expression of specific genes. In addition to silencing selected targets, RNAi reagents may induce various side effects, including immune responses. To determine the molecular markers of immune response activation when using RNAi reagents, we analyzed the results of experiments gathered in the RNAimmuno (v 2.0) and GEO Profiles databases. To better characterize and compare cellular responses to various RNAi reagents in one experimental system, we designed a reagent series in corresponding siRNA, D-siRNA, shRNA and sh-miR forms. To exclude sequence-specific effects the reagents targeted 3 different transcripts (Luc, ATXN3 and HTT). We demonstrate that RNAi reagents induce a broad variety of sequence-non-specific effects, including the deregulation of cellular miRNA levels. Typical siRNAs are weak stimulators of interferon response but may saturate the miRNA biogenesis pathway, leading to the downregulation of highly expressed miRNAs, whereas plasmid-based reagents induce known markers of immune response and may alter miRNA levels and their isomiR composition.

CANTATAdb: A Collection of Plant Long Non-Coding RNAs.

Long non-coding RNAs (lncRNAs) represent a class of potent regulators of gene expression that are found in a wide array of eukaryotes; however, our knowledge about these molecules in plants is still very limited. In particular, a number of model plant species still lack comprehensive data sets of lncRNAs and their annotations, and very little is known about their biological roles. To meet these shortcomings, we created an online database of lncRNAs in 10 model plant species. The lncRNAs were identified computationally using dozens of publicly available RNA sequencing (RNA-Seq) libraries. Expression values, coding potential, sequence alignments as well as other types of data provide annotation for the identified lncRNAs. In order to better characterize them, we investigated their potential roles in splicing modulation and deregulation of microRNA functions. The data are freely available for searching, browsing and downloading from an online database called CANTATAdb (http://cantata.amu.edu.pl, http://yeti.amu.edu.pl/CANTATA/).

miRNEST 2.0: a database of plant and animal microRNAs.

Ever growing interest in microRNAs has immensely populated the number of resources and research papers devoted to the field and, as a result, it becomes more and more demanding to find miRNA data of interest. To mitigate this problem, we created miRNEST database (http://mirnest.amu.edu.pl), an integrative microRNAs resource. In its updated version, named miRNEST 2.0, the database is complemented with our extensive miRNA predictions from deep sequencing libraries, data from plant degradome analyses, results of pre-miRNA classification with HuntMi and miRNA splice sites information. We also added download and upload options and improved the user interface to make it easier to browse through miRNA records.

mirEX: a platform for comparative exploration of plant pri-miRNA expression data.

mirEX is a comprehensive platform for comparative analysis of primary microRNA expression data. RT-qPCR-based gene expression profiles are stored in a universal and expandable database scheme and wrapped by an intuitive user-friendly interface. A new way of accessing gene expression data in mirEX includes a simple mouse operated querying system and dynamic graphs for data mining analyses. In contrast to other publicly available databases, the mirEX interface allows a simultaneous comparison of expression levels between various microRNA genes in diverse organs and developmental stages. Currently, mirEX integrates information about the expression profile of 190 Arabidopsis thaliana pri-miRNAs in seven different developmental stages: seeds, seedlings and various organs of mature plants. Additionally, by providing RNA structural models, publicly available deep sequencing results, experimental procedure details and careful selection of auxiliary data in the form of web links, mirEX can function as a one-stop solution for Arabidopsis microRNA information. A web-based mirEX interface can be accessed at http://bioinfo.amu.edu.pl/mirex.

Primate and rodent specific intron gains and the origin of retrogenes with splice variants.

Retroposition, a leading mechanism for gene duplication, is an important process shaping the evolution of genomes. Retrogenes are also involved in the gene structure evolution as a major player in the process of intron deletion. Here, we demonstrate the role of retrogenes in intron gain in mammals. We identified one case of "intronization," the transformation of exonic sequences into an intron, in the primate specific retrogene RNF113B and two independent "intronization" events in the retrogene DCAF12L2, one in the common ancestor of primates and rodents and another one in the rodent lineage. Intron gain resulted from the origin of new splice variants, and both genes have two transcript forms, one with retained intron and one with the intron spliced out. Evolution of these genes, especially RNF113B, has been very dynamic and has been accompanied by several additional events including parental gene loss, secondary retroposition, and exaptation of transposable elements.